AN ABSTRACT OF THE DISSERTATION OF
Zhixing Wang for the degree of Doctor of Philosophy in Pharmacy
presented on June 5, 2012.
Title: Transcriptional Regulation Of Mouse Epidermal Permeability
Barrier Development And Homeostasis By Ctip2
Abstract approved________________________________________
Arup Kumar Indra
Skin is the largest organ in the body that protects the organism from
environmental, chemical and physical traumas of each passing day.
The protective skin epidermal permeability barrier (EPB) is formed
within the exterior layers of the epidermis, which are regularly sloughed
off and repopulated by movement of inner cells. The epidermal
permeability barrier is established during in utero development and
maintained through lifetime. Impaired epidermal barrier formation is one
of the major features of several dermatoses such as psoriasis and
atopic dermatitis.
Chicken ovalbumin upstream promoter transcription factor (COUP-TF)interacting protein 2 (Ctip2), also known as Bcl11b, is a C2H2 zinc finger
protein expressed in many organs and tissues. It has been shown to
regulate the development of thymocyte, tooth and corticospinal motor
neurons. Ctip2 is highly expressed in mouse epidermis during skin
organogenesis and in adulthood. It is crucial for epidermal homeostasis
and protective barrier formation in developing mouse embryos.
Germline (Ctip2- null mice) and selective ablation of Ctip2 in mouse
epidermis (Ctip2ep-/- mice) leads to increased transepidermal water loss
(TEWL), impaired epidermal proliferation and terminal differentiation as
well as altered lipid distribution during embryogenesis. Sphingolipids
account for ~50% of total skin lipids by weight and are crucial
components of epidermal barrier. We have recently identified Ctip2 as a
key regulator of skin lipid metabolism. Germline deletion of Ctip2 in
mouse embryos leads to altered lipid composition in the developing
mouse epidermis by modulating the expression levels of key enzymes
involved in lipid metabolism (bio-synthesis and catabolism). We also
demonstrated that Ctip2 is recruited to the promoter regions of several
genes involved in the ceramide and sphingomyelin biosynthesis
pathways and could directly regulate their expression. Thus, we have
identified Ctip2 as a key regulator of several lipid metabolizing genes
and
hence
epidermal
sphingolipid
biosynthesis
during
skin
development.
To study the role of Ctip2 in adult skin homeostasis, we have utilized
Ctip2ep-/- mouse model in which Ctip2 is selectively deleted in epidermal
keratinocytes. We showed that keratinocytic ablation of Ctip2 leads to
atopic dermatitis (AD)-like skin inflammation, characterized by alopecia,
pruritus and scaling, as well as high infiltration of T lymphocytes and
immune cells. We have also observed increased expression of Th2type cytokines and chemokines in the mutant skin, as well as systemic
immune responses that share similarity with human AD patients.
Furthermore, we discovered that thymic stromal lymphopoietin (TSLP)
expression is significantly upregulated in the mutant epidermis as early
as postnatal day 1 and Ctip2 was recruited to the promoter region of the
TSLP gene in mouse epidermal keratinocytes. The results suggest that
upregulation of TSLP expression in the Ctip2ep-/- epidermis could be due
to a derepression of gene transcription in absence of Ctip2. Thus, our
data demonstrated a cell-autonomous role of Ctip2 in barrier
maintenance and epidermal homeostasis in adult skin, as well as a
non-cell autonomous role of keratinocytic Ctip2 in suppressing skin
inflammatory responses by regulating the expression of Th2-type
cytokines in adult mouse skin. Present results establish an initiating
role of epidermal TSLP in AD pathogenesis via a novel repressive
regulatory mechanism mediated by Ctip2 in mouse epidermal
keratinocytes.
Altogether, our study indicates that Ctip2 could be involved in a diverse
range of biological events in skin including barrier formation,
maintenance and epidermal homeostasis. Ctip2 appears to be a master
regulator in skin barrier functions by directly regulating the transcription
of a subset of genes involved in lipid metabolism and inflammatory
responses.
©Copyright by Zhixing Wang
June 5, 2012
All Rights Reserved
Transcriptional Regulation Of Mouse Epidermal Permeability Barrier
Development And Homeostasis By Ctip2
by
Zhixing Wang
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Doctor of Philosophy
Presented June 5, 2012
Commencement June, 2013
Doctor of Philosophy dissertation of Zhixing Wang presented on June 5,
2012.
APPROVED:
_______________________________________
Major Professor, representing Pharmacy
_______________________________________
Dean of the College of Pharmacy
_______________________________________
Dean of the Graduate School
I understand that my dissertation will become part of the permanent
collection of Oregon State University libraries. My signature below
authorizes release of my dissertation to any reader upon request.
Zhixing Wang, Author
DEDICATION
I would like to dedicate my thesis to my parents.
CONTRIBUTION OF AUTHORS
Dr. Arup Kumar Indra helped design research experiments and Zhixing
Wang primarily performed the research. Zhixing Wang and Dr. Arup
Kumar Indra analyzed most of the data and wrote the manuscripts.
Chapter 2: Zhixing Wang performed most of the experiments and
analyzed the data. Jay Kirkwood and Alan Taylor performed Mass
spectrometry experiments. Drs. Jan F. Stevens, Mark Leid and Gitali
Ganguli-Indra provided valuable suggestions and criticism for the
manuscript.
Chapter 3: Zhixing Wang performed most of the experiments. Dr. Lingjuan Zhang assisted with sample collection, and contributed to Figure
3.7, 3.8, 3.10 and 3.13. Dr. Gunjan Guha did ELISA in Figure 3.9. Dr.
Chrissa Kioussi provided the transgenic mice. Drs. Mark Leid and Gitali
Ganguli-Indra provided valuable suggestions during the course of the
conducted research.
TABLE OF CONTENTS
Page
Chapter 1 General Introduction .............................................................. 2
1.1 Skin homeostasis and human health ............................................ 3
1.1.1 The structure and morphogenesis of mammalian skin ........... 3
1.1.2 Skin permeability barrier formation ......................................... 4
1.1.3 Lipids in epidermal permeability barrier .................................. 5
1.1.4 Impaired barrier and skin inflammatory disorders................... 6
1.2 COUP-TF interacting proteins ....................................................... 9
1.2.1 Nuclear receptors (NRs) ......................................................... 9
1.2.2 Chicken ovalbumin upstream promoter transcription factors
(COUP-TFs)................................................................................... 10
1.2.3 COUP-TF interacting proteins .............................................. 11
1.2.4 COUP-TF interacting protein 1 (Ctip1) ................................. 12
1.2.5 COUP-TF interacting protein 2 (Ctip2) ................................. 13
1.3 The role of Ctip2 in skin development and homeostasis ............ 15
1.3.1 Ctip2 expression in mouse developing skin.......................... 15
1.3.2 Ctip2 expression in adult mouse skin ................................... 16
1.3.3 Ctip2 in epidermal barrier formation ..................................... 16
1.3.4 Ctip2 in epidermal surface lipid distribution .......................... 18
1.3.5 Ctip2 and cornified envelop (CE) formation .......................... 18
1.4 CTIP2 and skin diseases ............................................................ 19
1.4.1 Expression of CTIP2 in human head and neck squamous cell
carcinoma (HNSCC) ...................................................................... 19
1.4.2 Role of Ctip2 in cutaneous wound healing ........................... 21
1.5 Research objectives .................................................................... 23
1.6 References .................................................................................. 31
TABLE OF CONTENTS (Continued)
Page
Chapter 2 Transcription factor Ctip2 controls epidermal lipid metabolism
and regulates expression of genes involved in sphingolipid biosynthesis
during skin development ...................................................................... 40
2.1 Abstract ....................................................................................... 41
2.2 Introduction ................................................................................. 42
2.3 Materials and methods ................................................................ 45
2.4 Results ........................................................................................ 51
2.4.1 Altered profile of sphingolipids in the developing epidermis of
Ctip2-null mice ............................................................................... 52
2.4.2 Ctip2 directly regulates expression of a subset of genes
involved in lipid metabolism in the developing skin ....................... 54
2.5 Discussion ................................................................................... 58
2.6 References .................................................................................. 78
Chapter 3 Selective Ablation of Ctip2/Bcl11b in Epidermal
Keratinocytes Triggers Atopic Dermatitis-like Skin Inflammatory
Responses in Adult Mice ...................................................................... 84
3.1 Abstract ....................................................................................... 84
3.2 Introduction ................................................................................. 87
3.3 Materials and Methods ................................................................ 90
3.4 Results ........................................................................................ 97
3.4.1 Selective ablation of Ctip2 in epidermal keratinocytes leads to
altered epidermal proliferation, differentiation and spontaneous
dermatitis in adult skin ................................................................... 97
3.4.2 Preferential induction of Th2-type cytokines and chemokines
in adult Ctip2ep-/- skin ................................................................... 100
TABLE OF CONTENTS (Continued)
Page
3.4.3 TSLP is a direct target of Ctip2 in epidermal keratinocytes 102
3.4.4 Systemic inflammatory responses in older Ctip2ep-/- mice .. 103
3.5 Discussion ................................................................................. 105
3.6 References ................................................................................ 132
Chapter 4 General Conclusion ........................................................... 138
Bibliography .................................................................................... 146
LIST OF FIGURES
Figure
Page
Figure 1.1 Schematic representation of epidermal permeability barrier.
(Adapted from Segre et al., 2006). ................................................ 24
Figure 1.2 Amino acid sequence, diagram of amino acid sequence
alignment of CTIP1 and CTIP2. (Taken from Avram et al., 2000) . 25
Figure 1.3 Expression of Ctip2 in the mouse fetal skin. (Adapted from
Golonzhka et al., 2007).................................................................. 27
Figure 1.4 Ctip2 mutant mice exhibit permeability barrier defects and
epidermal hypoplasia. (Adapted from Golonzhka et al., 2007) ...... 29
Figure 1.5 Ctip2 is required for the formation and integrity of cornified
envelope in mouse epidermis. ....................................................... 30
Figure 2.1 Liquid chromatography tandem mass-spectrometry
(LC/MS/MS) analyses of mouse epidermal saturated ceramides
during skin development. ............................................................... 63
Figure 2.2 Liquid chromatography tandem mass-spectrometry
(LC/MS/MS) analyses of mouse epidermal unsaturated ceramides
during skin development. ............................................................... 64
Figure 2.3 LC/MS/MS analyses of mouse epidermal saturated
sphingomyelins during skin development. ..................................... 65
Figure 2.4 LC/MS/MS analyses of mouse epidermal unsaturated
sphingomyelins during skin development. ..................................... 66
Figure 2.5 LC/MS/MS analyses of mouse epidermal sphingosines and
sphinganines during skin development.......................................... 67
Figure 2.6 UPLC/MS/MS analyses of mouse epidermal cholesterol and
fatty acids during skin development............................................... 68
LIST OF FIGURES (Continued)
Figure
Page
Figure 2.7 Ctip2 regulates the expression of genes encoding lipid
metabolizing enzymes during skin development. .......................... 69
Figure 2.8 Relative expression of genes encoding lipid-metabolizing
enzymes during skin development. ............................................... 70
Figure 2.9 Immunoblot analyses of lipid metabolizing enzymes in the
developing murine epidermis. ........................................................ 71
Figure 2.10 Ctip2 interacts directly with the promoter region of lipid
metabolizing genes. ....................................................................... 72
Figure 2.11 ChIP analyses on murine keratinocytes for lipid
metabolizing genes. ....................................................................... 74
Figure 2.12 Schematic representation of Ctip2 mediated differential
regulation of genes encoding different enzymes of the ceramide
synthesis pathways........................................................................ 75
Figure 3.1 Ctip2ep-/- mice develop a chronic skin lesions and epidermal
hyperplasia. ................................................................................. 114
Figure 3.2 Ctip2ep-/- mice develop enhanced epidermal proliferation. 116
Figure 3.3 Ablation of Ctip2 leads to increased epidermal terminal
differentiation. .............................................................................. 117
Figure 3.4 Enhanced eosinophil and mast cell infiltrates in dorsal skin of
Ctip2ep-/- adult mice. ..................................................................... 118
Figure 3.5 Characterization of inflammatory cell infiltrates in dorsal skin
of WT and in Ctip2ep-/- adult mice. ................................................ 119
LIST OF FIGURES (Continued)
Figure
Page
Figure 3.6 Increased inflammatory cell infiltrate in Ctip2ep-/- adult mice.
..................................................................................................... 121
Figure 3.7 Th2-dependent cytokine and chemokine expression levels in
WT and Ctip2ep-/- skin. ................................................................. 123
Figure 3.8 Relative expression levels of cytokines and chemokines in
WT and Ctip2ep-/- skin. ................................................................. 125
Figure 3.9 Systemic immunological abnormalities in Ctip2ep-/- adult
mice. ............................................................................................ 126
Figure 3.10 Ctip2ep-/- adult mice exhibited systemic immune responses.
..................................................................................................... 127
Figure 3.11 Immunological abnormalities of spleen and lymph node in
Ctip2ep-/- adult mice. ..................................................................... 128
Figure 3.12 Relative expression levels of RXRa and genes involved in
Notch signaling pathway. ............................................................. 129
Figure 3.13 Relative expression levels of FLG in 1w and 2w mice
epidermis and dermis. ................................................................. 130
LIST OF TABLES
Table
Page
Table 2.1 Primer sequences used in q-PCR assays. ........................... 76
Table 2.2 Primer sequences used in ChIP assays............................... 77
Table 3.1 List of primers used for RT-qPCR and ChIP assays .......... 131
Transcriptional Regulation Of Mouse Epidermal Permeability Barrier
Development And Homeostasis By Ctip2
2
Chapter 1
General Introduction
3
1.1 Skin homeostasis and human health
1.1.1 The structure and morphogenesis of mammalian skin
Skin is the largest organ in the body that protects the organism from
environmental, chemical and physical traumas of each passing day
(Lippens et al., 2009). Skin is composed of epidermis and dermis,
which are separated by a basement membrane of extracellular matrix
(Fuchs and Raghavan, 2002). The epidermis is a stratified epithelium
that forms the uppermost compartment of the skin and is composed of
four layers: basal, spinous, granular and stratum corneum (Fuchs and
Raghavan, 2002). Keratinocytes are the most abundant cell type in the
epidermis, that move from proliferative cells in the basal layer through
the granular layer to form cornified envelope, finally to stratum corneum
as flattened, dead cells (Candi et al., 2005) (Figure 1.1).
The mouse epidermis develops from a single‐layered multipotent
embryonic ectoderm, which is highly proliferative (Mack et al., 2005).
Skin proliferation markers keratin 5 (K5) and keratin 14 (K14)
expressions were observed as early as embryonic day 9.5 (E9.5),
facilitating the stratification events that lead to the formation of the
periderm (E9‐E12), which overlies the ectoderm (Byrne et al., 2003;
Mack et al., 2005). After a series of proliferation and terminal
differentiation events, the epidermis is fully formed by E18.5, consisting
4
of three live morphologically distinct living layers (basal, spinous, and
granular) and a dead (cornified) layer (Byrne et al., 2003; Mack et al.,
2005) (Figure 1.1).
1.1.2 Skin permeability barrier formation
Barrier function is mainly conferred by the outermost layer of the
epidermis, the stratum corneum (Hardman et al., 1998). The process of
terminal differentiation starts when basal cells withdraw from the cell
cycle and detach from the basement membrane. In the spinous layer,
the cells are reinforced by a cytoskeletal framework of keratin filaments.
Lamellar bodies in the granular layer are the reservoir of lipids. Keratins
bundled with macrofibrils though filaggrin, and cornified envelopes
(CEs) are assembled by precursor proteins underneath plasma
membranes such as transglutaminase (TGM). As the cell membrane
disintegrates, transglutaminase crosslink the CE proteins irreversibly,
creating tough insoluble sac surrounding the keratin fibers.
Finally,
lipids are extruded onto the CE scaffold and the epidermal permeability
barrier is formed (Segre, 2006). Once built, this barrier has a “brick and
mortar” structure, with keratin macrofibrils and CEs as bricks and the
lipids as the mortar to seal together the CEs (Elias, 1983; Nemes and
Steinert, 1999) (Figure 1.1).
5
1.1.3 Lipids in epidermal permeability barrier
The exterior layer of skin, the stratum corneum (SC), plays a critical role
in skin permeability barrier formation and maintenance. Mammalian SC
contains a large number of lipids, such as ceramides, sphingomyelins,
cholesterols and fatty acids (Elias and Menon, 1991; Ishikawa et al.,
2010). Ceramides (CERs) are the most abundant lipid types in the SC
(50% by weight) and can be divided into at least 11 species (Oda et al.,
2008; Ishikawa et al., 2010). It has been shown that reduced content of
CERs leads to epidermal water loss as well as skin epidermal
dysfunctions, and causes diseases such as atopic dermatitis (AD)
(Imokawa, 2009; Ishikawa et al., 2010). Spingomyelins are another
large group of epidermal sphingolipids, which have been reported as
precursors of ceramides (Schmuth et al., 2000; Uchida et al., 2000;
Holleran et al., 2006). Cholesterols and fatty acids are also major skin
lipids, while cholesterol 3-sulfate (CSO4) is a ubiquitous metabolite of
cholesterol, which is involved in cornified envelope formation by
interacting with transglutaminase1 (Nemes et al., 2000). A large variety
of skin fatty acids are synthesized in epidermis, and disruption of the
skin permeability barrier leads to a rapid and marked increase in fatty
acid synthesis by upregulating the activity and mRNA levels of several
key enzymes involved in fatty acid biosynthesis (Feingold, 2007, 2009).
6
Furthermore, following acute barrier disruption, the recovery of
permeability barrier function is delayed, correlating with reduced fatty
acid synthesis (Feingold, 2007). Moreover, the elongation of fatty acids
is also critical as mice deficient in elongation of very-long-chain fatty
acid-like 4 (Elovl4) have a severely disrupted permeability barrier and
die perinatally (Cameron et al., 2007; Li et al., 2007; Vasireddy et al.,
2007).
1.1.4 Impaired barrier and skin inflammatory disorders
The epidermal permeability barrier is established during in utero
development
and
maintained
throughout
the
lifetime.
Impaired
epidermal barrier is one of the major features of several dermatoses
such as psoriasis and atopic dermatitis, which are two of the most
common inflammatory skin disorders (Ghadially et al., 1996; Segre,
2006).
Psoriasis is a chronic inflammatory skin disease that shows distinct
erythrosquamous silvery scaly skin either in the areas of inflection or
the entire body (Segre, 2006). Psoriasis has a 2%-3% of prevalence in
western populations, affecting mostly adults. Psoriatic skin is
characterized by inflammation, hyperproliferation, and abnormal
7
differentiation. Psoriasis was initially identified as a Th1-dependent
disease due to the observation of T helper type 1 (Th1) cell secreted
cytokines such as interleukin-1 (IL-1), tumor necrosis factor alpha
(TNFα) and interferon gamma (INFG) (Gudjonsson et al., 2010).
However, studies performed in psoriatic lesions have also identified
cytokines such as IL-17 and IL-22 that are produced by Th17 cells
(Roberson and Bowcock, 2010). The causes of psoriasis have not yet
been fully understood; however, genes involved in skin barrier
formation such as loricrin (LOR), involucrin (IVL), the small proline rich
proteins (SPRRs) and late cornified envelope proteins (LCEs) were
altered in the skin of psoriasis patients, indicating a correlation between
psoriasis and skin barrier defects (Roberson and Bowcock, 2010).10%30% of psoriasis patients develop psoriatic arthritis, which causes
destruction of joints if not treated aggressively (Nograles et al., 2009).
Psoriasis also increases the risk of heart diseases and stroke (Gelfand
et al., 2006, 2009).
Atopic dermatitis (AD) is a chronic, genetically predisposed skin
inflammatory disease that starts at early childhood with a prevalence of
10%-20% in children and 1%-3% in adults (Elias and Menon, 1991;
Ishikawa et al., 2010). Clinical features of AD include skin xerosis,
8
pruritus and eczematoid skin lesion (Leung and Bieber, 2003). AD is
characterized by both skin barrier deficiencies and immunological
responses (Scharschmidt and Segre, 2008). In AD, a defective skin
barrier is thought to permit the penetration of allergens and induces the
interactions of the allergens with immune cells, promoting the
subsequent release of pro-inflammatory cytokines and chemokines and
elevation of IgE level (Cork et al., 2009). The molecular pathways
involved in AD pathogenesis remain unclear. Distinct from psoriasis,
there have been many reports on the connections between atopic
dermatitis and Th2 inflammatory pathways. Pro-Th2 cytokines such as
IL-4, IL-13, IL-5, and IL10 are elevated in AD patients (B. Brandt and
Sivaprasad, 2011). One possible candidate that initiates inflammatory
responses in AD is thymic stromal lymphopoietin (TSLP), which is a
member of hematopoietic cytokine family and highly expressed in skinderived epithelial cells and also in fibroblasts and dendritic cells
(Comeau and Ziegler, 2010; Ziegler, 2010; De Monte et al., 2011;
Kashyap et al., 2011; Yamada et al., 2012). TSLP signals through a
heterodimeric receptor formed by TSLP receptor (TSLPR) as well as IL7 receptor alpha (IL7Ra) (Comeau and Ziegler, 2010). TSLP is a key
keratinocyte-derived cytokine, whose expression was greatly increased
in the epidermis of a group of acute and chronic atopic dermatitis
9
patients (Soumelis et al., 2002). In mouse models with skin barrier
deficiencies, mutation of genes (e.g. RXRα, VDR or Notch1) that are
involved in barrier functions also leads to TSLP induction, subsequently
causing AD-like phenotypes (Li, 2005, 2006; Demehri et al., 2008;
Ziegler, 2010).
1.2 COUP-TF interacting proteins
1.2.1 Nuclear receptors (NRs)
Nuclear receptors (NRs) are a class of proteins that interact with steroid
and thyroid hormones, as well as certain other proteins, to regulate
transcription in response to ligands. By regulating the expression of
specific genes, NRs control diverse aspects of the organism such as
embryonic development and adult homeostasis (Mangelsdorf et al.,
1995; Robinson-Rechavi et al., 2003).
NRs have the ability to bind to DNA and regulate the expression of
adjacent genes when activated by ligands; hence, they are classified as
members of the transcription factor family of proteins (Evans, 1988;
Olefsky, 2001). NRs are divided into two broad classed according to
their mechanism of action. Type I NRs are located in the cytosol in the
absence of the ligand. Hormone binding facilitates the dissociation of
10
heat shock protein (HSP) from NR, allowing NR dimerization and
translocation to the nucleus to bind to genomic DNA. The NR/DNA
complex recruits other proteins to regulate transcription and eventually
the cell functions. Members of Type I NRs include androgen receptor
(AR),
estrogen
receptor
(ER),
glucocorticoid
receptor
(GR),
progesterone receptor, as well as orphan receptors (Linja, 2004). In
contrast to Type I, Type II NRs are located in the nucleus regardless of
the presence of ligands. Without ligands, Type II NRs bind to the corepressor proteins; however, ligand binding leads to dissociation of corepressor and recruitment of the co-activator proteins to regulate gene
expression. Retinoic acid receptors (RAR α, β and γ), retinoid X
receptors (RXR α, β and γ) and thyroid hormone receptor (TR) are
members of the Type II NRs (Mangelsdorf et al., 1995; Klinge et al.,
1997; 1999).
1.2.2 Chicken ovalbumin upstream promoter transcription factors
(COUP-TFs)
Chicken ovalbumin upstream promoter transcription factors (COUPTFs) were originally identified to bind to the upstream promoter region
of the ovalbumin gene to activate its transcription (Pastorcic et al.,
1986; Sagami et al., 1986; Wang et al., 1987; Pereira et al., 1995).
11
They are members of the Type I NR superfamily and classified as
orphan receptors as no ligand has been identified for COUP-TFs to
date (Pereira et al., 1995; Kruse et al., 2008).
COUP-TFs are generally considered as transcription repressors with
different mechanisms of action. For example, COUP-TF has been
shown to bind promiscuously to a variety of different repeats including
the AGGTCA motif and hormone response elements of VDR, TR and
RAR, leading to silencing of basal transcription activity (Tsai, 1997). It’s
also well known that COUP-TF forms heterodimers with RXRs, which is
a universal partner for other nuclear receptors such as VDR, RAR,
PPAR and other orphan receptors (Tsai, 1997). This mechanism may
involve the recruitment of nuclear receptor repressor (NCoR) and
silencing mediator for retinoid and thyroid hormone receptor (SMRT),
which bind to RAR and TR in the absence of ligands (Tsai, 1997). Other
possible mechanisms involve active repression and transrepression
(Tsai, 1997).
1.2.3 COUP-TF interacting proteins
COUP-TF interacting proteins (CTIPs) were observed to interact
directly with COUP-TF family members (COUP-TFI/NR2F1, COUPTFII/ARP1/NR2F2 and COUP-TFIII/EAR2/NR2F6) in yeast and in vitro
12
(Avram et al., 2000). CTIP1 (BCL11A, EVI9) and CTIP2 (BCL11B, RIT1b) are highly related members of the CTIP family, which share high
sequence identity over the entire protein, and contain several
conserved C2H2 zinc finger motifs (Avram et al., 2000) (Figure 1.2).
They have been shown to repress the transcription of genes harbouring
COUP-TF regions by binding to the canonical GC box (Avram et al.,
2002). As mediators of transcriptional repression, their activity has been
found to be insensitive to reversal by trichostatin A (TSA), an inhibitor of
histone deacetylases (HDACs) ( Avram et al., 2000; Topark-Ngarm et
al., 2006).
1.2.4 COUP-TF interacting protein 1 (Ctip1)
Developmental studies in mice have shown that expression of Ctip1
starts as early 10.5 days post-coitum embryos (P10.5) in a diffused
pattern in forelimb bud, branchial arches and CNS (Avram et al., 2000;
Leid et al., 2004). Its expression became more restricted at later stages
at E12.5 and E14.5 with the highest expression in the developing
forebrain (Leid et al., 2004). CNS expression of Ctip1 remains strong
with low diffuse expression throughout the E18.5 fetus (Leid et al.,
2004).
13
In humans and mice, CTIP1 is also expressed in several hematopoietic
tissues such as bone marrow, splenic B and T cells, monocytes and
germinal B cells (Liu et al., 2003). Several reports indicated the
important role of CTIP1 as a myeloid or B cell proto-oncogene in mice
and humans as it is a common site of retroviral integration in myeloid
leukemia (Nakamura et al., 2000; Satterwhite, 2001; Uda et al., 2008).
CTIP1 is also required for normal lymphoid development as well as
human fatal hemoglobin expression (Liu et al., 2003; Sankaran et al.,
2008).
1.2.5 COUP-TF interacting protein 2 (Ctip2)
Both mouse Ctip2 and human CTIP2 gene contains 4 exons with the
majority of its open reading frame (ORF) encoded by exon 4 (Figure
1.2). The expression of Ctip2 during mouse development shares a
similar pattern with Ctip1. It is expressed diffusely at E10.5 and
becomes more restricted at E12.5 in the same region as Ctip1, except
in the cerebellar domain as well as mandibular and nasal mesenchyme
(Leid et al., 2004). A relatively high expression level of Ctip2 was
observed to be expressed in thymus at E14.5 when most of the thymic
cells are CD4-CD8-. The thymus becomes the most dominant organ
that expresses Ctip2 at E18.5. Ctip2 expression has been detected in
14
the developing mice skin and in adult mice and human skin (Golonzhka
et al., 2007; Ganguli-Indra et al., 2009a) Ctip2 expression was found to
be upregulated in the skin of human AD patients and allergic contact
dermatitis (ACD) patients (Ganguli-Indra et al., 2009a)
Ctip2 has been reported to interact with nucleosome remodelling and
deacetylation (NuRD) complex with histone deacetylase activity and
repress the transcription of target genes such as p57KIP2 (ToparkNgarm et al., 2006). However, new evidence suggested the role of
Ctip2 as a transcriptional activator. One-third of all Ctip2-regulated
genes in thymocytes are activated by Ctip2 (Kastner et al., 2010).
Ablation of Ctip2 in mouse embryonic skin leads to downregulation of
several genes involved in skin lipid homeostasis (Wang et al, 2012,
submitted). Hence, Ctip2 could also activate transcription under certain
biological conditions.
As Ctip2 null mice die perinatally, several studies have investigated the
possible causes of the lethality. Ctip2 null mice show defects in
thymocyte development and rearranged T cell receptors (TCR)
signalling, suggesting Ctip2 is required in differentiation and survival of
T lymphocytes (Wakabayashi et al., 2003). Ctip2 also plays a critical
role in the development of corticospinal motor neuron (CSMN) axonal
projections to the spinal cord in vivo (Arlotta et al., 2005). Thymocytes
15
isolated from Ctip2 null newborn mice exhibit increased vulnerability to
DNA replication stress and damage, which leads to increased apoptosis
(Kamimura et al., 2007; Karanam et al., 2010).
Recent reports
suggested crucial roles of Ctip2 in tooth and skin development
(Golonzhka et al., 2007, 2008, 2009).
1.3 The role of Ctip2 in skin development and homeostasis
1.3.1 Ctip2 expression in mouse developing skin
Ctip2 expression is detected in the ectoderm as early as E10.5 in the
outermost layer of skin. Some of the positive cells also express keratin
14 (K14), which is the marker of the basal layer (Golonzhka et al.,
2007). Embryonic basal layer is formed at E12.5, in which every cell is
found to express Ctip2 (Golonzhka et al., 2007). Strong expression of
Ctip2 is observed at E14.5, mainly in the basal layer and suprabasal
layer of the epidermis. Further investigation of Ctip2 expression was
performed on E16.5 and E18.5 mouse skin. Ctip2 expression remains
high in the basal layer at both stages with small groups of positive cells
in the suprabasal layer. Also, Ctip2 expression was found in hair
follicles and hair bulbs at later developmental stages with a few positive
16
cells in the dermis (Golonzhka et al., 2007).
Ctip2 expression is
overlapping with cell proliferation marker Ki67 positive signals in the
epidermis and hair follicles at majority of developmental stages,
indicating that Ctip2 positive cells are highly proliferative during skin
development (Golonzhka et al., 2007) (Figure 1.3).
1.3.2 Ctip2 expression in adult mouse skin
Similar to embryonic skin, in 8-10 week-old mouse skin, Ctip2 is
expressed in most of the cells in the basal layer, and some cells in the
suprabasal layer and hair follicles.
However, Ctip2 can be barely
detected in the dermal compartment. Compared with E18.5 embryos,
the expression level is much lower in the adult skin (Golonzhka et al.,
2007). Ctip2 is also expressed in the bulge region of adult mouse hair
follicles, which harbour stem cells and contribute to the generation of
hair follicle and inter-follicular epidermis (Golonzhka et al., 2007).
1.3.3 Ctip2 in epidermal barrier formation
As Ctip2 null mice die after birth, it’s likely that these mice have
epidermal defects, together with other developmental abnormality. An
X-gal diffusion assay was performed to determine the barrier function at
17
E17.5 and E18.5. Ctip2 null mice, but not wildtype, show strong X-gal
staining at E17.5, especially on the ventral surface and head, indicating
a delayed dorsal to ventral barrier establishment in the null mice
(Golonzhka et al., 2008) (Figure 1.4.A). Similar assays performed at
E18.5 didn’t reveal any significant difference between wildtype and null
mice (Figure 1.4.A). Trans-epidermal water loss (TEWL) studies
indicate that Ctip2 null embryos have significant higher water loss
compared with wildtype at E17.5 and E18.5 (Figure 1.4.B). Ctip2 null
mice also exhibited reduced epidermal thickness at E17.5 with defects
in proliferation and differentiation (Golonzhka et al., 2008) (Figure
1.4.D-E). Real-time quantitive PCR (RT-qPCR) studies performed on
wildtype and Ctip2 null mice revealed downregulation of genes involved
in epidermal terminal differentiation and barrier formation in the null
mice, such as transglutaminase-1 (Tgase1), Gata3, and caspase-14
(Golonzhka et al., 2008). Hence, Ctip2 regulates epidermal barrier
formation during embryonic development by modulating the expression
of genes involved in barrier formation and terminal differentiation
(Golonzhka et al., 2008).
18
1.3.4 Ctip2 in epidermal surface lipid distribution
By Nile Red staining, dense and evenly distributed neutral lipids were
stained in the wildtype fetus along the cornified layer, while uneven and
thinner neutral lipid layer was observed in the Ctip2 null mice at E17.5
(Golonzhka et al., 2008) (Figure 1.4.C). The ultrastructure of the
epidermis also revealed defects in lipid disc formation, loosely packed
corneocytes, as well as disorganized intercellular lamellar body
membranes in the cornified layer of Ctip2 null mice compared with
wildtype (Golonzhka et al., 2008). These results indicated impaired lipid
distribution following ablation of Ctip2 during development (Golonzhka
et al., 2008).
The defects in neutral lipid distribution and irregular lamellar body
formation in the mutant mice could be due to changed gene expression
profile. Several genes involved in lipid homeostasis were studied with
RT-qPCR. Dgat2, Elovl4, eLox3 and Alox12b were downregulated in
the null embryos at E17.5, suggesting that Ctip2 could regulate the
expression of lipid homeostasis genes, thus contributing to lipid barrier
formation during development (Golonzhka et al., 2008).
1.3.5 Ctip2 and cornified envelop (CE) formation
Ctip2 also has an important role in epidermal barrier formation by
regulating the formation and integrity of CEs (Figure 1.5, unpublished
19
data). CE suspension was sonicated for different lengths of time and
the morphology of the CEs were monitored using a phase contrast
microscope. The mutant CEs were less in number, morphologically
abnormal and irregular in shape compared to the wildtypes. Also, the
mutant CEs were more fragile during sonication, and were completely
destroyed after 2 minutes of sonication. Thus, Ctip2 may regulate the
epidermal barrier by involving in the formation of functional and integral
CEs.
1.4 CTIP2 and skin diseases
1.4.1 Expression of CTIP2 in human head and neck squamous cell
carcinoma (HNSCC)
Head and neck cancer is the 6th most common cancer in the world. In
United States, head and neck cancer accounts for less than 5% of all
cancers and less than 3% of all cancer deaths. The most common type
of head and neck cancer is head and neck squamous cell carcinoma
(HNSCC) (Hunter et al., 2005). Disappointingly, the survival rate of this
disease cannot be improved even with aggressive and multidisciplinary
treatment and medicine currently available. The localization of CTIP2 in
20
human HNSCC was studied by immunohistochemical staining (GanguliIndra et al., 2009b). In tumor adjacent normal epithelium tissue, CTIP2
is exclusively observed in the nucleus of basal layer keratinocytes. In
dysplasia samples, the expression of CTIP2 is much stronger than
normal tissue, with extended expression in the spinous layer. In welldifferentiated tumors, CTIP2 expresses in basal layer of the keratinized
horn pearl. Moderately and poorly differentiated tumors showed
homologous and strong CTIP2 staining (Ganguli-Indra et al., 2009b).
Overall, increased expression of CTIP2 correlated well with the
progression of carcinoma (Ganguli-Indra et al., 2009b). CTIP2
expression has been reported to be linked to poor differentiation status
of the tumor (Ganguli-Indra et al., 2009b).
The expression level of CTIP2 in human cell lines confirmed above
results. Low level of CTIP2 is found in primary human epidermal
keratinocytes
(Hek-a)
and
in
spontaneously
immortalized
non-
tumorigenic human skin keratinocytes (Hacat), while high expression of
CTIP2 has been detected in head and neck carcinoma cell lines (SCC4,
SCC25, etc) and other carcinomas (Ganguli-Indra et al., 2009b).
Altogether, results indicate that CTIP2 expression is linked to HNSCC
progression.
21
1.4.2 Role of Ctip2 in cutaneous wound healing
Cutaneous wound healing is a highly organized process involving
crosstalk between different cell types, epidermal barrier homeostasis
recovery, as well as re-epithelialization carried out by cell migration and
proliferation (Gurtner et al., 2008). Various pathways regulate this
process with key factors such as integrins, stem cell markers, growth
factors, cytokines as well as other regulatory proteins (Werner et al.,
2007; Barrientos et al., 2008; Liang et al., 2012).
Tape stripping creates a model for mechanical skin injury in mouse
skin. It induces transient epidermal hyperplasia with altered epidermal
proliferation and differentiation (Oyoshi et al., 2010). Another model to
study more several mechanical injuries is to create an excisional wound
in mouse skin and monitor the healing process by measurement of the
gap between the migratory tongues from the wound site (Wong et al.,
2011). In wildtype mice, the expression level of Ctip2 increases after
24-48 hours post tape stripping, and 7-11 days post wounding, mainly
expressed in epidermal basal layer as well as post-mitotic suprabasal
layers during later phases of wound healing (Liang et al., 2012).
Since Ctip2 null mice die after birth, a new mouse line was generated in
which Ctip2 is selectively deleted in mouse epidermal keratinocytes
(Ctip2ep-/-) using Cre-LoxP strategies and K14/Cre deletor mice (Indra et
22
al., 1999; Golonzhka et al., 2008). Ctip2ep-/- mice exhibit increased
epidermal proliferation and terminal differentiation in unwounded skin.
After wounds were created, the mutant mice show significantly delayed
wound healing with lower re-epithelialization efficiency compared with
wildtype. Also, Ctip2ep-/- mice have thick and blunted migratory tongues
in the wound margin while the wildtypes have thinner ones. The mutant
mice also have impaired keratinocyte activation and altered cell
proliferation and differentiation, which could contribute to the delayed
cutaneous wound healing (Liang et al., 2012). Altogether, Ctip2 plays a
critical role in mouse skin wound healing and re-epithelialization by
regulating keratinocyte activation, proliferation and differentiation in a
cell-autonomous manner(Liang et al., 2012).
During mice skin wound healing, the expression of alpha SMA which is
responsible for development of extracellular matrix (ECM) was reduced
in the mutant on day 5 after wounding. Several stem cell markers, such
as K15, CD133 as well as CD34 were significantly downregulated in the
Ctip2ep-/- mice 5 days post wounding. These results indicate aberrant
expression levels of regulatory proteins in the mouse cutaneous wound
healing process in the absence of Ctip2 (Liang et al., 2012).
23
1.5 Research objectives
In the study described under Chapter 2, we have evaluated how Ctip2
regulates lipid composition in mouse epidermis in connection to the role
of Ctip2 in epidermal permeability barrier formation. The molecular
mechanisms of Ctip2 mediated regulation of sphingolipids biosynthesis
were also addressed.
In Chapter 3, our study focused on the regulation of adult skin barrier
maintenance and homeostasis by Ctip2. The role of Ctip2 in atopic
dermatitis-like mouse skin inflammatory disease was studied. Key
factors involved in this disease progression were identified.
24
Figure 1.1 Schematic representation of epidermal permeability
barrier. (Adapted from Segre et al., 2006).
Epidermal keratinocytes undergo a series of proliferation and
differentiation from basal cells to spinous cells to enucleated granular
cells, resulting finally in differentiated squames in the stratum corneum,
which is composed of keratin macrofibrils and cross-linked cornified
envelopes (CEs). Tight junction also plays an important role in
epidermal permeability barrier.
25
Figure 1.2 Amino acid sequence, diagram of amino acid sequence
alignment of CTIP1 and CTIP2. (Taken from Avram et al., 2000)
(A) and (B) Amino acid sequence of CTIP1 and CTIP2, respectively.
The conserved cysteine and histidine in zinc finger motifs for both
proteins are underlined.
(C) Schematic diagram of CTIP1 and CTIP2 amino acid alignment.
Black boxes indicate homologous regions between CTIP1 and CTIP2,
and percentage of identity is listed.
26
27
Figure 1.3 Expression of Ctip2 in the mouse fetal skin. (Adapted
from Golonzhka et al., 2007)
(A) Highly expressed Ctip2 (green) was observed in the ectoderm at
E10.5 (upper panel) and E12.5 (lower panel) and is co-localized with
the expression of K14 (red).
(B) Ctip2 expression was mainly in the basal cells and upper layers of
the epidermis of E14.5 (upper panel), E16.5 (middle panel) and E18.5
embryos (lower panel). Ctip2 (red) was co-stained with K14 (basal
layer) and K10 (suprabasal layer) (in red). At E16.5 and E18.5 stages of
development CTIP2 is highly expressed in the basal layer of epidermis
as well as in the dermis and hair follicles. All sections were
counterstained with DAPI (in blue). Images were taken at 40X
magnification. EC: ectoderm, M: mesoderm, E: epidermis, D: dermis, B:
basal cell layer, SB: suprabasal cell layers, HB: hair bulb, HF: hair
follicle.
28
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29
Figure 1.4 Ctip2 mutant mice exhibit permeability barrier defects
and epidermal hypoplasia. (Adapted from Golonzhka et al., 2007)
(A) X-gal diffusion assay performed on wildtype and Ctip2 null mice at
E17.5 and E18.5.
(B) Transepidermal water loss (TEWL) measurement of E17.5 and
E18.5 embryonic dorsal and ventral skin. *P<0.05, # not statistically
significant.
(C) Nile red staining of wildtype and Ctip2 null mice at E18.5. Yellow
ribbon indicates neutral lipid layer. Yellow arrows indicate loss of
neutral lipid in the Ctip2 null epidermis. Scale bar: 50 µm. E, epidermis;
D, dermis.
(D) Immunohistochemical staining of wildtype and null mice dorsal skin
at E18.5 with proliferation markers Ki67 (red), K14 (red) and early
differentiation marker K10 (red).
(E) Immunohistochemical staining of wildtype and null mice dorsal skin
at E18.5 with terminal differntiation markers Filaggrin (red), Involucrin
(red) and Loricrin (red). All sections were counterstained with DAPI
(blue). Scale bar: 100 µm.
30
Figure 1.5 Ctip2 is required for the formation and integrity of cornified
envelope in mouse epidermis.
Pictures represent the morphology of cornified envelope extracted from
wildtype and Ctip2ep-/- after different time of sonication.
31
1.6 References
Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., and
Macklis, J.D. (2005). Neuronal Subtype-Specific Genes that Control
Corticospinal Motor Neuron Development In Vivo. Neuron 45, 207–221.
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D.J., Ishmael, J.E.,
and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger
proteins implicated in transcriptional repression mediated by chicken
ovalbumin upstream promoter transcription factor (COUP-TF) orphan
nuclear receptors. J Biol Chem 275, 10315–10322.
Avram, D., Fields, A., Senawong, T., Topark-Ngarm, A., and Leid, M.
(2002). COUP-TF (chicken ovalbumin upstream promoter transcription
factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding
protein. Biochem J 368, 555–563.
B. Brandt, E., and Sivaprasad, U. (2011). Th2 Cytokines and Atopic
Dermatitis. Journal of Clinical & Cellular Immunology 02,.
Barrientos, S., Stojadinovic, O., Golinko, M.S., Brem, H., and TomicCanic, M. (2008). PERSPECTIVE ARTICLE: Growth factors and
cytokines in wound healing. Wound Repair and Regeneration 16, 585–
601.
Byrne, C., Hardman, M., and Nield, K. (2003). Covering the limb-formation of the integument. J Anat 202, 113–123.
Cameron, D.J., Tong, Z., Yang, Z., Kaminoh, J., Kamiyah, S., Chen, H.,
Zeng, J., Chen, Y., Luo, L., and Zhang, K. (2007). Essential role of
Elovl4 in very long chain fatty acid synthesis, skin permeability barrier
function, and neonatal survival. Int. J. Biol. Sci. 3, 111–119.
Candi, E., Schmidt, R., and Melino, G. (2005). The cornified envelope:
a model of cell death in the skin. Nat. Rev. Mol. Cell Biol. 6, 328–340.
Comeau, M.R., and Ziegler, S.F. (2010). The influence of TSLP on the
allergic response. Mucosal Immunol 3, 138–147.
Cork, M.J., Danby, S.G., Vasilopoulos, Y., Hadgraft, J., Lane, M.E.,
Moustafa, M., Guy, R.H., MacGowan, A.L., Tazi-Ahnini, R., and Ward,
32
S.J. (2009). Epidermal Barrier Dysfunction in Atopic Dermatitis. Journal
of Investigative Dermatology 129, 1892–1908.
Demehri, S., Liu, Z., Lee, J., Lin, M.-H., Crosby, S.D., Roberts, C.J.,
Grigsby, P.W., Miner, J.H., Farr, A.G., and Kopan, R. (2008). NotchDeficient Skin Induces a Lethal Systemic B-Lymphoproliferative
Disorder by Secreting TSLP, a Sentinel for Epidermal Integrity. PLoS
Biology 6, e123.
Elias, P.M. (1983). Epidermal lipids, barrier function,
desquamation. J. Invest. Dermatol. 80 Suppl, 44s–49s.
and
Elias, P.M., and Menon, G.K. (1991). Structural and lipid biochemical
correlates of the epidermal permeability barrier. Adv. Lipid Res 24, 1–
26.
Evans, R.M. (1988). The steroid and thyroid hormone receptor
superfamily. Science 240, 889–895.
Feingold, K.R. (2007). Thematic review series: skin lipids. The role of
epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid
Res 48, 2531–2546.
Feingold, K.R. (2009). The outer frontier: the importance of lipid
metabolism in the skin. J. Lipid Res. 50 Suppl, S417–422.
Fuchs, E., and Raghavan, S. (2002). Getting under the skin of
epidermal morphogenesis. Nat Rev Genet 3, 199–209.
Ganguli-Indra, G., Liang, X., Hyter, S., Leid, M., Hanifin, J., and Indra,
A.K. (2009a). Expression of COUP-TF-interacting protein 2 (CTIP2) in
human atopic dermatitis and allergic contact dermatitis skin.
Experimental Dermatology 18, 994–996.
Ganguli-Indra, G., Wasylyk, C., Liang, X., Millon, R., Leid, M., Wasylyk,
B., Abecassis, J., Indra, A.K., and Indra, A. (2009b). CTIP2 expression
in human head and neck squamous cell carcinoma is linked to poorly
differentiated tumor status. PLoS ONE 4, e5367.
Gelfand, J.M., Dommasch, E.D., Shin, D.B., Azfar, R.S., Kurd, S.K.,
Wang, X., and Troxel, A.B. (2009). The Risk of Stroke in Patients with
Psoriasis. Journal of Investigative Dermatology 129, 2411–2418.
33
Gelfand, J.M., Neimann, A.L., Shin, D.B., Wang, X., Margolis, D.J., and
Troxel, A.B. (2006). Risk of Myocardial Infarction in Patients With
Psoriasis. JAMA: The Journal of the American Medical Association 296,
1735–1741.
Ghadially, R., Reed, J.T., and Elias, P.M. (1996). Stratum Corneum
Structure and Function Correlates with Phenotype in Psoriasis. Journal
of Investigative Dermatology 107, 558–564.
Golonzhka, O., Leid, M., Indra, G., and Indra, A.K. (2007). Expression
of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during
development and in adulthood. Gene Expr Patterns 7, 754–760.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J.-M., Campbell,
A.L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M., and Indra,
A.K. (2008). Dual Role of COUP-TF-Interacting Protein 2 in Epidermal
Homeostasis and Permeability Barrier Formation. J Investig Dermatol
129, 1459–1470.
Golonzhka, O., Metzger, D., Bornert, J.-M., Bay, B.K., Gross, M.K.,
Kioussi, C., and Leid, M. (2009). Ctip2/Bcl11b controls ameloblast
formation during mammalian odontogenesis. Proc. Natl. Acad. Sci.
U.S.A. 106, 4278–4283.
Gudjonsson, J.E., Ding, J., Johnston, A., Tejasvi, T., Guzman, A.M.,
Nair, R.P., Voorhees, J.J., Abecasis, G.R., and Elder, J.T. (2010).
Assessment of the Psoriatic Transcriptome in a Large Sample:
Additional Regulated Genes and Comparisons with In Vitro Models. J
Investig Dermatol 130, 1829–1840.
Gurtner, G.C., Werner, S., Barrandon, Y., and Longaker, M.T. (2008).
Wound repair and regeneration. Nature 453, 314–321.
Hardman, M.J., Sisi, P., Banbury, D.N., and Byrne, C. (1998). Patterned
acquisition of skin barrier function during development. Development
125, 1541–1552.
Holleran, W.M., Takagi, Y., and Uchida, Y. (2006). Epidermal
sphingolipids: metabolism, function, and roles in skin disorders. FEBS
Lett. 580, 5456–5466.
34
Hunter, K.D., Parkinson, E.K., and Harrison, P.R. (2005). Opinion:
Profiling early head and neck cancer. Nature Reviews Cancer 5, 127–
135.
Imokawa, G. (2009). A possible mechanism underlying the ceramide
deficiency in atopic dermatitis: Expression of a deacylase enzyme that
cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.
Journal of Dermatological Science 55, 1–9.
Indra, A.K., Warot, X., Brocard, J., Bornert, J.M., Xiao, J.H., Chambon,
P., and Metzger, D. (1999). Temporally-controlled site-specific
mutagenesis in the basal layer of the epidermis: comparison of the
recombinase activity of the tamoxifen-inducible Cre-ER(T) and CreER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327.
Ishikawa, J., Narita, H., Kondo, N., Hotta, M., Takagi, Y., Masukawa, Y.,
Kitahara, T., Takema, Y., Koyano, S., Yamazaki, S., et al. (2010).
Changes in the Ceramide Profile of Atopic Dermatitis Patients. Journal
of Investigative Dermatology 130, 2511–2514.
Kamimura, K., Mishima, Y., Obata, M., Endo, T., Aoyagi, Y., and
Kominami, R. (2007). Lack of Bcl11b tumor suppressor results in
vulnerability to DNA replication stress and damages. Oncogene 26,
5840–5850.
Kastner, P., Chan, S., Vogel, W.K., Zhang, L.-J., Topark-Ngarm, A.,
Golonzhka, O., Jost, B., Le Gras, S., Gross, M.K., and Leid, M. (2010).
Bcl11b represses a mature T-cell gene expression program in immature
CD4(+)CD8(+) thymocytes. Eur. J. Immunol. 40, 2143–2154.
Karanam, N.K., Grabarczyk, P., Hammer, E., Scharf, C., Venz, S.,
Gesell-Salazar, M., Barthlen, W., Przybylski, G.K., Schmidt, C.A., and
Völker, U. (2010). Proteome analysis reveals new mechanisms of
Bcl11b-loss driven apoptosis. J. Proteome Res. 9, 3799–3811.
Kashyap, M., Rochman, Y., Spolski, R., Samsel, L., and Leonard, W.J.
(2011). Thymic stromal lymphopoietin is produced by dendritic cells. J.
Immunol. 187, 1207–1211.
Klinge, C.M., Bodenner, D.L., Desai, D., Niles, R.M., and Traish, A.M.
(1997). Binding of type II nuclear receptors and estrogen receptor to full
and half-site estrogen response elements in vitro. Nucleic Acids Res.
25, 1903–1912.
35
Kruse, S.W., Suino-Powell, K., Zhou, X.E., Kretschman, J.E., Reynolds,
R., Vonrhein, C., Xu, Y., Wang, L., Tsai, S.Y., Tsai, M.-J., et al. (2008).
Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic
Acid–Activated Receptor. PLoS Biology 6, e227.
Leid, M., Ishmael, J.E., Avram, D., Shepherd, D., Fraulob, V., and
Dolle, P. (2004). CTIP1 and CTIP2 are differentially expressed during
mouse embryogenesis. Gene Expr Patterns 4, 733–739.
Leung, D.Y., and Bieber, T. (2003). Atopic dermatitis. The Lancet 361,
151–160.
Li, M. (2005). Retinoid X receptor ablation in adult mouse keratinocytes
generates an atopic dermatitis triggered by thymic stromal
lymphopoietin. Proceedings of the National Academy of Sciences 102,
14795–14800.
Li, M. (2006). Topical vitamin D3 and low-calcemic analogs induce
thymic stromal lymphopoietin in mouse keratinocytes and trigger an
atopic dermatitis. Proceedings of the National Academy of Sciences
103, 11736–11741.
Li, W., Sandhoff, R., Kono, M., Zerfas, P., Hoffmann, V., Ding, B.C.-H.,
Proia, R.L., and Deng, C.-X. (2007). Depletion of ceramides with very
long chain fatty acids causes defective skin permeability barrier
function, and neonatal lethality in ELOVL4 deficient mice. Int. J. Biol.
Sci. 3, 120–128.
Liang, X., Bhattacharya, S., Bajaj, G., Guha, G., Wang, Z., Jang, H.-S.,
Leid, M., Indra, A.K., and Ganguli-Indra, G. (2012). Delayed Cutaneous
Wound Healing and Aberrant Expression of Hair Follicle Stem Cell
Markers in Mice Selectively Lacking Ctip2 in Epidermis. PLoS ONE 7,
e29999.
Linja, M.J. (2004). Expression of Androgen Receptor Coregulators in
Prostate Cancer. Clinical Cancer Research 10, 1032–1040.
Lippens, S., Hoste, E., Vandenabeele, P., Agostinis, P., and Declercq,
W. (2009). Cell death in the skin. Apoptosis 14, 549–569.
Liu, P., Keller, J.R., Ortiz, M., Tessarollo, L., Rachel, R.A., Nakamura,
T., Jenkins, N.A., and Copeland, N.G. (2003). Bcl11a is essential for
normal lymphoid development. Nature Immunology 4, 525–532.
36
Mack, J.A., Anand, S., and Maytin, E.V. (2005). Proliferation and
cornification during development of the mammalian epidermis. Birth
Defects Res C Embryo Today 75, 314–329.
Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schütz, G.,
Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., et al.
(1995). The nuclear receptor superfamily: the second decade. Cell 83,
835–839.
De Monte, L., Reni, M., Tassi, E., Clavenna, D., Papa, I., Recalde, H.,
Braga, M., Di Carlo, V., Doglioni, C., and Protti, M.P. (2011). Intratumor
T helper type 2 cell infiltrate correlates with cancer-associated fibroblast
thymic stromal lymphopoietin production and reduced survival in
pancreatic cancer. J. Exp. Med. 208, 469–478.
Nakamura, T., Yamazaki, Y., Saiki, Y., Moriyama, M., Largaespada,
D.A., Jenkins, N.A., and Copeland, N.G. (2000). Evi9 encodes a novel
zinc finger protein that physically interacts with BCL6, a known human
B-cell proto-oncogene product. Mol. Cell. Biol. 20, 3178–3186.
Nemes, Z., Demény, M., Marekov, L.N., Fésüs, L., and Steinert, P.M.
(2000). Cholesterol 3-sulfate interferes with cornified envelope
assembly by diverting transglutaminase 1 activity from the formation of
cross-links and esters to the hydrolysis of glutamine. J. Biol. Chem.
275, 2636–2646.
Nemes, Z., and Steinert, P.M. (1999). Bricks and mortar of the
epidermal barrier. Exp. Mol. Med. 31, 5–19.
Nograles, K.E., Brasington, R.D., and Bowcock, A.M. (2009). New
insights into the pathogenesis and genetics of psoriatic arthritis. Nature
Clinical Practice Rheumatology 5, 83–91.
Oda, Y., Uchida, Y., Moradian, S., Crumrine, D., Elias, P.M., and Bikle,
D.D. (2008). Vitamin D Receptor and Coactivators SRC2 and 3
Regulate Epidermis-Specific Sphingolipid Production and Permeability
Barrier Formation. J Investig Dermatol 129, 1367–1378.
Olefsky, J.M. (2001). Nuclear receptor minireview series. J. Biol. Chem.
276, 36863–36864.
Oyoshi, M.K., Larson, R.P., Ziegler, S.F., and Geha, R.S. (2010).
Mechanical injury polarizes skin dendritic cells to elicit a T(H)2
37
response by inducing cutaneous thymic stromal lymphopoietin
expression. J. Allergy Clin. Immunol. 126, 976–984, 984.e1–5.
Pastorcic, M., Wang, H., Elbrecht, A., Tsai, S.Y., Tsai, M.J., and
O’Malley, B.W. (1986). Control of transcription initiation in vitro requires
binding of a transcription factor to the distal promoter of the ovalbumin
gene. Mol. Cell. Biol. 6, 2784–2791.
Pereira, F.A., Qiu, Y., Tsai, M.J., and Tsai, S.Y. (1995). Chicken
ovalbumin upstream promoter transcription factor (COUP-TF):
expression during mouse embryogenesis. J. Steroid Biochem. Mol.
Biol. 53, 503–508.
Roberson, E.D.O., and Bowcock, A.M. (2010). Psoriasis genetics:
breaking the barrier. Trends in Genetics 26, 415–423.
Robinson-Rechavi, M., Escriva Garcia, H., and Laudet, V. (2003). The
nuclear receptor superfamily. J. Cell. Sci. 116, 585–586.
Sagami, I., Tsai, S.Y., Wang, H., Tsai, M.J., and O’Malley, B.W. (1986).
Identification of two factors required for transcription of the ovalbumin
gene. Mol. Cell. Biol. 6, 4259–4267.
Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel,
B., Mikkola, H.K.A., Hirschhorn, J.N., Cantor, A.B., and Orkin, S.H.
(2008). Human Fetal Hemoglobin Expression Is Regulated by the
Developmental Stage-Specific Repressor BCL11A. Science 322, 1839–
1842.
Satterwhite, E. (2001). The BCL11 gene family: involvement of BCL11A
in lymphoid malignancies. Blood 98, 3413–3420.
Scharschmidt, T.C., and Segre, J.A. (2008). Modeling atopic dermatitis
with increasingly complex mouse models. J. Invest. Dermatol. 128,
1061–1064.
Schmuth, M., Man, M.Q., Weber, F., Gao, W., Feingold, K.R., Fritsch,
P., Elias, P.M., and Holleran, W.M. (2000). Permeability barrier disorder
in Niemann-Pick disease: sphingomyelin-ceramide processing required
for normal barrier homeostasis. J. Invest. Dermatol. 115, 459–466.
Segre, J.A. (2006). Epidermal barrier formation and recovery in skin
disorders. J Clin Invest 116, 1150–1158.
38
Soumelis, V., Reche, P.A., Kanzler, H., Yuan, W., Edward, G., Homey,
B., Gilliet, M., Ho, S., Antonenko, S., Lauerma, A., et al. (2002). Human
epithelial cells trigger dendritic cell–mediated allergic inflammation by
producing TSLP. Nature Immunology.
Topark-Ngarm, A., Golonzhka, O., Peterson, V.J., Barrett, B., Martinez,
B., Crofoot, K., Filtz, T.M., and Leid, M. (2006). CTIP2 associates with
the NuRD complex on the promoter of p57KIP2, a newly identified
CTIP2 target gene. J Biol Chem 281, 32272–32283.
Tsai, S.Y. (1997). Chick Ovalbumin Upstream Promoter-Transcription
Factors (COUP-TFs): Coming of Age. Endocrine Reviews 18, 229–240.
Uchida, Y., Hara, M., Nishio, H., Sidransky, E., Inoue, S., Otsuka, F.,
Suzuki, A., Elias, P.M., Holleran, W.M., and Hamanaka, S. (2000).
Epidermal sphingomyelins are precursors for selected stratum corneum
ceramides. J. Lipid Res. 41, 2071–2082.
Uda, M., Galanello, R., Sanna, S., Lettre, G., Sankaran, V.G., Chen,
W., Usala, G., Busonero, F., Maschio, A., Albai, G., et al. (2008).
Genome-wide association study shows BCL11A associated with
persistent fetal hemoglobin and amelioration of the phenotype of thalassemia. Proceedings of the National Academy of Sciences 105,
1620–1625.
Vasireddy, V., Uchida, Y., Salem, N., Jr, Kim, S.Y., Mandal, M.N.A.,
Reddy, G.B., Bodepudi, R., Alderson, N.L., Brown, J.C., Hama, H., et
al. (2007). Loss of functional ELOVL4 depletes very long-chain fatty
acids (> or =C28) and the unique omega-O-acylceramides in skin
leading to neonatal death. Hum. Mol. Genet. 16, 471–482.
Wakabayashi, Y., Watanabe, H., Inoue, J., Takeda, N., Sakata, J.,
Mishima, Y., Hitomi, J., Yamamoto, T., Utsuyama, M., Niwa, O., et al.
(2003). Bcl11b is required for differentiation and survival of alphabeta T
lymphocytes. Nat Immunol 4, 533–539.
Wang, L.H., Tsai, S.Y., Sagami, I., Tsai, M.J., and O’Malley, B.W.
(1987). Purification and characterization of chicken ovalbumin upstream
promoter transcription factor from HeLa cells. J. Biol. Chem. 262,
16080–16086.
39
Werner, S., Krieg, T., and Smola, H. (2007). Keratinocyte-fibroblast
interactions in wound healing. J Invest Dermatol 127, 998–1008.
Wong, V.W., Sorkin, M., Glotzbach, J.P., Longaker, M.T., and Gurtner,
G.C. (2011). Surgical Approaches to Create Murine Models of Human
Wound Healing. Journal of Biomedicine and Biotechnology 2011, 1–8.
Yamada, T., Saito, H., Kimura, Y., Kubo, S., Sakashita, M., Susuki, D.,
Ito, Y., Ogi, K., Imoto, Y., and Fujieda, S. (2012). CpG-DNA suppresses
poly(I:C)-induced TSLP production in human laryngeal arytenoid
fibroblasts. Cytokine 57, 245–250.
Ziegler, S.F. (2010). The role of thymic stromal lymphopoietin (TSLP) in
allergic disorders. Current Opinion in Immunology 22, 795–799.
(1999). A unified nomenclature system for the nuclear receptor
superfamily. Cell 97, 161–163.
40
Chapter 2
Transcription factor Ctip2 controls epidermal lipid metabolism and
regulates expression of genes involved in sphingolipid
biosynthesis during skin development
Zhixing Wang, Jay S. Kirkwood, Alan W. Taylor, Jan F. Stevens,
Mark Leid, Gitali Ganguli-Indra and Arup K. Indra
Journal of Investigative Dermatology
In revision 41
2.1 Abstract
The stratum corneum is composed of a continuous sheath of proteinenriched corneocytes embedded in an intercellular matrix enriched in
nonpolar lipids organized as lamellar layers and give rise to the
epidermal permeability barrier (EPB). EPB defects play an important
role in the pathophysiology of skin diseases such as eczema. The
transcriptional control of skin lipid metabolism is poorly understood. We
have discovered that mouse lacking a transcription factor COUP-TF
interacting protein 2 (Ctip2) exhibit EPB defects including altered
keratinocyte terminal differentiation, delayed skin barrier development
and interrupted neutral lipid distribution in the epidermis. We adapted
herein a targeted lipidomics approach using mass spectrometry, and
have determined that Ctip2-/- mice (germline deletion of Ctip2 gene)
display altered composition of major epidermal lipids such as ceramides
and sphingomyelins compared to the wildtype at different stages of skin
development. Interestingly, expressions of several genes involved in
skin sphingolipid biosynthesis and metabolism were altered in mutant
skin. Ctip2 was found to be recruited to the promoter region of a subset
of those genes, suggesting their possible direct regulation by Ctip2. Our
Ctip2 mouse model exhibiting EPB defects can be utilized to screen for
new drugs for treatment and recovery of barrier dysfunctions.
42
2.2 Introduction
The exterior layer of skin, stratum corneum (SC), plays a critical role in
skin permeability barrier formation and maintenance (Fuchs and
Raghavan, 2002; Elias, 2004). It forms a protective barrier against
external stress and environmental insults. Skin barrier defects are
caused not only by altered levels of protein(s) involved in keratinocytes
terminal differentiation, cross-linking of cornified envelops (CEs), but
also by impaired formation and maintenance of skin lipid barrier (Elias
and Menon, 1991; Nemes and Steinert, 1999; Feingold, 2007).
Mammalian SC contains a large number of lipids, such as ceramides,
sphingomyelins, cholesterols and fatty acids (Elias and Menon, 1991;
Ishikawa et al., 2010). Ceramides (CERs) are the most abundant lipid
types in SC (50% by weight) and can be divided into at least 11 species
(Oda et al., 2008; Ishikawa et al., 2010). It has been shown that
reduced content of CERs leads to epidermal water loss as well as skin
epidermal dysfunctions, and causes diseases such as atopic dermatitis
(AD) or eczema (Imokawa, 2009; Ishikawa et al., 2010). Spingomyelins
are another large group of epidermal sphingolipids, which have been
reported as precursors of ceramides (Schmuth et al., 2000; Uchida et
al., 2000; Holleran et al., 2006). Cholesterols and fatty acids are also
major skin lipids, and cholesterol 3-sulfate (CSO4) is a ubiquitous
43
metabolite of cholesterol, which is involved in cornified envelope
formation by interacting with transglutaminase1 (Nemes et al., 2000).
Ceramide biosynthesis is regulated at multiple levels. Four pathways
have been reported so far, de novo pathway, sphingomyelinase
pathway, salvage pathway and exogenous ceramide recycling pathway
(Kitatani et al., 2008). In de novo pathway, serine and fatty acyl-CoA
are the initiators of a series of reactions.
Ceramide synthases 1-6
(Lass1-Lass6) play an important role since each of them has
specificities on synthesizing certain ceramides (Mizutani et al., 2009).
Other
enzymes
such
as
palmitoyltransferases
(Sptlc1-3),
dihydroceramide desaturase (Degs1) and ketodihydro-sph reductase
(Kdsr) are also key enzymes in de novo pathway.
The salvage
pathway has been reported recently as a complex mechanism,
commencing as the catabolism of complex sphingolipids into
glucosylceramid by glucosylceramide synthase (Ugcg), and followed by
the formation of ceramide by acid beta-glucosidase (Gba2) (Ogretmen,
2002; Becker, 2004). These ceramides can be broken down into
sphingosines by acid ceramidase (Asah1 and Asah2), which are reused
to produce ceramides.
In sphingomyelinase pathway, ceramide is
formed by hydrolysis of sphingomyelins, and the reverse reaction is
controlled by sphingomyelin synthase (Marchesini and Hannun, 2004).
44
Exogenous short chain ceramide can also be utilized to generate
sphingosine, thus leads to the synthesis of endogenous long chain
ceramides (Ogretmen, 2002; Sultan et al., 2006).
COUP-TF-interacting protein 2 (Ctip2), also known as Bcl11b, is a C2H2
zinc finger protein expressed in many organs and tissues including
developing mouse epidermis (Avram et al., 2000, 2002; Golonzhka et
al., 2008). It has been reported that Ctip2 null mice exhibit altered
epidermal proliferation and late terminal differentiation, as well as
barrier defects during embryogenesis (Golonzhka et al., 2008). The
barrier defects were correlated with altered surface lipid distribution in
the absence of Ctip2 (Golonzhka et al., 2008).
Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has
been used to characterize ceramide species in mouse and human
stratum corneum (Masukawa et al., 2008; Park et al., 2009; Ishikawa et
al., 2010; Janssens et al., 2011; van Smeden et al., 2011). Here we
adopted the LC/MS/MS methodology to elucidate the CER [NS],
sphingmyelin and sphingosine profile in wildtype and Ctip2 null (Ctip2-/-)
embryonic skin epidermis. The expression levels of genes encoding
various enzymes involved in sphingolipid metabolism were analyzed by
RT-qPCR and immuno-blotting in wildtype and Ctip1-null skin.
Furthermore, Ctip2 recruitment to the promoter regions of several
45
genes that were altered in Ctip2-/- mice was evaluated by chromatin
immunoprecipitation (ChIP) analyses. Results indicated that Ctip2
control sphingolipid metabolism in the developing skin by directly or
indirectly regulating expression of a subset of genes involved in the
sphingolipid bio-synthesis pathways.
2.3 Materials and methods
Mice
Ctip2
null (Ctip2-/-) mice were generated by floxing exon 4 of Ctip2
locus, which encodes for three-fourth of open reading frame (ORF)
(Golonzhka et al., 2008). OSU IACUC approval was obtained for animal
experiments.
Lipid extraction
Fetus from E16.5 to E18.5 and neonatal mice were collected and
genotyped (Golonzhka et al., 2008). A minimum of three wildtype and
Ctip2-/- fetuses were taken from the same litter and compared between
littermates. A minimum of three litters were analyzed from each
developmental stages. Skin was isolated, weighed and incubated
overnight
in
CnT-07
medium
(CELLnTEC,
Bern,
Switzerland)
46
containing 1 mg ml−1 dispase (Gibco, Grand Island, NY) at 4 °C to
dissociate epidermis.
Raw skin lipid was extracted using Bligh and Dyer method (Folch et al.,
1957; Bligh and Dyer, 1959). Briefly, skin epidermis was incubated in
extraction buffer (chloroform: methanol: water (1:2:0.8), 10-15mg
tissue/1ml extraction buffer) overnight at room temperature. Fresh
extraction buffer was added to epidermis samples the second day and
old extraction buffer was saved. Samples were sonicated for 5 cycles,
30 seconds each, and the suspension was shaked for 30min in room
temperature. After centrifuged at 2000rpm for 10min, the pellets were
discarded and the extraction buffer was combined. Chloroform and
water were added to extraction buffer in a ratio of extraction buffer:
chloroform: water (7.6: 2: 2). After mixing and centrifuging, upper phase
was discarded and the lower phase (chloroform) was washed twice with
chloroform: methanol: water (1: 1: 0.9). Lower phase was dried in
nitrogen, weighed and saved in -20°C.
LC-MS/MS
Raw skin lipid was further purified as in previous reports (Merrilljr et al.,
2005; Monette et al., 2011). 10µl sphingolipid internal standard mixture
(Avanti, Alabaster, Alabama) and 1mg raw lipid were added to 1.5ml of
47
chloroform: methanol (1:2) and sonicated in the bath sonicator for 30
seconds. After 2 hours of incubation at 48 °C, the samples were cooled
on ice. Samples were then incubated for 1 hour at 37°C with 75μl 1M
KOH in methanol to remove the free fatty acids and triacylglycerol, and
cooled on ice. 12μl of glacial acetic acid was added to neutralize the
pH. Phase separation was achieved by adding 2ml chloroform and 4ml
water and samples were gently vortexed. After centrifuge for 15 min at
2000rcf, the chloroform layer was aspirated and dried under nitrogen.
The samples were reconstituted in 200μl chloroform: methanol (3:1)
and diluted 1:4 with acetonitrile: methanol: acetic acid (97:2:1) with
5mM ammonium acetate.
LC-tandem
mass
spectrometry
was
performed
and
relative
quantification was based on skin weight and sphingolipid standards
(Monette et al., 2011). In brief, lipids were separated by HPLC using a
Supelco Discovery column (2mm x 50mm; Sigma-Aldrich) with
300μl/min flow rate. Mobile phase A: methanol: water (60:40) and
mobile phase B: methanol: chloroform (60:40) both contained 0.2%
(v/v) formic acid and 10 mM ammonium acetate. The column was preequilibrated at 100% phase A and 5μl sample was injected; 100%
mobile phase A was maintained for 1min, followed by a linear increase
to 40% mobile phase B in a 7 min period; followed by a linear increase
48
to 70% mobile phase B in next 6 min, which was maintained for the
remainder (6min) of the 20 min run.
Sample detection was performed using a triple-quadrupole mass
spectrometer operated in positive mode (Applied Biosystems/ MDS
Sciex, API300) with multiple reaction monitoring. Quantitation was
based on comparison to a synthetic sphingolipid standard mixture
(Monette et al., 2010).
UPLC-MS/MS
Ultra high pressure liquid chromatography was performed on a
Shimadzu Nexera system (Shimadzu, Columbia, MD) coupled to a
hybrid quadrupole-time of flight mass spectrometer (TripleTOFTM 5600,
AB SCIEX) with raw skin lipids. Chromatographic separations were
carried out on a Brownlee Analytical C18 column (50 × 2.1 mm, 1.9
mm; PerkinElmer, Boston, MA). The flow rate was 0.35 mL/min and
mobile phases consisted of water (A) and acetonitrile (B), both with
0.1% formic acid. Elution gradient was as follows: 0 min, 25% B; 2 min,
55% B; 7 min, 100 % B; 9 min, 100% B. The column was equilibrated 3
minutes prior to each injection. Column temperature was held at 40 °C
and the injection volume was 3 µL.
49
Mass spectrometry was performed on an AB SCIEX TripleTOFTM 5600
equipped with an electrospray ionization source. The instrument was
operated in information dependent MS/MS acquisition mode with the
collision energy set at 30 V and a collision energy spread of 10 V. TOF
MS acquisition time was 0.15 seconds and MS/MS acquisition time was
0.10 seconds. Scan range was 70-1000 m/z for TOF MS and 40-1000
m/z for MS/MS. Ion source gas 1 and 2 and curtain gas (all nitrogen)
were set at 50 and 40 and 25, respectively. Source temperature was
set at 500 °C and IonSpray voltage at 5.5/-5.5 kV. A set of standards
was run periodically throughout the batch to assess system variance
and stability. The instrument was automatically calibrated every five
runs.
Stearic acid, palmitic acid, cholesterol, and cholesterol-3-sulfate were
identified using authentic standards with accurate mass measurements,
isotope distribution, MS/MS fragmentation, and retention time.
RT-qPCR
RNA was extracted and cDNA was synthesized from fetus and neonatal
skin (Indra et al., 2005b; Wang et al., 2011). Real-time PCR was
performed on an ABI 7500 Real-Time PCR system using SYBR green
methodology (Indra et al., 2005a, 2005b). Relative gene expression
50
analysis of the RT-qPCR data was performed using Gapdh as an
internal control, and the specific primers were indicated in Table 2.1
(Wang et al., 2011). All assays were performed in triplicates.
Western blotting
Protein was extracted from mouse skin biopsies and western blotting
was performed due to previous reports using specific antibodies against
Lass2, Gba2 and eLox3. (Wang et al., 2011; Santa Cruz Biotechnology,
Santa Cruz, CA). Briefly, skin samples were homogenized, and proteins
were extracted with a lysis buffer (150mM NaCl, 50mM Tris pH 7.5,
5mM EDTA, and 1% Nonidet P-40, 0.1% SDS, 0.5% sodium
deoxycholate) containing protease inhibitors (2µg/ml Aprotinin, 2µg/ml
Leupeptin, 100µg/ml Pefabloc and 1µg/ml Pepstatin). Equal amounts
of protein extract (25 µg) from each lysate were resolved using SDS
polyacrylamide-gel electrophoresis and transferred onto a nitrocellulose
membrane. Blots were blocked overnight with 5% nonfat dry milk and
incubated with specific antibodies. All western blot experiments were
done in triplicates, using a minimum of three biological replicates from
each group of mice.
Chromatin immunoprecipitation (ChIP)
51
ChIP assay was performed according to Hyter et al., 2010 with minor
modifications. Briefly, mouse keratinocytes were isolated from neonatal
wildtype and Ctip2-/- mice skin epidermis and fixed in 1% formaldehyde.
Each ChIP started with 3x106 cells, which are sonicated and
immunoprecipitated with 2µg anti-Ctip2 antibody (Abcam, Cambridge,
MA) overnight at 4°C. Rat IgG was used as control. 25ml protein G
beads (Invitrogen, Carlsbad, CA) were added to each ChIP and
incubated at 4°C for four hours. DNA was uncrosslinked at 65°C
overnight and purified by QIAquick PCR Purification Kit (Qiagen,
Germantown, MD). DNA was amplified using ABI-7500 Real-time PCR
machine with specific primers indicated in Supplementary table 2.
Statistics
Statistical significance of differences between groups was analyzed
using GraphPad Prism software using student’s unpaired t-test. All
statistical analyses were performed in a double-blinded manner.
2.4 Results
52
2.4.1 Altered profile of sphingolipids in the developing epidermis
of Ctip2-null mice
We hypothesized that impaired epidermal permeability barrier in the
Ctip2-null (Ctip2-/- ) mice could be at least in part due to altered skin
lipid
composition. In order to determine the lipid composition of
embryonic skin, wildtype and Ctip2-null epidermis were isolated at
different developmental stages, E16.5, E17.5 E18.5 and neonatal (P0).
Lipidomic studies were then performed using LC/MS/MS technique and
absolute quantities of the different skin sphingolipids were analyzed (for
details
see
materials
and
methods).
Both
ceramides
and
sphingomyelins were abundant during mouse skin development,
especially during the earlier stages of skin development (E16.5, E17.5)
(Figure 2.1-2.4).
Ceramide C14:0 level remained very low from E16.5-P0. Long chain
ceramide C16:0 was the most abundant saturated ceramide at E16.5
and was 2-fold higher in the mutant epidermis compared with wildtype
(Figure 2.1 A). Significant induction of ceramide C18:0 was observed in
the E16.5 mutant skin (Figure 2.1 A). The amount of long chain
ceramides (C16:0, C18:0, C20:0 and C22:0) was in a very low
abundance at E17.5-E18.5, with no discernable difference observed
between wildtype and mutant skin; however, their quantities are larger
at P0, each having a ~2-fold reduction in the Ctip2-/- skin (Figure 2.1 B-
53
D). At E17.5 and E18.5, very long chain ceramide C26:0 became the
dominant subtype in mouse skin, modestly reduced at E17.5 and 2-fold
decreased at E18.5 in Ctip2-/- skin (Figure 2.1 B-C). Modest decrease of
ceramide C24:0 was also observed in Ctip2-/- epidermis at P0 (Figure
2.1 D). Unsaturated ceramides were less abundant compared with
saturated ceramides in the wild type epidermis. Long chain unsaturated
ceramide C16:1 was significantly reduced at E16.5 in the mutant
epidermis, unaffected at E17.5 and E18.5, with a 2-fold decrease in P0
mutant epidermis (Figure 2.2 A-D).
Among saturated sphingomyelins, long chain sphingomyelins C16:0,
C20:0 and C22:0 were the most abundant subtypes in embryonic
epidermis at most of the stages, which were highly induced in the
mutant skin, especially at E17.5 and E18.5 (Figure 2.3 B and C). Very
long chain sphingomyelins (C24:1, C26:1, C28:1) were also increased
in the mutant skin at E16.5-E18.5 (Figure 2.3 A-C).
unsaturated
sphingomyelins
were
less
Interestingly,
predominant
in
all
developmental stages; however, they showed the similar trend as
saturated ones, with higher amount in embryonic mutant epidermis. As
early as E16.5, long chain unsaturated sphingomyelin C24:1 was
significantly elevated in Ctip2-/- skin (Figure 2.4 A). Higher quantities of
54
long chain sphingomyelins C20:1, C22:1, and C24:1 were noted in the
mutant epidermis at E17.5 and E18.5 (Figure 2.4 B-D).
The profiles of sphingosine and sphinganine were also determined by
LC/MS/MS. Compared with ceramides and sphingomyelins, the
quantity of free sphingoid bases were smaller in embryonic skin, with no
significant change observed between wildtype and mutant epidermis at
all developmental stages (Figure 2.5 A-D).
We also performed UPLC/MS/MS analyses to determine the relative
amount of cholesterols and fatty acids. Levels of cholesterol and
cholesterol 3-sulfate were significantly reduced in the epidermis of
Ctip2-/- mice at E16.5, but were unaltered at all later timepoints (Figure
2.6 A-B). Although the amount of palmitic acid and stearic acid was
similar between wildtype and null skin at E16.5-E18.5, their levels
doubled in the P0 Ctip2-/- epidermis compared to the wildtype epidermis
(Figure 2.6 C-D). Altogether, our results indicate that epidermal lipid
composition is altered in absence of transcriptional regulator Ctip2.
2.4.2 Ctip2 directly regulates expression of a subset of genes
involved in lipid metabolism in the developing skin
Previous studies have identified four major pathways involved in
ceramide
synthesis:
de
novo
pathway,
salvage
pathway,
sphingomyelinase pathway and exogenous ceramide-recycling pathway
55
(Kitatani et al., 2008; Mizutani et al., 2009). We hypothesized that
altered sphingolipid profiles in mutant mice could be due to altered
expression of genes encoding proteins involved in the sphingolipid
metabolism. To test that, we evaluated the expression of genes
involved in the different ceramide biosynthesis pathways by RT-qPCR
analyses in the wild type control and mutant skin (for details see
materials and methods). Expressions of ceramide synthase 3 (Lass3),
palmitoyltransferases (Sptlc1 and Sptlc3) were downregulated at E16.5
and E17.5, while ceramide synthases Lass1 and Lass2 were
significantly reduced at E18.5 in Ctip2-/- skin (Figure 2.7 A-C). Lass6
was consistently downregulated from E16.5 to E18.5 in the mutant skin
(Figure 2.7 A-C). Thus, genes encoding enzymes in de novo pathway
were altered in ablation of Ctip2. Similarly, transcript levels of enzymes
involved in sphingomyelinase pathway such as sphingomyelinases
(Smpd1 and Smpd2) were significantly downregulated at E18.5,
whereas sphingomyelin synthase (Sgms2) level was upregulated at
E16.5 in null embryos compared to the wildtypes (Figure 2.7 D-F).
Expression of glucosylceramide synthase (Ugcg), which turns complex
sphingolipids into glucosylcerarimde, was also reduced at E18.5 while
that of acid beta-glucosidase (Gba2), which is involved in salvage
pathway, was downregulated in skin of null mice from E16.5 to E18.5
56
(Figure 2.7 D-F). Ceramidases are involved in the breaking down of
short-chain ceramide, facilitating its conversion to sphingosine. In
particular, reduced expression of acid ceramidase 2 (Asah 2) and
alkaline ceramidase 1 (Acer1) was observed in all developmental
stages in the mutant epidermis (Figure 2.7 G-I). Levels of all other
members of acid ceramidases (Asah 1 and Asah 3) and alkaline
ceramidases (Acer 2 and Acer 3) were unaltered in the mutants at all
stages of development (Figure 2.7 G-I). Similarly, sphingosine kinases
(Sphk1, Sphk2) are responsible for the disposal of sphingosine to
sphingosine-1-phosphate, whose expression level was reduced at
E16.5 in the mutant epidermis (Figure 2.7 G). No significant changes in
the relative mRNA levels of several other enzymes involved in ceramide
synthesis pathways was observed between wildtype and Ctip2-/embryonic skin (Figure 2.7 G-I, 2.8 A-C). Furthermore, protein levels of
Lass2, Gba2 and eLox3 were significantly reduced in the Ctip2 null skin
compared with the wildtype skin at E18.5 (Figure 2.9 A-B) (Golonzhka
et al., 2008). Taken together, our results indicate that expression of a
subset of genes encoding enzymes involved in the sphingolipid
biosynthesis pathways was altered during skin organogenesis in
absence of Ctip2.
57
To investigate whether some of the lipid-metabolizing genes, which are
dysregulated in the mutant skin, are direct targets of transcriptional
regulator Ctip2, we conducted in vivo chromatin immunoprecipitation
(ChIP) assay on neonatal (P0) mouse epidermal keratinocyte extracts.
The promoter regions of several genes encoding lipid-metabolizing
enzymes such as Lass1-3, 6, Sptlc1-3, Smpd1-2, Ugcg, Gba2, and
eLox3 (Golonzhka et al., 2008), whose expressions were altered in the
Ctip2-/- skin, were selected and primers were designed to determine
possible Ctip2 binding within -6 kb upstream of transcription starting
sites (TSS). Our results indicated that Ctip2 was recruited within -1 kb
promoter region of Lass2, Gba2 and eLox3, as well as between 5-6 kb
promoter region of eLox3 relative to TSS (Figure 2.10 A-C). However,
we did not observe the direct recruitment of Ctip2 to the promoter
regions of several other lipid-metabolizing genes (Figure 2.11 A-K). Our
results indicate that Ctip2 is directly recruited to the promoter region of
a subset of lipid metabolizing genes and possibly regulates their
expression in the developing epidermis.
Altogether, results suggest
that Ctip2 directly or indirectly regulates expression of a subset of lipid
metabolizing genes and control epidermal sphingolipid biosynthesis
during skin organogenesis.
58
2.5 Discussion
The establishment of an effective physical barrier protecting the living
organism from its surrounding environment is the key function of
mammalian skin (Hoffjan and Stemmler, 2007). The transport of most
substances across the stratum corneum (SC) takes place through the
lipid bilayer, suggesting its essential role for the epidermal barrier
function (Jungersted et al., 2008). The major SC lipids, ceramides,
cholesterol and fatty acids have distinct roles in maintaining normal
barrier functions (Elias and Menon, 1991; Ishikawa et al., 2010).
Ceramides as well as cholesterol were shown to have an influence on
(and are influenced by) the integrity of the SC, while free fatty acids
play a major role in the lipid bilayer formation and pH maintenance
(Baroni et al., 2012). We have identified Ctip2 as a key regulator of skin
lipid metabolism, which plays an important role in establishment of
epidermal permeability barrier (EPB) during development. Ablation of
Ctip2 leads to altered lipid composition in the developing mouse
epidermis, such as reduced ceramide at E17.5-P0, as well as increased
sphingomyelins
at
E16.5-E18.5.
Ctip2
regulate
sphingolipid
homeostasis in mouse embryonic skin by modulating the expression of
several key enzymes involved in lipid metabolism. Here we
demonstrate that transcription factor Ctip2 is recruited to the promoter
59
regions of a subset of genes (elox3, Lass2 and Gba2) involved in the
sphingolipid biosynthesis pathways and could directly regulate their
expression (Figure 2.12).
Skin barrier defects are caused not only by altered levels of protein(s)
involved in keratinocytes terminal differentiation, cross-linking of
cornified envelops (CEs), formation of intercellular junctions, but also by
impaired formation and maintenance of skin lipid barrier (Furuse et al.,
2002; Kirschner and Brandner, 2012). Our previous studies have shown
that Ctip2 regulate keratinocyte proliferation and late terminal
differentiation in a cell-autonomous manner; the ultrastructural analyses
of the epidermis have revealed defects in lipid disc formation, loosely
packed corneocytes, as well as disorganized intercellular lamellar body
membranes in Ctip2 null mice (Golonzhka et al., 2008 and unpublished
data). In this study, we have focused on the role of Ctip2 in controlling
epidermal lipid barrier by modulating lipid metabolism. Herein, we have
applied a mass spectrometry method to systemically detect and profile
ceramides,
sphingomyelins,
sphingosines
and
sphinganines
simultaneously in murine epidermis. As a crucial member of stratum
corneum
lipids,
there
are
few
reports
on
profiling
of
skin
sphingomyelins. Our present data confirm deregulated levels of both
long chain and very long chain saturated and unsaturated ceramides in
60
the mutant epidermis at different stages of skin development. Along the
same line, overall levels of long chain and very long chain saturated
and unsaturated sphingomyelins were significantly enhanced in the
mutants at all stages analyzed. In the present study, we have
specifically looked at ceramide [NS] but were unable to perform massspectrometry study on other subclasses such as NP, NH, AS, AP, AH,
EOS, EOP and EOH, due to the lack of available deuterated ceramide
internal standards for each subclasses. Additional analyses for the
different subclasses will be performed in future using specific internal
standard for each subclass.
Our results indicate that altered expression(s) of ceramide synthases 16
(Lass
1-6),
sphingomyelinases
(Smpd1
and
Smpd2),
glucosylceramide synthase (Ugcg) and that of acid beta-glucosidase
(Gba2) at different stages of skin development contribute to an overall
decrease in the epidermal ceramide levels in the mutant embryonic
skin. Lass 1-6 have been reported to be involved in UVB-induced
apoptosis and their expression was increased after UVB irradiation in
normal human keratinocytes, but reduced in aged skin (Uchida et al.,
2003; Jensen et al., 2005). A recent study has reported that Lass3
mutant mice die after birth due to increased skin barrier defects,
characterized by increased TEWL, loss of ultra-long chain ceramides as
61
well as non-functional cornified envelopes (Jennemann et al., 2011).
Upregulation of sphingomyelin synthase (Sgms2) in Ctip2-/- mice may
partially account for increased amount of sphingomyelins observed at
later stages in the mutant skin (Figure 2.3 B-C(Wang et al., 2011)).
Furthermore, loss of Gba2, whose expression is altered in the mutant
epidermis, has been reported to lead to barrier defects in Gaucher’s
disease patients (Holleran et al., 1994). Similarly, eLox3 as well as its
substrate 12R-lipoxygenase (12R-LOX) are involved in the formation of
oxidized ceramides in mouse epidermis; 12R-LOX deficient mice die
shortly after birth due to severe barrier abnormalities with absence of
protein-bound EOS ceramide (Zheng et al., 2011). Interestingly, we
have reported earlier that eLox3 expression is downregulated in the
Ctip2-/- embryonic skin (Golonzhka et al., 2008). Here we have
furthered our studies and have shown that Ctip2 could directly regulate
the expression of eLox3, Gba2 and Lass2 in mouse embryonic skin by
recruitment to their promoter regions. Altogether, we have identified
Ctip2 as a key regulator of several lipid metabolizing genes and hence
epidermal sphingolipid biosynthesis during skin development.
Sphingolipids are key components in skin barrier homeostasis, as well
as in other cellular processes such as cell cycle arrest, apoptosis and
stress responses (Hannun, 1996; Ogretmen and Hannun, 2004).
62
Ceramide is a central metabolite, whose biosynthesis is regulated at
multiple levels with spatial separation of enzymes involved in ceramide
formation (Futerman and Riezman, 2005; Kitatani et al., 2008).
It
remains unclear that if alteration of the enzymes in one of the pathways
could have an effect on other pathways, and finally modulate the
biological functions. It would be interesting to systemically compare the
lipid composition in healthy subjects vs patients with skin disorders
such as atopic dermatitis and psoriasis. It is likely possible that besides
Ctip2, other regulatory pathways are also involved in controlling
ceramide biosynthesis. B-cell lymphoma/leukemia 11A (Bcl11a/Ctip1) is
a homolog of Ctip2, which was found to be upregulated in Ctip2 null
mice at E18.5 (Golonzhka et al., 2008). The role of Ctip1 in epidermal
barrier development and maintenance is still unclear. Further
investigation about the expression pattern and distribution of Ctip1, as
well as its role in lipid barrier homeostasis in mouse epidermis will be
performed utilizing Ctip1-/- mice.
63
E16.5
150
100
N-acyl chain length
28
100
28
26
24
22
28
26
24
N-acyl chain length
20
50
0
22
20
18
16
14
*
WT
Ctip2-/-
18
50
150
16
*
100
D
14
WT
Ctip2-/-
Ceramide Content
(pmol/mg skin)
Ceramide Content
(pmol/mg skin)
26
P0
150
0
24
N-acyl chain length
E18.5
C
22
0
20
50
18
26
24
22
*
20
18
16
0
*
WT
Ctip2-/-
200
16
100
250
14
150
50
B
Ceramide Content
(pmol/mg skin)
WT
Ctip2-/-
14
Ceramide Content
(pmol/mg skin)
200
28
A
E17.5
N-acyl chain length
Figure 2.1 Liquid chromatography tandem mass-spectrometry
(LC/MS/MS) analyses of mouse epidermal saturated ceramides
during skin development.
Epidermal sphingolipids were extracted from skin of (A) E16.5; (B)
E17.5; (C) E18.5 embryos and (D) P0 wildtype and Ctip2-/- mice.
Amount of saturated ceramides was determined according to their Nacyl chain length using LC/MS/MS analyses. Statistical analyses were
performed by student’s unpaired t-test using Graphpad Prism software.
*p<0.05.
64
E16.5
15
WT
Ctip2-/-
10
N-acyl chain length
N-acyl chain length
28:1
26:1
WT
Ctip2-/-
30
20
28:1
26:1
24:1
22:1
20:1
0
18:1
10
16:1
28:1
26:1
24:1
22:1
20:1
18:1
16:1
5
40
14:1
10
D
Ceramide Content
(pmol/mg skin)
WT
Ctip2-/-
14:1
Ceramide Content
(pmol/mg skin)
24:1
P0
15
0
22:1
N-acyl chain length
E18.5
C
20:1
0
18:1
5
16:1
26:1
24:1
22:1
20:1
18:1
0
16:1
5
14:1
Ceramide Content
(pmol/mg skin)
10
B
14:1
WT
Ctip2-/-
*
Ceramide Content
(pmol/mg skin)
15
28:1
A
E17.5
N-acyl chain length
Figure 2.2 Liquid chromatography tandem mass-spectrometry
(LC/MS/MS) analyses of mouse epidermal unsaturated ceramides
during skin development.
Epidermal sphingolipids were extracted from skin of (A) E16.5; (B)
E17.5; (C) E18.5 embryos and (D) P0 wildtype and Ctip2-/- mice.
Amount of unsaturated ceramides was determined according to their Nacyl chain length using LC/MS/MS analyses. Statistical analyses were
performed by student’s unpaired t-test using Graphpad Prism software.
*p<0.05.
65
E16.5
100
50
N-acyl chain length
28
26
24
22
0
20
28
26
24
22
20
18
16
*
50
18
20
100
16
*
WT
Ctip2-/-
14
**
40
150
Sphingomyelin Content
(pmol/mg skin)
WT
Ctip2-/-
**
14
Sphingomyelin Content
(pmol/mg skin)
28
P0
D
60
0
26
N-acyl chain length
E18.5
C
24
N-acyl chain length
*
22
0
28
26
24
22
20
18
0
16
*
150
20
500
WT
Ctip2-/-
200
18
1000
250
16
*
B
14
WT
Ctip2-/-
Sphingomyelin Content
(pmol/mg skin)
1500
14
Sphingomyelin Content
(pmol/mg skin)
A
E17.5
N-acyl chain length
Figure 2.3 LC/MS/MS analyses of mouse epidermal saturated
sphingomyelins during skin development.
Epidermal sphingolipids were extracted from skin of (A) E16.5; (B)
E17.5; (C) E18.5 embryos and (D) P0 wildtype and Ctip2-/- mice.
Amount of saturated sphingomyelins was determined according to their
N-acyl chain length using LC/MS/MS analyses. Statistical analyses
were performed by student’s unpaired t-test using Graphpad Prism
software. *p<0.05; **p<0.005.
66
E16.5
60
40
N-acyl chain length
N-acyl chain length
15
WT
Ctip2-/-
10
28:1
26:1
24:1
22:1
20:1
18:1
0
16:1
5
14:1
28:1
26:1
24:1
22:1
20:1
18:1
16:1
2
Sphingomyelin Content
(pmol/mg skin)
*
14:1
Sphingomyelin Content
(pmol/mg skin)
D
WT
Ctip2-/-
***
0
28:1
P0
6
4
26:1
N-acyl chain length
E18.5
C
24:1
**
20
0
28:1
26:1
24:1
22:1
20:1
16:1
0
18:1
**
*
80
22:1
100
WT
Ctip2-/-
20:1
200
100
18:1
300
B
16:1
WT
Ctip2-/-
14:1
*
Sphingomyelin Content
(pmol/mg skin)
400
14:1
Sphingomyelin Content
(pmol/mg skin)
A
E17.5
N-acyl chain length
Figure 2.4 LC/MS/MS analyses of mouse epidermal unsaturated
sphingomyelins during skin development.
Epidermal sphingolipids were extracted from skin of (A) E16.5; (B)
E17.5; (C) E18.5 embryos and (D) P0 wildtype and Ctip2-/- mice.
Amount of unsaturated sphingomyelins was determined according to
their N-acyl chain length using LC/MS/MS analyses. Statistical analyses
were performed by student’s unpaired t-test using Graphpad Prism
software. *p<0.05; **p<0.005; ***p<0.001.
67
E16.5
A
E17.5
WT
Ctip2-/-
B
25
Lipid Content
(pmol/mg skin)
Lipid Content
(pmol/mg skin)
6
4
2
WT
Ctip2-/-
20
15
10
5
0
C
16 18 20
16 18 20
Sphingosine
Sphinganine
16 18 20
16 18 20
Sphingosine
Sphinganine
N-acyl chain length
N-acyl chain length
E18.5
P0
15
WT
Ctip2-/-
D
10
Lipid Content
(pmol/mg skin)
Lipid Content
(pmol/mg skin)
0
10
5
WT
Ctip2-/-
8
6
4
2
0
16 18 20
16 18 20
Sphingosine
Sphinganine
N-acyl chain length
0
16 18 20
16 18 20
Sphingosine
Sphinganine
N-acyl chain length
Figure 2.5 LC/MS/MS analyses of mouse epidermal sphingosines
and sphinganines during skin development.
Epidermal sphingosines and sphinganines were measured in the skin of
(A) E16.5, (B) E17.5, (C) E18.5 embryos and (D) P0 wildtype and
Ctip2-/- mice. The amount of skin sphingosines and sphinganines were
determined according to their N-acyl chain length by LC/MS/MS
analyses, quantified utilizing sphingosine and sphinganine standards,
and expressed as pmol/ mg of skin (for details see methods).
Statistical analyses were performed by student’s unpaired t-test using
Graphpad Prism software. The results depicted in this figure are
representative of at least two additional studies that have been
conducted using different litters of mice of identical genotypes.
68
200
P0
E18.5
100
P0
E18.5
stearic acid
300
WT
Ctip2-/-
200
*
100
0
P0
300
E17.5
0
E18.5
WT
Ctip2-/-
400
peak area /skin dry weight (mg)
500
0
5000
D
palmitic acid
*
10000
E17.5
P0
E18.5
E17.5
0
E16.5
50
*
15000
E16.5
100
WT
Ctip2-/-
20000
E17.5
150
cholesterol 3-sulfate
25000
E16.5
WT
Ctip2-/-
peak area /skin dry weight (mg)
200
C
peak area /skin dry weight (mg)
B
cholesterol
E16.5
peak area /skin dry weight (mg)
A
Figure 2.6 UPLC/MS/MS analyses of mouse epidermal cholesterol
and fatty acids during skin development.
Epidermal lipids were extracted from skin of E16.5, E17.5, E18.5
embryos and P0 wildtype and Ctip2-/- mice. Amount of (A) cholesterol,
(B) cholesterol 3-sulfate, (C) palmitic acid and (D) stearic acid were
determined using UPLC/MS/MS analyses. Statistical analyses were
performed by student’s unpaired t-test using Graphpad Prism software.
*p<0.05; **p<0.005.
69
*
*
0.5
*
**
Acer3
Acer1
Acer2
Sphk2
0.0
1.0
*
**
**
0.0
*
*
Lass1
Lass2
**
Sptlc3
Lass6
Sptlc1
Lass3
WT
0.0
WT
1.5
Ctip2-/-
1.0
*
*
Smpd2
*
0.5
Smpd1
2.0
1.5
Gba2
WT
Ctip2-/-
1.0
0.5
Ugcg
WT
0.0
Sgms2
Relative Expression Level
Relative Expression Level
Sptlc3
Lass6
Sptlc1
Lass3
Lass2
WT
Lass1
Ctip2-/-
0.5
0.5
*
*
*
0.0
Acer3
1.0
I
WT
1.5
Ctip2-/-
1.0
WT
Asah1
Asah2
Sphk1
Sphk2
Acer1
Acer2
Ctip2-/-
0.0
Relative Expression Level
H
WT
*
Acer2
Acer3
Gba2
Ugcg
Sgms2
Smpd2
Smpd1
WT
1.5
Asah2
Sphk1
Relative Expression Level
0.0
0.5
1.5
Gba2
*
*
Acer1
*
0.5
1.0
Ugcg
1.0
Ctip2-/-
Sgms2
1.5
*
WT
F
WT
1.5
Smpd2
Ctip2-/-
*
0.0
Sphk1
Sphk2
WT
2.0
*
0.5
E
Relative Expression Level
Sptlc3
Lass6
Sptlc1
Lass3
Lass2
WT
Lass1
0.0
WT
Asah1
Relative Expression Level
*
**
1.0
WT
*
0.5
Ctip2-/-
Smpd1
*
1.5
E18.5
C
WT
WT
1.0
D
G
Relative Expression Level
Ctip2-/-
Relative Expression Level
Relative Expression Level
1.5
E17.5
B
WT
Asah1
Asah2
E16.5
A
Figure 2.7 Ctip2 regulates the expression of genes encoding lipid
metabolizing enzymes during skin development.
Expression of (A, B, C) ceramide synthases (Lass1-Lass3, Lass6),
serine
palmitoyltransferases
(Sptlc1,
Sptlc3);
(D,
E,
F)
sphingomyeinases (Smpd1, Smpd2), sphingomyelin synthase 2
(Sgms2), glucosylceramide synthase (Ugcg) and acid beta-glucosidase
(Gba2); (G, H, I) N-acylsphingosine amidohydrolases (acid
ceramidases: Asah1, Asah2), sphingosine kinases (Sphk1, Sphk2) and
alkaline ceramidases (Acer1, Acer2) was analyzed by RT-qPCR using
specific primers indicated in table S1. All values represent relative
transcript level after normalization with Gapdh transcripts. Statistical
analyses were performed by student’s unpaired t-test using Graphpad
Prism software. *p<0.05; **p<0.005. The results depicted in this figure
are representative of at least three additional studies that have been
conducted using numerous litters of mice of identical genotypes.
70
E16.5
1.0
Sgms1
Kdsr
Figure 2.8 Relative expression of genes encoding
Wang
et al., 2012
metabolizing enzymes Figure
duringS3,
skin
development.
Degs1
Lass5
0.0
Sptlc2
0.5
WT
Degs1
Sgms1
Kdsr
Lass5
Sptlc2
0.0
WT
0.5
WT
Ctip2-/-
1.5
Lass4
1.0
Relative Expression Level
C
WT
Ctip2-/-
1.5
Lass4
Degs1
Sgms1
Kdsr
Lass5
Sptlc2
Lass4
0.5
Relative Expression Level
1.0
0.0
E18.5
B
WT
Ctip2-/-
1.5
WT
Relative Expression Level
A
E17.5
lipid-
Expression
of
ceramide
synthases
(Lass4-5),
serine
palmitoyltransferase 2 (Sptlc2), dihydroceramide desaturase (Degs1),
Ketodihydro-sph reductase (Kdsr) and sphingomyelin synthase 1
(Sgms1) was studied at (A) E16.5, (B) E17.5 and (C) E18.5 stages of
development by RT-qPCR using specific primers indicated in table S1.
Values represent relative transcript level after normalization with Gapdh
transcripts. Statistical analyses were performed by student’s unpaired ttest using Graphpad Prism software.
71
Figure 2.9 Immunoblot analyses of lipid metabolizing enzymes in
the developing murine epidermis.
(A) Western blotting analyses was performed on protein extracted from
E18.5 wildtype (WT) and Ctip2-/- mice epidermis to study the expression
level of eLox3, Gba2 and Lass2 using specific antibodies. β-actin was
used as an internal control. (B) Protein levels of eLox3, Gba2 and
Lass2 were quantified in wildtype and mutant epidermis by
densitometry analyses of western blots. All data was normalized by the
corresponding β-actin levels. Statistical analyses were performed by
student’s unpaired t-test using Graphpad Prism software. *p<0.05. The
results depicted in this figure are representative of at least three
additional studies that have been conducted using different litters of
mice of identical genotypes.
72
8
6
4
30
**
10
0
0
di
st
al
pr
ox
im
al
3'
-U
TR
5
di
st
al
pr
ox
im
al
3'
-U
TR
2
15
eLox3
IgG
Ctip2
IgG
Ctip2
**
20
10
**
0
di
st
al
pr
ox
im
al
3'
-U
TR
*
IgG
Ctip2
C
Gba2
Relative input %
10
Relative input %
B
Lass2
Relative input %
A
Figure 2.10 Ctip2 interacts directly with the promoter region of
lipid metabolizing genes.
Chromatin immunoprecipitation (ChIP) assay was performed on freshly
isolated neonatal mouse skin keratinocytes using anti-Ctip2 antibody
and results were analyzed by qPCR using primers indicated in table S2.
Rat IgG was used as a control. Ctip2 was recruited to the promoter
regions of (A) Lass2, (B) Gba2 and (C) eLox3. Statistical analyses were
performed by student’s unpaired t-test using Graphpad Prism software.
*p<0.05; **p<0.005.
0.20
0.15
0.10
0.05
0.00
0.0
Smpd3
H
IgG
Ctip2
0.3
0.2
0.1
0.0
IgG
Ctip2
4
Dgat2
K
3
2
1
0
0.0
E Smpd1
F
IgG
Ctip2
1.5
1.0
0.5
0.0
Sgms2
I
IgG
Ctip2
3
2
1
0
Aloxe12b
IgG
Ctip2
pr
ox
im
al
0.5
pr
ox
im
al
di
st
al
1.0
Relative input %
IgG
Ctip2
Relative input %
pr
ox
im
al
C
pr
ox
im
al
0.5
2.0
di
st
al
1.0
1.5
di
st
al
IgG
Ctip2
Lass3
Relative input %
Ugcg
pr
ox
im
al
di
st
al
0.0
pr
ox
im
al
0.4
di
st
al
0.5
Relative input %
1.0
B
pr
ox
im
al
0.5
di
st
al
1.5
Relative input %
IgG
Ctip2
Relative input %
pr
ox
im
al
Lass1
di
st
al
J
pr
ox
im
al
di
st
al
Relative input %
1.5
Relative input %
G
pr
ox
im
al
di
st
al
Relative input %
D
pr
ox
im
al
di
st
al
Relative input %
A
di
st
al
Relative input %
73
Lass6
3
IgG
Ctip2
2
1
0
Smpd2
2.0
IgG
Ctip2
1.5
1.0
0.5
0.0
Elovl4
2.5
IgG
Ctip2
2.0
1.5
1.0
0.5
0.0
74
Figure 2.11 ChIP analyses on murine keratinocytes for lipid
metabolizing genes.
Chromatin immunoprecipitation (ChIP) assay was performed on freshly
isolated primary keratinocytes from neonatal mouse skin using antiCtip2 antibody and results were analyzed by qPCR using specific
primers indicated in table S2. Rat IgG was used as a control. Ctip2 was
not recruited to distal and proximal promoter regions of (A) Lass1, (B)
Lass2, (C) Lass6, (D) Ugcg, (E) Smpd1, (F) Smpd2, (G) Smpd3, (H)
Sgms2, (I) Elovl4, (J) Dgat2 and (K) Aloxe12b.
75
de novo pathway
Salvage pathway
palmitoyl-CoA
L-serine
complex
sphingolipids
Sptlc1 (E16.5-E17.5)
Sptlc2
Sptlc3 (E16.5)
glucosyl-cer
ketodihydro-sph
!"
Kdsr
dihydro-sph
Lass4
Lass5
Lass6
(E16.5-E18.5)
Gba2 (E16.5, E17.5)
Ctip2
Lass1 (E18.5)
Lass2 (E18.5)
Lass3 (E16.5)
dihydro-cer
Exogenous
ceramide recycling
Ugcg (E18.5)
Ceramide
!"
Degs1
Asah1
Asah2 (E16.5-E18.5)
Ceramide
Lass/CerSs
Short chain
ceramide
Asah1
Asah2 (E16.5-E18.5)
Acer1 (E16.5-E18.5)
Acer2
Acer3
Sphingosine
Asahs
Acers
Smpd1 (E16.5-E18.5)
Smpd2 (E18.5)
Sgms1
Sgms2 (E16.5)
Sphk1 (E16.5)
Sphk2 (E16.5)
sphingomyelin
Sphingomyelinase
pathway
Sphingosine1-phosphate
Figure S5, Wang et al., 2012
Figure 2.12 Schematic representation of Ctip2 mediated
differential regulation of genes encoding different enzymes of the
ceramide synthesis pathways.
The scheme indicates metabolic pathways for ceramide biosynthesis:
de novo pathway, salvage pathway, sphingomyelinase pathway and
exogenous ceramide recycling. Genes involved in these pathways are
indicated. Stages at which altered gene expression were observed are
indicated within brackets. Red: downregulation of gene expression;
green: upregulation of gene expression. Ctip2 positively regulates Gba2
and Lass2 expressions by direct recruitment to their promoter regions.
Expression of ceramide synthases 1-6 (Lass 1-6), serine
palmitoyltransferases 1-3 (Sptlc 1-3), dihydroceramide desaturase
(Degs1), Ketodihydro-sph reductase (Kdsr), glucosylceramide synthase
(Ugcg), acid beta-glucosidase (Gba2), sphingomyelin synthases 1-2
(Sgms1-2), sphingomyeinases 1-2 (Smpd1, Smpd2),N-acylsphingosine
amidohydrolases (acid ceramidases: Asah1, Asah2), alkaline
ceramidases 1-2 (Acer1, Acer2), sphingosine kinases 1-2 (Sphk1,
Sphk2) are indicated for the different pathways.
76
Table 2.1 Primer sequences used in q-PCR assays.
77
Table 2.2 Primer sequences used in ChIP assays.
78
2.6 References
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D.J., Ishmael, J.E.,
and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger
proteins implicated in transcriptional repression mediated by chicken
ovalbumin upstream promoter transcription factor (COUP-TF) orphan
nuclear receptors. J Biol Chem 275, 10315–10322.
Avram, D., Fields, A., Senawong, T., Topark-Ngarm, A., and Leid, M.
(2002). COUP-TF (chicken ovalbumin upstream promoter transcription
factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding
protein. Biochem J 368, 555–563.
Baroni, A., Buommino, E., De Gregorio, V., Ruocco, E., Ruocco, V.,
and Wolf, R. (2012). Structure and function of the epidermis related to
barrier properties. Clinics in Dermatology 30, 257–262.
Becker, K.P. (2004). Selective Inhibition of Juxtanuclear Translocation
of Protein Kinase C II by a Negative Feedback Mechanism Involving
Ceramide Formed from the Salvage Pathway. Journal of Biological
Chemistry 280, 2606–2612.
Bligh, E.G., and Dyer, W.J. (1959). A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37, 911–917.
Elias, P.M. (2004). The epidermal permeability barrier: from the early
days at Harvard to emerging concepts. J. Invest. Dermatol 122, xxxvi–
xxxix.
Elias, P.M., and Menon, G.K. (1991). Structural and lipid biochemical
correlates of the epidermal permeability barrier. Adv. Lipid Res 24, 1–
26.
Feingold, K.R. (2007). Thematic review series: skin lipids. The role of
epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid
Res 48, 2531–2546.
Folch, J., Lees, M., and Sloane Stanley, G.H. (1957). A simple method
for the isolation and purification of total lipides from animal tissues. J.
Biol. Chem. 226, 497–509.
79
Fuchs, E., and Raghavan, S. (2002). Getting under the skin of
epidermal morphogenesis. Nat Rev Genet 3, 199–209.
Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani,
Y., Noda, T., Kubo, A., and Tsukita, S. (2002). Claudin-based tight
junctions are crucial for the mammalian epidermal barrier: a lesson from
claudin-1-deficient mice. J. Cell Biol. 156, 1099–1111.
Futerman, A.H., and Riezman, H. (2005). The ins and outs of
sphingolipid synthesis. Trends Cell Biol. 15, 312–318.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J.-M., Campbell,
A.L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M., and Indra,
A.K. (2008). Dual Role of COUP-TF-Interacting Protein 2 in Epidermal
Homeostasis and Permeability Barrier Formation. J Investig Dermatol
129, 1459–1470.
Hannun, Y.A. (1996). Functions of Ceramide in Coordinating Cellular
Responses to Stress. Science 274, 1855–1859.
Hoffjan, S., and Stemmler, S. (2007). On the role of the epidermal
differentiation complex in ichthyosis vulgaris, atopic dermatitis and
psoriasis. British Journal of Dermatology 157, 441–449.
Holleran, W.M., Ginns, E.I., Menon, G.K., Grundmann, J.U., Fartasch,
M., McKinney, C.E., Elias, P.M., and Sidransky, E. (1994).
Consequences of beta-glucocerebrosidase deficiency in epidermis.
Ultrastructure and permeability barrier alterations in Gaucher disease.
J. Clin. Invest. 93, 1756–1764.
Holleran, W.M., Takagi, Y., and Uchida, Y. (2006). Epidermal
sphingolipids: metabolism, function, and roles in skin disorders. FEBS
Lett. 580, 5456–5466.
Hyter, S., Bajaj, G., Liang, X., Barbacid, M., Ganguli-Indra, G., and
Indra, A.K. (2010). Loss of nuclear receptor RXRα in epidermal
keratinocytes promotes the formation of Cdk4-activated invasive
melanomas. Pigment Cell Melanoma Res 23, 635–648.
Imokawa, G. (2009). A possible mechanism underlying the ceramide
deficiency in atopic dermatitis: Expression of a deacylase enzyme that
cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.
Journal of Dermatological Science 55, 1–9.
80
Indra, A.K., Dupe, V., Bornert, J.M., Messaddeq, N., Yaniv, M., Mark,
M., Chambon, P., and Metzger, D. (2005a). Temporally controlled
targeted somatic mutagenesis in embryonic surface ectoderm and fetal
epidermal keratinocytes unveils two distinct developmental functions of
BRG1 in limb morphogenesis and skin barrier formation. Development
132, 4533–4544.
Indra, A.K., Mohan, W.S., Frontini, M., Scheer, E., Messaddeq, N.,
Metzger, D., and Tora, L. (2005b). TAF10 is required for the
establishment of skin barrier function in foetal, but not in adult mouse
epidermis. Dev Biol 285, 28–37.
Ishikawa, J., Narita, H., Kondo, N., Hotta, M., Takagi, Y., Masukawa, Y.,
Kitahara, T., Takema, Y., Koyano, S., Yamazaki, S., et al. (2010).
Changes in the Ceramide Profile of Atopic Dermatitis Patients. Journal
of Investigative Dermatology 130, 2511–2514.
Janssens, M., van Smeden, J., Gooris, G.S., Bras, W., Portale, G.,
Caspers, P.J., Vreeken, R.J., Kezic, S., Lavrijsen, A.P.M., and
Bouwstra, J.A. (2011). Lamellar Lipid Organization and Ceramide
Composition in the Stratum Corneum of Patients with Atopic Eczema.
Journal of Investigative Dermatology 131, 2136–2138.
Jennemann, R., Rabionet, M., Gorgas, K., Epstein, S., Dalpke, A.,
Rothermel, U., Bayerle, A., van der Hoeven, F., Imgrund, S., Kirsch, J.,
et al. (2011). Loss of ceramide synthase 3 causes lethal skin barrier
disruption. Hum. Mol. Genet.
Jensen, J.-M., Förl, M., Winoto-Morbach, S., Seite, S., Schunck, M.,
Proksch, E., and Schütze, S. (2005). Acid and neutral
sphingomyelinase, ceramide synthase, and acid ceramidase activities
in cutaneous aging. Exp. Dermatol 14, 609–618.
Jungersted, J.M., Hellgren, L.I., Jemec, G.B.E., and Agner, T. (2008).
Lipids and skin barrier function – a clinical perspective. Contact
Dermatitis 58, 255–262.
Kirschner, N., and Brandner, J.M. (2012). Barriers and more: functions
of tight junction proteins in the skin. Ann. N. Y. Acad. Sci. 1257, 158–
166.
81
Kitatani, K., Idkowiak-Baldys, J., and Hannun, Y.A. (2008). The
sphingolipid salvage pathway in ceramide metabolism and signaling.
Cell. Signal 20, 1010–1018.
Marchesini, N., and Hannun, Y.A. (2004). Acid and neutral
sphingomyelinases: roles and mechanisms of regulation. Biochem. Cell
Biol. 82, 27–44.
Masukawa, Y., Narita, H., Shimizu, E., Kondo, N., Sugai, Y., Oba, T.,
Homma, R., Ishikawa, J., Takagi, Y., Kitahara, T., et al. (2008).
Characterization of overall ceramide species in human stratum
corneum. The Journal of Lipid Research 49, 1466–1476.
Merrilljr, A., Sullards, M., Allegood, J., Kelly, S., and Wang, E. (2005).
Sphingolipidomics: High-throughput, structure-specific, and quantitative
analysis of sphingolipids by liquid chromatography tandem mass
spectrometry. Methods 36, 207–224.
Mizutani, Y., Mitsutake, S., Tsuji, K., Kihara, A., and Igarashi, Y. (2009).
Ceramide biosynthesis in keratinocyte and its role in skin function.
Biochimie 91, 784–790.
Monette, J.S., Gómez, L.A., Moreau, R.F., Bemer, B.A., Taylor, A.W.,
and Hagen, T.M. (2010). Characteristics of the rat cardiac sphingolipid
pool in two mitochondrial subpopulations. Biochemical and Biophysical
Research Communications 398, 272–277.
Monette, J.S., Gómez, L.A., Moreau, R.F., Dunn, K.C., Butler, J.A.,
Finlay, L.A., Michels, A.J., Shay, K.P., Smith, E.J., and Hagen, T.M.
(2011). (R)-α-Lipoic acid treatment restores ceramide balance in aging
rat cardiac mitochondria. Pharmacological Research 63, 23–29.
Nemes, Z., Demény, M., Marekov, L.N., Fésüs, L., and Steinert, P.M.
(2000). Cholesterol 3-sulfate interferes with cornified envelope
assembly by diverting transglutaminase 1 activity from the formation of
cross-links and esters to the hydrolysis of glutamine. J. Biol. Chem.
275, 2636–2646.
Nemes, Z., and Steinert, P.M. (1999). Bricks and mortar of the
epidermal barrier. Exp. Mol. Med. 31, 5–19.
82
Oda, Y., Uchida, Y., Moradian, S., Crumrine, D., Elias, P.M., and Bikle,
D.D. (2008). Vitamin D Receptor and Coactivators SRC2 and 3
Regulate Epidermis-Specific Sphingolipid Production and Permeability
Barrier Formation. J Investig Dermatol 129, 1367–1378.
Ogretmen, B. (2002). Biochemical Mechanisms of the Generation of
Endogenous Long Chain Ceramide in Response to Exogenous Short
Chain Ceramide in the A549 Human Lung Adenocarcinoma Cell Line.
ROLE FOR ENDOGENOUS CERAMIDE IN MEDIATING THE ACTION
OF EXOGENOUS CERAMIDE. Journal of Biological Chemistry 277,
12960–12969.
Ogretmen, B., and Hannun, Y.A. (2004). Biologically active
sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer
4, 604–616.
Park, H., Haynes, C.A., Nairn, A.V., Kulik, M., Dalton, S., Moremen, K.,
and Merrill, A.H. (2009). Transcript profiling and lipidomic analysis of
ceramide subspecies in mouse embryonic stem cells and embryoid
bodies. The Journal of Lipid Research 51, 480–489.
Schmuth, M., Man, M.Q., Weber, F., Gao, W., Feingold, K.R., Fritsch,
P., Elias, P.M., and Holleran, W.M. (2000). Permeability barrier disorder
in Niemann-Pick disease: sphingomyelin-ceramide processing required
for normal barrier homeostasis. J. Invest. Dermatol. 115, 459–466.
van Smeden, J., Hoppel, L., van der Heijden, R., Hankemeier, T.,
Vreeken, R.J., and Bouwstra, J.A. (2011). LC/MS analysis of stratum
corneum lipids: ceramide profiling and discovery. J. Lipid Res. 52,
1211–1221.
Sultan, I., Senkal, C.E., Ponnusamy, S., Bielawski, J., Szulc, Z.,
Bielawska, A., Hannun, Y.A., and Ogretmen, B. (2006). Regulation of
the sphingosine-recycling pathway for ceramide generation by oxidative
stress, and its role in controlling c-Myc/Max function. Biochemical
Journal 393, 513.
Uchida, Y., Hara, M., Nishio, H., Sidransky, E., Inoue, S., Otsuka, F.,
Suzuki, A., Elias, P.M., Holleran, W.M., and Hamanaka, S. (2000).
Epidermal sphingomyelins are precursors for selected stratum corneum
ceramides. J. Lipid Res. 41, 2071–2082.
83
Uchida, Y., Nardo, A.D., Collins, V., Elias, P.M., and Holleran, W.M.
(2003). De novo ceramide synthesis participates in the ultraviolet B
irradiation-induced apoptosis in undifferentiated cultured human
keratinocytes. J. Invest. Dermatol. 120, 662–669.
Wang, Z., Coleman, D.J., Bajaj, G., Liang, X., Ganguli-Indra, G., and
Indra, A.K. (2011). RXRα ablation in epidermal keratinocytes enhances
UVR-induced DNA damage, apoptosis, and proliferation of
keratinocytes and melanocytes. J. Invest. Dermatol. 131, 177–187.
Zheng, Y., Yin, H., Boeglin, W.E., Elias, P.M., Crumrine, D., Beier, D.R.,
and Brash, A.R. (2011). Lipoxygenases Mediate the Effect of Essential
Fatty Acid in Skin Barrier Formation: A PROPOSED ROLE IN
RELEASING OMEGA-HYDROXYCERAMIDE FOR CONSTRUCTION
OF THE CORNEOCYTE LIPID ENVELOPE. Journal of Biological
Chemistry 286, 24046–24056.
84
Chapter 3
Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes
Triggers Atopic Dermatitis-like Skin Inflammatory Responses in
Adult Mice
Zhixing Wang, Ling-juan Zhang, Gunjan Guha, Kateryna
Kyrylkova, Chrissa Kioussi, Mark Leid, Gitali Ganguli-Indra, Arup
K. Indra
Submitted to PLoSOne
3.1 Abstract
85
Ctip2 is crucial for epidermal homeostasis and protective barrier
formation in developing mouse embryos. Selective ablation of Ctip2 in
mouse epidermis leads to increased transepidermal water loss (TEWL)
from skin, impaired epidermal proliferation and terminal differentiation
as well as altered lipid composition during development. However, little
is known about the role of Ctip2 in adult mice skin homeostasis. To
study the role of Ctip2 in adult skin homeostasis, we utilized a Ctip2ep-/mouse model in which Ctip2 is selectively deleted in epidermal
keratinocytes. Measurement of TEWL, followed by histological,
immuno-histochemical and RT-qPCR analyses on skin biopsies from
control and mutant mice revealed an important role of Ctip2 in barrier
maintenance and in regulating adult skin homeostasis. We have shown
that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like
skin inflammation, characterized by alopecia, pruritus and dermatitis, as
well as high infiltration of T lymphocytes and immune cells. We have
also observed increased expression of Th2-type cytokines and
chemokines in the mutant skin, as well as systemic immune responses
that share similarity with human AD patients. Furthermore, we
discovered that thymic stromal lymphopoietin (TSLP) expression is
significantly upregulated in the mutant epidermis as early as postnatal
day 1 and Ctip2 was recruited to the promoter region of the TSLP gene
86
in epidermal keratinocytes. The results suggest that upregulation of
TSLP expression in the Ctip2ep-/- epidermis could be due to a
derepression of gene transcription in absence of Ctip2. Thus, our data
demonstrated a cell-autonomous role of Ctip2 in barrier maintenance
and epidermal homeostasis in adult skin, as well as a non-cell
autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory
responses by regulating the expression of Th2-type cytokines in adult
mouse skin. Present results establish an initiating role of epidermal
TSLP in AD pathogenesis via a novel repressive regulatory mechanism
mediated by Ctip2.
87
3.2 Introduction
Mammalian skin forms the first defense barrier in the body for
protecting against physical injuries, ultraviolet radiation, bacterial
infections as well as excessive loss of water (Fuchs and Raghavan,
2002). The most abundant cell type in epidermis is keratinocytes, which
forms four layers: basal, spinous, granular and stratum corneum (Fuchs
and Raghavan, 2002; Proksch et al., 2008). Atopic dermatitis (AD) is a
chronic, inflammatory disease of the skin that starts at early childhood.
AD patients are genetically predisposes to the disease, which has a
prevalence of 10%-20% in children and 1%-3% in adults (Leung and
Bieber, 2003; Tokura, 2010). Clinical features of AD include skin
xerosis, pruritus and eczematoid skin lesions (Leung and Bieber, 2003).
AD is characterized by both skin barrier deficiencies and immunological
responses (Scharschmidt and Segre, 2008). In atopic dermatitis, a
defective skin barrier is thought to permit the penetration of allergens
and induces the interactions of the allergens with immune cells,
promoting the subsequent release of pro-inflammatory cytokines and
chemokines and elevation of IgE level (Cork et al., 2009).
The molecular pathways involved in AD pathogenesis remain unclear.
There have been many reports on the connections between atopic
88
dermatitis and Th2 inflammatory pathways (Leung and Bieber, 2003;
Biedermann et al., 2004; B. Brandt and Sivaprasad, 2011). Pro-Th2
cytokines, such as IL-4, IL-13, IL-5, and IL10, are elevated in AD
patients (B. Brandt and Sivaprasad, 2011). One possible candidate that
initiates inflammatory responses in AD is thymic stromal lymphopoietin
(TSLP), which is a member of hematopoietic cytokine family and highly
expressed in skin-derived epithelial cells, fibroblasts, and dendritic cells
(Comeau and Ziegler, 2010; Ziegler, 2010; Kashyap et al., 2011). TSLP
signals through a heterodimeric receptor formed by TSLP receptor
(TSLPR) as well as IL-7 receptor alpha (IL7Ra) (Comeau and Ziegler,
2010). TSLP is a key keratinocyte-derived cytokine, whose expression
was greatly increased in the epidermis of a group of acute and chronic
atopic dermatitis patients (Soumelis et al., 2002). Mice that are deficient
of nuclear receptors (NRs) retinoid X receptor a (RXRa), vitamin D
receptor (VDR) or Notch1 in epidermal keratinocytes, exhibit epidermal
permeability barrier (EPB) defects, express elevated levels of TSLP in
skin, and subsequently develop AD-like phenotypes (Li, 2005, 2006;
Demehri et al., 2008; Ziegler, 2010).
Chicken ovalbumin upstream promoter transcription factor (COUP-TF)
interacting protein 2 (Ctip2), also known as Bcl11b, is a C2H2 zinc finger
89
protein that is crucial in the development of central nervous and
immune system (Avram et al., 2000; Arlotta et al., 2005; Topark-Ngarm
et al., 2006; Kastner et al., 2010). Ctip2 is also expressed in human and
mouse adult epidermis (Golonzhka et al., 2007; Ganguli-Indra et al.,
2009). Studies with mice harboring germline deletion and epidermal
specific ablation of Ctip2 revealed its critical role(s) in epidermal
proliferation and terminal differentiation, as well as barrier formation
during mouse embryonic development in both cell autonomous and
non-cell autonomous ways (Golonzhka et al., 2008). Ctip2 was shown
to be a “master regulator” in skin homeostasis by interacting with the
promoter regions of many genes involved in skin development such as
eLox3, Gba2, Lass2 and Notch1 (Wang et al., 2012, submitted). Since
Ctip2 null mice die at birth, the function(s) of keratinocytic Ctip2 in adult
mice EPB maintenance as well as in epidermal homeostasis is
unknown.
Here we report a novel role of Ctip2 in regulating epidermal
homeostasis and skin inflammation by selective Cre-recombinase
mediated ablation of Ctip2 gene in epidermal keratinocytes of adult
mice skin (Indra et al., 1999; Dassule et al., 2000). We show that
ablation of Ctip2 in adult mice epidermal keratinocytes results in
impaired EPB maintenance, increased epidermal hyperplasia and a
90
severe form of AD-like skin inflammation that becomes disseminated
with progression of the disease, all of which are very similar to AD in
humans. Our present data indicates that keratinocytic Ctip2 plays a
crucial role in triggering skin inflammatory responses by regulating the
expression of genes encoding Th2-type cytokines in adult mouse skin.
The results presented herein establish an initiating role of epidermal
TSLP in AD pathogenesis and demonstrate a key anti-inflammatory role
of Ctip2, which strongly represses TSLP expression in wild-type skin.
3.3 Materials and Methods
Mice
The function of Ctip2 in adult mice skin homeostasis was characterized
using Ctip2ep-/- mice that selectively lacked Ctip2 in epidermal
keratinocytes after Cre-recombinase mediated deletion of Ctip2 gene in
keratinocytes using K14-Cre deleter mice (Indra et al., 1999; Dassule et
al., 2000; Golonzhka et al., 2008). Ctip2 floxed mice (Ctip2
L2/L2
, WT)
were used as controls (Indra et al., 1999; Golonzhka et al., 2008). 8 to
10 mice from multiple litters were used in each group at each time
point. Mice were maintained in a temperature/humidity-controlled facility
91
with a 12-hour light/dark cycle. OSU IACUC approval was obtained for
animal experiments.
Transepidermal water loss (TEWL) measurement
Transepidermal water loss was measured using the calibrated
Tewameter TM300 with Multi Probe Adapter (CK electronic GmbH,
Köln, Germany) in accordance with manufacture operating instructions.
Data was expressed in g/m2h, and represents the mean ± S.E.M. for 8
independent animals (8 measurements each animal) of each genotype.
Statistical analysis was performed using Graphpad Prism software with
student’s unpaired t-test.
Histological analysis
Skin biopsies were fixed in 4% paraformaldehyde overnight and
embedded in paraffin blocks. 5mm paraffin sections were sectioned
using a Leica RM2255 microtome (Bannockburn, IL). Hematoxylin and
eosin staining was performed according to general protocols (Wang et
al., 2011).
Combined eosinophil/mast cell stain (C.E.M) was performed using a
commercial kit according to the manufacturer’s protocol (American
MasterTech, Lodi, CA). Briefly, slides were deparaffinized with xylene
92
and hydrated through alcohols. After washed in running tap water,
slides were placed in astra blue staining solution for 30 minutes and
rinsed in tap water. Slides were put in vital new red staining solution for
another 30 minutes and rinsed with tap water. After that, slides were
placed in modified Mayer’s hematoxylin for 15-30 seconds and rinsed
again with tap water for 2 minutes. Finally, dehydration of slides was
performed with 3 changes of absolute alcohol and xylene before
mounting. Mast cells showed bright blue color while eosinophils were
red. Nuclei were stained blue.
Toluidine blue staining was performed as described (Li, 2005).
Toluidine blue working solution was prepared by mixing 5ml 1%
toluidine blue EtOH solution (Sigma-Aldrich, St. Louis, MO) with 45ml
1% sodium chloride (pH2.3). Slides were deparaffinized and hydrated in
distilled water. Sections were stained with toluidine blue working
solution (pH2.0-2.5) for 2-3 minutes before washed in 3 changes of
distilled water. Slides were dehydrated through alcohols and xylenes
and mounted with resinous mounting medium.
Immunohistochemistry
IHC staining of paraffin and frozen sections was described previously
(Wang et al., 2011; Liang et al., 2012). In brief, paraffin sections were
93
deparaffinized and antigen retrieval was performed with pH6.0 citrate
buffer and 95-100° C for 20 minutes and cooled down. For frozen
sections, slides were fixed with cold acetone for 20 minutes and
allowed for air dry. Slides were washed with 0.1% PBST and blocked
with 10% normal goat serum (Vector Laboratories, Builingame, CA) for
30 minutes before incubated with primary antibodies overnight at 4°C.
Fluorescently labeled secondary antibodies were applied to slides for
an hour at room temperature. Nuclei were visualized with DAPI. After
the final washes, slides were dehydrated and mounted. Images were
captured at X20 magnification using a Carl Zeiss Axio Imager Z1
fluorescent microscope and AxioCam camera. Data were analyzed and
quantified using Adobe Photoshop and ImageJ software. Multiple IHC
fields were randomly chosen and 10-15 such fields per slide were
counted independently in a double-blinded manner. All experiments in
each category were repeated in triplicates.
The concentrations of antibodies used were as follows: Ki67 (Abcam,
Cambridge, MA, 1:500), K14 (Abcam, 1:1000), K10 (Abcam, 1:1000),
Filaggrin (Abcam, 1:1000), Loricrin (Abcam, 1:500), F4/80 (Biolegend,
San Diego, CA, 1:250), CD11b (Abcam, 1:100), CD45 (Abcam, 1:500),
Ly6g (Abcam, 1:250), CD11c (BD Pharmingen, San Jose, CA, 1:100),
RXRα (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), Notch1
94
(Cell Signaling technology, Danvers, MA, 1:500), Cy3 goat anti rabbit
(Jackson ImmunoResearch Laboratories Inc, West Grove, PA, 1:500),
Cy3 goat anti rat (Jackson ImmunoResearch, 1:500), Cy2 goat anti rat
(Jackson ImmunoResearch, 1:500), Cy3 goat anti mouse (Jackson
ImmunoResearch, 1:1000).
RT-qPCR
RNA extraction and cDNA preparation were performed as described
(Indra et al., 2005b). Real-time PCR was performed on an ABI 7500
Real-Time PCR system using SYBR Green methodology using specific
sets of primers as indicated in Supplementary Table1 (Indra et al.,
2005a, 2005b). Relative gene expression analysis of the RT-qPCR data
was performed using HPRT as an internal control. All assays were
performed in triplicates.
ELISA
ELISA kits for detecting mouse IL-4, IL-13, TNFα and TSLP were
obtained from eBioscience, Inc. (San Diego, CA, USA). Mouse IgE
ELISA kit was procured from Bethyl Laboratories, Inc. (Montgomery,
TX, USA), while CCL17 immunoassay kit was purchased from R&D
Systems, Inc. (Minneapolis, MN, USA).
95
1-month and 4-month-old mice (6 WT and 6 Ctip2ep-/- from each age
group) were selected for studying secondary systemic response to
Ctip2ep-/- genotype. Blood plasma was isolated from the mice following
the method of Argmann and Auwerx (Argmann and Auwerx, 2006).
ELISA was performed for estimation of the levels of IL-4, IL-13, IgE,
CCL17, TNFα and TSLP using kits following the respective
manufacturer’s instructions. All samples were assayed in triplicates.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed according to Hyter et al., 2010 with minor
modifications
(Hyter
et
al.,
2010).
Briefly,
mouse
epidermal
keratinocytes from 1-day postnatal wildtype mice skin were isolated and
fixed in 1% formaldehyde. Cell lysate from 3x106 keratinocytes (each
ChIP) were obtained after incubation with hypotonic buffer (10mM
Tris/HCl, pH7.5, 10mM NaCl, 3mM MgCl2 and 0.5% NP40). Nuclei
were suspended in ChIP sonication buffer (50mM Hepes, pH7.9,
150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium
deoxycholate)
with
0.2%
SDS.
Nuclei
were
sonicated
and
immunoprecipitated with 2mg anti-Ctip2 antibody (Abcam, Cambridge,
MA) overnight at 4°C. Rat IgG was used as a control. 25ml preequilibrated protein G beads (Invitrogen, Carlsbad, CA) were added to
96
each ChIP and incubated for four hours at 4°C. After washing twice with
ChIP sonication buffer, buffer A (50mM Hepes, 500mM NaCl, 1mM
EDTA, 1% Triton X-100, 0.1% SDS and 0.1% sodium deoxycholate),
buffer B (20mM Tris/HCl, pH8.0, 1mM EDTA, 250mM LiCl, 0.5% NP40
and 0.5% sodium deoxycholate) and once with TE buffer, DNA was
uncrosslinked at 65°C overnight and purified by QIAquick PCR
Purification Kit (Qiagen, Germantown, MD). All the buffers used in the
study were added with PMSF and protease inhibitors before use. DNA
was amplified using ABI-7500 Real-time PCR machine with specific
primers indicated in Supplementary Table 1. Primers were designed
from proximal (-200bp) and distal (-1kb) promoter regions of TSLP to
study the possible interaction between Ctip2 and TSLP, and primers
selected from 3’-UTR region was used as a control.
Statistics
Statistical significance of differences between groups was analyzed
using GraphPad Prism software using student’s unpaired t-test. All
statistical analyses were performed in a double-blinded manner.
97
3.4 Results
3.4.1 Selective ablation of Ctip2 in epidermal keratinocytes leads
to altered epidermal proliferation, differentiation and spontaneous
dermatitis in adult skin
The function of Ctip2 in adult mice skin homeostasis was characterized
using Ctip2ep-/- mice that selectively lacked Ctip2 in epidermal
keratinocytes after Cre-recombinase mediated deletion of Ctip2 gene in
keratinocytes using K14-Cre deleter mice (Indra et al., 1999; Dassule et
al., 2000). At approximately 8-10 weeks after birth, 67% Ctip2ep-/- mice
(n=18) developed dry and scaly skin and 17% mutant mice also
developed spontaneous lesions on the dorsal skin at the same age,
which were absent in wildtype (WT) mice (Fig 3.1 A). The severity of all
of these abnormalities worsened with age (Figure 3.1 A). By 4 months
of age, almost all the mutants (89%, n=18) developed spontaneous
dermatitis that occurred predominantly on the dorsal skin, in the neck
region, and on the face (Figure 3.1 A). Furthermore, progressive
alopecia was evident in most of the 4 month-old Ctip2ep-/- mice (~85%)
(Figure 3.1 A). We hypothesized that the skin lesions observed in the
mutant mice could be due to impaired function of the skin barrier. To
test this hypothesis, trans-epidermal water loss (TEWL) was measured
in wildtype and Ctip2ep-/- mice. Interestingly, the mutant mice showed
98
increased TEWL as early as 2 weeks of age, and the values were ~10fold higher than wildtype mice at 4 months of age (Figure 3.1 B).
Histological analysis of hematoxylin- and eosin- (H&E) stained skin
sections revealed significant epidermal hyperplasia in Ctip2ep-/- mice as
early as 2 weeks of age (Figures 3.1 C and D).
In order to determine if increased epidermal hyperplasia could be due
to
altered
keratinocyte
proliferation,
immunohistochemical
(IHC)
analyses for proliferation marker Ki67 and keratin 14 (K14) were
performed on control and mutant skin (Figures 3.2 A and D). The
percentage of Ki67-positive cells was significantly higher in Ctip2ep-/epidermis compared to the wild-type littermates at all time-points
(Figure 3.2 A and D). Similarly, the expression level of K14 in the
mutant epidermis was prominently increased in Ctip2ep-/- mice at 2 and
4 months of age (Figure 3.2 B). Expression levels of the early
differentiation marker K10 were not significantly different between
wildtype and Ctip2ep-/- mice (Figure 3.2 C). Expression of terminal
differentiation markers, filaggrin and loricrin, were also upregulated in
Ctip2ep-/- mice at later time points (Figure 3.3 A-B). These results
indicate that selective, somatic ablation of Ctip2 in epidermal
keratinocytes leads to increased epidermal proliferation and altered
terminal differentiation during adulthood. Altogether, results suggest
99
that adult Ctip2ep-/- mice exhibited impaired barrier functions and
developed spontaneous dermatitis, which worsened as the mice aged.
3.4.2. Increased infiltration of inflammatory cells in adult Ctip2ep-/mice skin
Dermal infiltrates were notable in Ctip2ep-/- mice starting around 2
months of age, as revealed by histological analyses (Figure 3.1 C).
Combined eosinophil and mast cell (C.E.M.) staining revealed
increased eosinophils in the dermis of Ctip2ep-/- skin around 1 month of
age, while mast cells were most numerous in the mutant dermis at 4
months of age (Figures 3.4 A and B). IHC staining for T lymphocytes
revealed a two-fold increase in CD3+ T cells in the skin of 4 month-old
Ctip2ep-/- mice compared to the wild-type skin (Figures 3.5 A-B). A
similar increase in the number of CD4+ T cells was observed in 4
month-old mutant skin compared to the wildtype skin (Figure 3.5 C and
D). The number of macrophages in the dermis was significantly higher
in Ctip2ep-/- mice skin at 4 months of age (Figure 3.6 A-B), as were the
number of infiltrating neutrophils and monocytes (Figure 3.6 C-F). In
addition, CD45+ cell infiltration was observed in one month-old Ctip2ep-/mice skin, but this resolved by 4 months of age (Figure 3.6 G-H).
100
Taken together, these results demonstrate an increased inflammatory
response in Ctip2ep-/- skin that progressively increased with time.
3.4.2 Preferential induction of Th2-type cytokines and chemokines
in adult Ctip2ep-/- skin
Upregulated expression of cytokines and chemokines play a crucial role
in inflammatory skin diseases (Grewe et al., 1998; B. Brandt and
Sivaprasad, 2011). Differential expression profiles of cytokines and/or
chemokines orchestrate and determine the type and outcome of
inflammatory responses in mouse and human skin. We next determined
if
expression
of
pro-inflammatory
cytokines/chemokines
were
dysregulated in mutant skin. RT-qPCR analyses revealed a preferential
induction of Th2-type cytokines, including TSLP, CCL17, IL13, IL4, IL6,
and IL10 in mutant skin (Figures 3.7 A-E and 3.10 A). In contrast,
neither expression of Th1-type (IL1α, IL2, IL12a/b; Figure 3.10 B-E) nor
that of Th17-type (IL17a, IL18 and IL23; Figure 3.10 F-H) cytokines was
unaltered in mutant skin.
Time course analyses also revealed several distinct induction patterns
of cytokines and chemokines in the mutant skin. TSLP, a master
initiator of Th2 inflammatory responses, was induced ~30-fold at
postnatal day 1 (P1), and we observed a ~200-fold induction at P7
101
compared to control skin (Liu, 2006). Expression of TSLP remained
significantly high at all later time points (~1000-fold induction between 1
month to 4 months after birth; Figure 3.7 A). Interestingly, the high level
of TSLP expression was observed predominantly in the mutant
epidermis, whereas TSLP transcripts were barely detectable in the
dermal layer of the mutant skin (Figure 3.7 F). These data suggest that
TSLP may be an initial triggering cytokine that is induced in the
absence of Ctip2 in the epidermis.
Chemokine CCL17 is one of known downstream genes whose
expression can be stimulated by TSLP (Ziegler and Artis, 2010). CCL17
and IL13 transcripts were significantly induced in the mutant skin at
around 1 week and 2 weeks after birth, respectively. Induction of these
two genes was transient, peaking at 1 month, followed by a decrease of
expression during later time points (2 - 4 months; Figure 3.7 B and C).
In contrast to these early and transient inductions, we observed a
strong, late induction of cytokines IL4 and IL6 and chemokines CCL3
and CXCL2 in the mutant skin (Figure 3.7 D-E, 3.8 L-M). We also
observed a late onset of induction of Th1-type cytokines, including
TNFα and IFNγ, at 4 months of age (Figure 3.8 N-O). These results
suggest that deletion of Ctip2 in the epidermis leads to an increased
102
inflammatory response, which is characterized by a Th2-type
cytokine/chemokine profile.
3.4.3 TSLP is a direct target of Ctip2 in epidermal keratinocytes
Recent studies have revealed that ablation of RXRα and RXRβ in adult
murine keratinocytes leads to an AD-like phenotype triggered by
induction of TSLP (Li, 2005).. Similarly, TSLP is also elevated in Notch
signaling-deficient keratinocytes (deficient in either Notch1/2 or
recombining binding protein suppressor of hairless (RBP-j; Refs (Li,
2005; Demehri et al., 2008; Dumortier et al., 2010)). Thus, it is possible
that Ctip2 regulates TSLP expression via the nuclear receptor or Notch
signaling pathways in epidermis. To test that, we determined the
expression of key players in both the Notch and RXR signaling
pathways, including Notch1, Notch2, RBP-j, RXRα and RXRβ.
Expression of the corresponding transcripts was not affected by loss of
Ctip2 in the epidermis at both one and four months after birth (Figure
3.12 A-D, data not shown). Similarly, epidermal protein levels of Notch1
and RXRα were comparable between wildtype and mutants at all timepoints, suggesting an alternative mechanism of Ctip2-mediated
regulation of TSLP expression in the adult murine skin (Figure 3.12 EF).
103
In order to determine if Ctip2 directly regulates TSLP expression in
murine keratinocytes, we conducted chromatin immunoprecipitation
(ChIP) assays on neonatal mouse epidermal extracts. Primers were
designed from the proximal (-200bp) and distal (-1kb) promoter region
relative to the transcriptional start site of TSLP to determine the
possible interaction of Ctip2 with TSLP. We observed that Ctip2
interacted with the distal but not the proximal region of TSLP promoter,
suggesting Ctip2 may directly repress TSLP transcription in mouse
keratinocytes (Figure 3.7 G). Hence, ablation of Ctip2 in epidermal
keratinocytes could lead to upregulation of TSLP expression due a
derepression mechanism.
3.4.4 Systemic inflammatory responses in older Ctip2ep-/- mice
We hypothesized that chronic skin inflammation developed in Ctip2ep-/mice may trigger systemic immune responses in the mutant skin. To
test that we determined blood plasma concentrations of IgE as well as
various Th2-type cytokines/chemokines (IL-4, IL-13, CCL17 and TSLP)
and Th1-type cytokine TNF-α from 1-month and 4-months-old mice by
ELISA (Figure 3.9 A-F). Plasma level for all of the tested cytokines,
were observed to be higher in both 1-month and 4-month old mutant
mice (Figure 3.9 A-E), including IL4 and the related IL-13 (Figure 3.9 A,
104
B), CCL17 (Figure 3.9 C), TSLP (Figure 3.9 D), and TNFα (Figure 3.9
E). While circulating IgE levels were elevated in both wild-type and
mutant mice at four months of age, IgE levels in mutant mice were 2.5
times greater than those in wildtype mice (Figure 3.9 F).
Ctip2ep-/- mice exhibited inguinal lymphadenopathy smaller convoluted
thymus at four months of age (Figure 3.10 A, data not shown). The
mutant mice displayed progressive splenomegaly starting from one
month of age, with a 2-fold increase in spleen weight at the age of four
months (Figure 3.10 A-B). Histological studies of the spleens revealed
an increased cell infiltration in the 4-month-old mutant spleen in
comparison with wildtype (Figure 3.11 A, D, G, J). 4 month-old Ctip2ep-/mice exhibited increased eosinophil infiltration in the spleen and lymph
node compared with wildtype (Figure 3.11, B, E, H, K). Very few mast
cells were observed in both wildtype and mutant mice spleens and the
number of mast cells was larger in the lymph node, indistinguishable
between 4 month-old wildtype and mutant mice (Figure 3.11, C, F, I, L).
These results clearly indicate that Ctip2ep-/- mice display a systemic
immune response that may be driven by primarily TSLP expression
from keratinocytes.
105
3.5 Discussion
Epidermal keratinocytes have long been suspected to play a crucial role
in initiating and directing the immune response in inflammatory skin
diseases, such as AD. In the present report we have shown that skin of
adult Ctip2ep-/- mice, with an epidermal-specific deletion of the gene
encoding Ctip2, display histological and cellular features that are typical
of human AD. First, Ctip2ep-/- mice develop skin lesions, alopecia and
pruritus, which are major clinical features in patients afflicted with AD
(Leung and Bieber, 2003; Dubrac et al., 2010; Boguniewicz and Leung,
2011). Second, Ctip2ep-/- mice exhibit epidermal hyperplasia that is
characterized by increased epidermal proliferation and differentiation,
similar to what has been observed in AD patients (Proksch et al., 2008).
Third, the skin of Ctip2ep-/- mice is characterized by a dramatic
upregulation of Th2-type cytokines and chemokines, including TSLP,
CCL17, IL13 and IL4. TSLP, all of which were induced early in Ctip2ep-/epidermis, and are crucially involved in the initiation of pathogenesis in
human AD (Tokura, 2010; B. Brandt and Sivaprasad, 2011; Novak and
Leung, 2011). Finally, Ctip2ep-/- adult mice exhibit Th2-like systemic
immune syndrome that is similar to that found in most AD patients
(Spergel, 2003). Based on the phenotypic spectrum of Ctip2ep-/- adult
106
mice, it appears that this line may serve as a useful model for the study
of human AD.
Selective ablation of Ctip2 in the developing epidermis reduced
epidermal
proliferation
and
impaired
terminal
differentiation
of
keratinocytes (Golonzhka et al., 2008). Isolated Ctip2 null keratinocytes
also exhibit severe proliferation and differentiation defects in culture
(Zhang et al., submitted). To our surprise, in the postnatal stages, the
epidermis of Ctip2ep-/- adult mice exhibited increased proliferation and
differentiation compared with wild-type epidermis (Figure 3.1 C, 3.2,
3.3). A hyperplastic epidermis and a significantly higher percentage of
Ki67-positive cells were observed in the mutants as early as 1 week
after birth. Elevated proliferation and differentiation could be secondary
to impaired epidermal barrier functions caused by Ctip2 ablation.
Previous
studies
have
established
that
hyperproliferation
and
acanthosis (thickened epidermis) are compensatory responses to
impaired epidermal barrier (Proksch et al., 1991)
A number of candidate genes have been identified in association with
AD, however, may of these linkages have been called into question due
to insufficiently powered studies and/or heterogeneity of the disease
(Dubrac et al., 2010; Boguniewicz and Leung, 2011). In independent
107
studies, several genes such as Filaggrin (FLG), trans-acting T-cellspecific transcription factor (GATA3) and IL4 has been reported to be
involved in AD pathogenesis (Barnes, 2010). FLG deficiency alone
decreases stratum corneum hydration and leads to increased TEWL,
and has been correlated with AD in the highest number of studies
(Palmer et al., 2006; Barnes, 2010). It is worth mentioning that skin
barrier deficiency has also been reported in AD patients without FLG
mutation(s) or loss of expression (Jakasa et al., 2010) suggesting
existence of different subtypes of AD in humans. We have shown
previously that Ctip2 deficiency is associated with impaired terminal
differentiation and decreased FLG expression during development
(Golonzhka et al., 2008). Indeed, we have observed a ~50% reduction
of FLG expression in the epidermis of Ctip2ep-/- skin compared to that of
wildtype littermates around 1 week of age (Figure 3.13). However, no
significant difference in level of FLG expression was observed between
the wildtype and mutant epidermis at later timpoints from 2 weeks to 4
months of age possibly due to a compensatory upregulation of other
related factors such as Ctip1 (Figure 3.3A, 3.13). The lack of loss-ofFLG expression observed in Ctip2ep-/- mice at later stages, indicates
possible involvement of other factors to modulate barrier integrity.
108
TSLP, a keratinocyte-derived triad of cytokines, has been studied
intensively recently in the pathogenesis of allergic inflammation and
subsequent development of AD and other immune disorder (Soumelis
et al., 2002; Yoo, 2005; Liu, 2006). It is recognized as the master switch
for AD and other atopic diseases, such as asthma, as it plays a critical
role to induce the Th2-type allergic inflammation in AD pathogenesis
(Liu, 2006). Keratinocyte-derived TSLP participates in the inflammatory
cascade by activation of other immune cells, such as dendritic cells and
mast cells, and subsequent stimulation of release of Th2-related
cytokines/chemokines from these immune cells, eventually initiate
inflammatory a Th2 lymphocyte response (Soumelis et al., 2002; Liu,
2009; Comeau and Ziegler, 2010). In the present study, we have
observed that TSLP was the earliest known cytokine induced in Ctip2ep/-
mice and this induction was maintained in the mutants at all postnatal
stages of development. Expression of CCL17, a downstream target of
TSLP, was also significantly induced in the mutant mice at early
timepoints. The induction of TSLP was mainly restricted to the
epidermis, which correlates well with epithelial cells of the keratinocyte
lineage as major producers of TSLP in AD patients (Soumelis et al.,
2002). Hence, induction of TSLP following Ctip2 ablation in
109
keratinocytes could be a trigger factor that initiates and directs the Th2type inflammatory cascade observed in Ctip2ep-/- skin.
Transgenic mice over-expressing TSLP in keratinocytes develop ADlike symptoms, indicating that TSLP expression is sufficient to initiate
AD-like inflammatory responses (Yoo, 2005). Therefore, it remains
critical to address upstream triggers of TSLP expression and how TSLP
is regulated before and during AD pathogenesis. Transcriptional
regulation of TSLP by transcription factors (NF-κB, AP-1, AP-2α) and
nuclear receptors (RXR, RAR, VDR) were reported previously (Li, 2005,
2006; Lee and Ziegler, 2007; Lee et al., 2008; Takai, 2012). NF-κB and
AP-1 was shown to directly regulate TSLP expression by binding to 3.8kb of transcription initiation site in mouse and human lung epithelium
cells (Lee et al., 2008). Previous studies have identified presence of a
RARE (DR2) around ~ 1kb of the TSLP promoter region that may bind
to RXRα(β)-RAR heterodimers (Li, 2006). Since we have observed
Ctip2 recruitment around that region on the mTSLP promoter, it is
possible that NRs and Ctip2 could be a part of the same transcriptional
complex to regulate TSLP gene expression in mouse keratinocytes.
However, additional co-recruitment of Ctip2 along with NRs in further
distal region of the mTSLP gene promoter cannot be ruled out. Here,
we have identified a novel transcriptional regulatory mechanism(s) of
110
negative regulation of TSLP expression by Ctip2 in mouse epidermis.
Ctip2 appears to interact directly with and repress expression of the
TSLP promoter.
We have demonstrated a predominant upregulation of Th2 cytokines in
the Ctip2ep-/- skin at all timepoints, but a late surge of Th1 cytokines,
including IFNγ and TNF α , was also observed in the mutant skin at 4
months (Figure 3.8 N-O). Indeed, it has been demonstrated that Th2and Th1 type cytokines both contribute to different stages of the
development of skin lesions in AD patients (Grewe et al., 1998). Th2cell subsets are specifically activated during the initiation phase,
followed by the Th1-cell subset to maintain the persistence of the
chronic inflammatory response. Our work confirms that Th2- and Th1type immune responses are not mutually exclusive, especially at the
chronic phage of the inflammatory disease. The interaction between
different Th-cell subsets at the chronic stage may be a fruitful area for
future investigation of AD pathogenesis.
Systemic immune response related to elevated expression of IL-4, IL13,
CCL17 and IgE, observed in Ctip2ep-/- mice is evidently Th2 celldependent and humoral in nature. IL-4, for example, induces
differentiation of CD4+ T-cells into Th2 cells, and also inhibits
production of Th1 cells (Rang, 2003). Th2 cells secrete IL-13, which
111
together with IL-4, initiates a B lymphocyte-directed, humoral response
(Rang, 2003). IL-4 also upregulates MHC class II expression and
induces B-cell class switching to IgE [41, 42]. Furthermore, TSLP
stimulation of naïve CD4+ T-cells induces a specialized Th2
polarization, which results in production of downstream Th2-associated
pro-inflammatory cytokines, such as IL-13, which carry out humoral
immune processes (Ziegler and Artis, 2010). Simultaneously, CCL17 is
known to attract Th2 cells, thereby favoring humoral responses
(Nakayama et al., 2004). In summary, inflammation due to selective
Ctip2 ablation in mice epidermis is not restricted to the site of deletion;
rather Ctip2ep-/- mice show a chronic secondary Th2-dependent humoral
systemic inflammatory response.
In summary, our study reveals a novel role(s) of keratinocytic Ctip2 in
epidermal barrier maintenance and homeostasis, and inflammation in
adult mice. Ctip2 clearly modulates AD-like responses within the skin,
as well as systemic inflammatory responses. TSLP was strongly
induced in Ctip2ep-/- epidermis and likely causes increased dendritic
cells infiltration into lymph nodes, in which these cells interact with
CD4+ T cells to produce Th-2 inflammatory cytokines. Subsequently,
other immune cells such as macrophages, leukocytes, mast cells and
eosinophils could be attracted and recruited to the site of skin
112
inflammation, which become disseminated, leading to elevated
circulating levels of inflammatory mediators. This study identifies a new
mediator, Ctip2, that may be implicated in the etiology of AD. However,
several issues need to be resolved in the future. For example, it is
presently unclear if a defective skin barrier precedes inflammation and
enhanced cellular proliferation in the early stages of AD development.
Second, the contributory role of TSLP in AD and the mechanistic basis
of regulation of TSLP expression in keratinocytes have not been
identified. Finally, the role of Ctip1 in AD development in presence or
absence of Ctip2 could shed further lights in AD pathogenesis and
identify new avenues for therapeutic intervention. Our study highlights a
central role of epidermal keratinocytes in initiating and shaping the
immune response during the pathogenesis of AD-like skin diseases in
mice.
D
epidermal thickness (µm)
113
WT
Ctip2ep-/-
40
***
***
***
***
30
20
10
0
2w
1m
2m
4m
114
Figure 3.1 Ctip2ep-/- mice develop a chronic skin lesions and
epidermal hyperplasia.
(A) Gross morphology of 4 months old wildtype (WT) and Ctip2ep-/mice. The yellow arrowheads indicate lesion and alopecia of Ctip2ep-/mice in the back, face and neck, to be compared with the normal
appearance in a wildtype mouse. (B) Measurement of trans-epidermal
water loss (TEWL) from dorsal skin of wildtype and Ctip2ep-/- mice at
different time points. Statistical analyses were performed by student’s
unpaired t-test using GraphPad Prism software; ** P< 0.01, ***
P<0.001. (C) Hemotoxylin & Eosin stained 5µm thick paraffin sections
from dorsal skin of WT and Ctip2ep-/- mice at 1 week (1W), 2 weeks
(2w), 1month (1m), 2 months (2m) and 4 months (4m). Scale bar: 100
µm. Epidermis (E) and dermis (D) are indicated. (D) Measurement of
epidermal thickness of WT and Ctip2ep-/- mice skin. Statistical analyses
were performed by student’s unpaired t-test using GraphPad Prism
software;
*
P<0.05,
**
P<
0.005,
***
P<0.0001.
115
116
Figure 3.2 Ctip2ep-/proliferation.
mice
develop
enhanced
epidermal
Immunohistochemical staining of dorsal skin biopsies from WT and
Ctip2ep-/- mice was performed with antibodies directed against (A) Ki67,
(B) K14 and (C) K10 (all in red). All sections were counterstained with
DAPI (blue). Scale bar: 100 µm. Epidermis (E) and dermis (D) are
indicated. (D) Epidermal percent Ki67 positive cells in dorsal skin
sections of WT and mutant mice. Statistical analyses were performed
by student’s unpaired t-test using GraphPad Prism software; * P<0.05,
** P< 0.005.
117
Figure 3.3 Ablation of Ctip2 leads to increased epidermal terminal
differentiation.
Immunohistochemical staining of dorsal skin biopsies from WT and
Ctip2ep-/- mice was performed with antibodies directed against (A)
filaggrin and (B) loricrin (all in red). All sections were counterstained
with DAPI (blue). Scale bar: 100 µm. Epidermis (E) and dermis (D) are
indicated.
118
Figure 3.4 Enhanced eosinophil and mast cell infiltrates in dorsal
skin of Ctip2ep-/- adult mice.
(A) Combined eosinophil and mast cell (C.E.M) staining for eosinophils
(pink) and mast cells (blue). Black arrows point to eosinophils. Scale
bar: 50 µm. (B) Toluidine blue stained dorsal paraffin skin sections of
WT and Ctip2ep-/- mice. Mast cells stain intensive blue color. Scale bar:
100 µm.
119
Figure 3.5 Characterization of inflammatory cell infiltrates in dorsal
skin of WT and in Ctip2ep-/- adult mice.
(A) Immunohistochemical staining of dorsal skin biopsies from WT and
Ctip2ep-/- mice were performed with specific antibodies against CD3
(red). Yellow arrowhead indicates dermal infiltrates of CD3+ cells. Scale
bar: 100 µm. (B) Percent CD3+ T cells at 2m and 4m. (C)
Immunostaining of CD4+ T cells (red) in WT and mutant mice. Scale
bar: 100 µm. (D) Percent CD4+ T cells at 2m and 4m. All sections were
counterstained with DAPI (blue). Statistical analyses were performed by
student’s unpaired t-test using GraphPad Prism software; ** P< 0.005.
120
121
Figure 3.6 Increased inflammatory cell infiltrate in Ctip2ep-/- adult
mice.
(A) Immunohistochemical staining of dorsal skin biopsies from WT and
Ctip2ep-/- mice were performed with specific antibodies against F4/80
(green) to detect macrophage/dendritic cells. (B) Percent F4/80 positive
cells at 2m and 4m. (C) Immunostaining of neutrophils (green) using
anti-Ly6g antibody in WT and mutant mice skin. (D) Percent Ly6g
positive cells at 2m and 4m. (E) Immunostaining of
monocytes/macrophages (green) with anti-CD11b antibody in WT and
mutant mice skin. (F) Percent CD11b positive cells at 2m and 4m. (G)
Immunohistochemical staining of dorsal skin biopsies with antibody
against CD45 (green). (H) Percent CD45 positive cells at 1m and 2m.
All sections were counterstained with DAPI (blue). Scale bar (A, C, E
and G): 100 µm. Statistical analyses were performed by student’s
unpaired t-test using GraphPad Prism software; ** P< 0.005.
122
123
Figure 3.7 Th2-dependent cytokine and chemokine expression
levels in WT and Ctip2ep-/- skin.
(A-F) Quantitative RT-PCR (RT-qPCR) analyses of cytokines and
chemokines in the dorsal skin of 1day, 1 week, 2 weeks, 1month, 2
months and 4 months old WT and mutant mice using specific primers
as indicated in Supplementary Table1. Relative mRNA expression
levels of (A) TSLP, (B) CCL17, (C) IL13, (D) IL4, and (E) IL6 in Ctip2ep-/skin was compared to wildtype (WT) skin (set as 1.0). (F) RT-qPCR
analyses of TSLP mRNA levels in separated epidermis and dermis from
tail skin. All values represent relative transcript level after normalization
with HPRT transcripts. (G) Chromatin immunoprecipitation (ChIP)
assay was performed on freshly isolated neonatal mouse skin
keratinocytes using anti-Ctip2 antibody and results were analyzed by
qPCR using primers indicated in table S1. Rat IgG was used as a
control. Ctip2 was recruited to the distal promoter regions of TSLP.
Statistical analyses were performed by student’s unpaired t-test using
GraphPad Prism software; * P<0.05, ** P<0.01, *** P<0.001.
2
4m
0.0
1m
2
*
1
0
1m
4m
1.0
0.5
0.0
1m
4m
CCL20
WT
Ctip2ep-/-
500
***
400
300
200
5
4
3
2
1
0
*
1m
4m
CXCL2
Relative Expression Level
**
1.0
0.5
0.0
1m
1m
4m
4m
WT
Ctip2ep-/-
2.0
1.5
1.0
0.5
0.0
1m
4m
CCL22
5
WT
Ctip2ep-/-
4
*
3
2
1
0
1m
4m
IFN
1.5
*
1.0
0.5
0.0
1.0
0.5
0.0
1m
4m
1.5
1.0
0.5
0.0
1m
4m
CXCL10
O
WT
Ctip2ep-/-
5
4
*
3
2
1
0
1m
4m
TNF
Supplementary figure 3
4m
WT
Ctip2ep-/-
2.5
2.0
1.5
1.0
0.5
0.0
1m
4m
IL23
L
WT
Ctip2ep-/-
1m
H
IL18
WT
Ctip2ep-/-
2.0
WT
Ctip2ep-/-
IL12a
1.5
K
N
Relative Expression Level
Relative Expression Level
1.5
0.0
IL17a
WT
Ctip2ep-/-
2.0
0.5
G
*
1.5
J
M
Relative Expression Level
WT
Ctip2ep-/-
2.0
IL12b
I
1.0
2.0
IL2
Relative Expression Level
3
Relative Expression Level
Relative Expression Level
F
WT
Ctip2ep-/-
4
*
IL1
IL10
E
4m
Relative Expression Level
1m
Relative Expression Level
0
0.5
D
Relative Expression Level
**
**
1.0
WT
Ctip2ep-/-
1.5
Relative Expression Level
4
C
Relative Expression Level
**
WT
Ctip2ep-/-
1.5
Relative Expression Level
B
WT
Ctip2ep-/-
6
Relative Expression Level
A
Relative Expression Level
124
100
90
80
70
60
50
5
4
3
2
1
0
WT
Ctip2ep-/-
***
1m
4m
CCL3
125
Figure 3.8 Relative expression levels of cytokines and chemokines
in WT and Ctip2ep-/- skin.
The expression level of (A) IL10, (B) IL1α, (C) IL2, (D) IL12a, (E) IL12b,
(F) IL17a, (G) IL18, (H) IL23, (I) CCL20, (J) CCL22, (K) CXCL10, (L)
CCL3, (M) CXCL2, (N) IFNγ and (O) TNFα was studied with RT-qPCR
in 1-month and 4-month-old wildtype and Ctip2ep-/- dorsal skin. Values
represent relative transcript level after normalization with HPRT
transcripts. Statistical analyses were performed by student’s unpaired ttest using GraphPad Prism software; * P<0.05, ** P<0.01, *** P<0.001.
126
Figure 3.9 Systemic immunological abnormalities in Ctip2ep-/- adult
mice.
Serum concentration of (A) IL4, (B) IL13, (C) CCL17, (D) TSLP, (E)
TNFα and (F) IgE was measured by ELISA. Statistical analyses were
performed by student’s unpaired t-test using GraphPad Prism software;
* P<0.05, ** P< 0.01, *** P<0.005
127
Figure 3.10 Ctip2ep-/- adult mice exhibited systemic immune
responses.
(A) Gross morphological comparison of spleen, lymph node (LN) and
liver in 4-months-old WT and Ctip2ep-/- mice. (B) Spleen weight of WT
and Ctip2ep-/- mice at different months of age is indicated. Statistical
analyses were performed by student’s unpaired t-test using GraphPad
Prism software; * P<0.05, ** P< 0.01, *** P<0.005
128
Figure 3.11 Immunological abnormalities of spleen and lymph
node in Ctip2ep-/- adult mice.
(A, D) Hemotoxylin & Eosin stained 5µm thick paraffin sections from
spleen of WT and Ctip2ep-/- mice at 4m. (B, C, E, F) C.E.M staining for
eosinophils (pink) and mast cells (blue) in 4 month-old mice spleen
sections. (G, J) Hemotoxylin & Eosin stained 5µm thick paraffin
sections of WT and Ctip2ep-/- mice lymph node at 4m. (H, I, K, L) C.E.M
staining for eosinophils (pink) and mast cells (blue) in 4 month-old mice
lymph node. Black arrows point to eosinophils. Scale bar: 50 µm
129
Figure 3.12 Relative expression levels of RXRa and genes involved
in Notch signaling pathway.
The expression level of (A) Notch1, (B) Notch2, (C) Rbp-j and (D)
RXRα was studied with RT-qPCR in 1-month and 4-month-old wildtype
and Ctip2ep-/- dorsal skin. Values represent relative transcript level after
normalization with GAPDH transcripts. Statistical analyses were
performed by student’s unpaired t-test using GraphPad Prism software.
(E) Immunohistochemical staining of dorsal skin biopsies with antibody
against Notch1 (red) and (F) RXRα (red). Scale bar: 100 µm. All
sections (in E & F) were counterstained with DAPI (blue).
130
Figure 3.13 Relative expression levels of FLG in 1w and 2w mice
epidermis and dermis.
The expression of FLG in (A) 1-week-old and (B) 2-week-old wildtype
and Ctip2ep-/- skin by RT-qPCR. Values represent relative transcript
level after normalization with HPRT transcripts. Statistical analyses
were performed by student’s unpaired t-test using GraphPad Prism
software; * P<0.05, ** P<0.01, *** P<0.001.
131
Table 3.1 List of primers used for RT-qPCR and ChIP assays
Gene
Sense
Antisense
CCL3
CTCCCAGCCAGGTGTCATTTT
CTTGGACCCAGGTCTCTTTGG
CCL11
TCCACAGCGCTTCTATTCCT
CTATGGCTTTCAGGGTGCAT
CCL17
TACCATGAGGTCACTTCAGATGC
GCACTCTCGGCCTACATTGG
CCL20
TCACCTCTGCAGCCAGGCAGA
TCTTAGGCTGAGGAGGTTCACAGC
CCL22
CCGTCACCCTCTGCCATCACG
GACCTGCCTGGGATCGGCAC
CXCL10
CCACGTGTTGAGATCATTGCCACG
ATCCATCGCAGCACCGGGGT
CXCL2
AGAAGTCATAGCCACTCTCAAGGGC
AGCAGCCCAGGCTCCTCCTTT
HPRT
GTTAAGCAGTACAGCCCCAAA
AGGGCATATCCAACAACAAACTT
IL1a
TCACCTTCAAGGAGAGCCG
ATCTGGGTTGGATGGTCTCTT
IL2
TGAGCAGGATGGAGAATTACAGG
GTCCAAGTTCATCTTCTAGGCAC
IL4
GAGCCATATCCACGGATGCGAC
ATGCGAAGCACCTTGGAAGCCC
IL5
CAGCTGTCCGCTCACCGAGCT
TTTCCACAGTACCCCCACGGACAG
IL6
ACAAAGCCAGAGTCCTTCAGAGAGA
AGCCACTCCTTCTGTGACTCCAG
IL10
GGCGCTGTCATCGATTTCTCCCC
GGCCTTGTAGACACCTTGGTCTTGG
IL12a
CTGTGCCTTGGTAGCATCTATG
GCAGAGTCTCGCCATTATGATTC
IL12b
AGTGTGAAGCACCAAATTACTCC
CCCGAGAGTCAGGGGAACT
IL13
TGCTTGCCTTGGTGGTCTCGC
GCGGCCAGGTCCACACTCCA
IL17a
ACGCGCAAACATGAGTCCAGGG
TGAGGGATGATCGCTGCTGCCT
IL18
GTGAACCCCAGACCAGACTG
CCTGGAACACGTTTCTGAAAGA
IL23a
GAACGCACATGCACCAGCGG
TGCAAGCAGAACTGGCTGTTGTCC
TNFa
ACTTCGGGGTGATCGGTCCCC
TGGTTTGCTACGACGTGGGCTAC
TSLP
ACGGATGGGGCTAACTTACAA
AGTCCTCGATTTGCTCGAACT
RXRa
GATATCAAGCCGCCACTAGG
TGTTGTCTCGGCAGGTGTAG
RXRb
CACCTCTTACCCCTTCAGCA
GAGCGACACTGTGGAGTTGA
Notch1
TCAATGCCGTGGATGACCTA
CCTTGTTGGCTCCGTTCTTC
Notch2
GAGAAAAACCGCTGTCAGAATGG
GGTGGAGTATTGGCAGTCCTC
Rbpj
AGTTGCACAGAAGTCTTACGG
CCTATTCCAATAAACGCACAGGG
TSLP-ChIP
proximal
TSLP-ChIP
distal
TSLP-ChIP
3’-UTR
ATCTTAACCCAACCCACCAT
CTAGGGGAGGAACAGCTTCT
CCGTAGGCGTTTAGGTGTTA
CAAAGACTGTGCTCGGGTAT
TATTGCAAATCCAGCTGTCA
TTTCCAAAAGTGCTCACAAAA
132
3.6 References
Argmann, C.A., and Auwerx, J. (2006). Collection of blood and plasma
from the mouse. Curr Protoc Mol Biol Chapter 29, Unit 29A.3.
Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., and
Macklis, J.D. (2005). Neuronal Subtype-Specific Genes that Control
Corticospinal Motor Neuron Development In Vivo. Neuron 45, 207–221.
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D.J., Ishmael, J.E.,
and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger
proteins implicated in transcriptional repression mediated by chicken
ovalbumin upstream promoter transcription factor (COUP-TF) orphan
nuclear receptors. J Biol Chem 275, 10315–10322.
B. Brandt, E., and Sivaprasad, U. (2011). Th2 Cytokines and Atopic
Dermatitis. Journal of Clinical & Cellular Immunology 02,.
Barnes, K.C. (2010). An update on the genetics of atopic dermatitis:
Scratching the surface in 2009. Journal of Allergy and Clinical
Immunology 125, 16–29.e11.
Biedermann, T., Röcken, M., and Carballido, J.M. (2004). TH1 and TH2
lymphocyte development and regulation of TH cell-mediated immune
responses of the skin. J. Investig. Dermatol. Symp. Proc. 9, 5–14.
Boguniewicz, M., and Leung, D.Y.M. (2011). Atopic dermatitis: a
disease of altered skin barrier and immune dysregulation. Immunol.
Rev. 242, 233–246.
Comeau, M.R., and Ziegler, S.F. (2010). The influence of TSLP on the
allergic response. Mucosal Immunol 3, 138–147.
Cork, M.J., Danby, S.G., Vasilopoulos, Y., Hadgraft, J., Lane, M.E.,
Moustafa, M., Guy, R.H., MacGowan, A.L., Tazi-Ahnini, R., and Ward,
S.J. (2009). Epidermal Barrier Dysfunction in Atopic Dermatitis. Journal
of Investigative Dermatology 129, 1892–1908.
Dassule, H.R., Lewis, P., Bei, M., Maas, R., and McMahon, A.P. (2000).
Sonic hedgehog regulates growth and morphogenesis of the tooth.
Development 127, 4775–4785.
Demehri, S., Liu, Z., Lee, J., Lin, M.-H., Crosby, S.D., Roberts, C.J.,
Grigsby, P.W., Miner, J.H., Farr, A.G., and Kopan, R. (2008). Notch-
133
Deficient Skin Induces a Lethal Systemic B-Lymphoproliferative
Disorder by Secreting TSLP, a Sentinel for Epidermal Integrity. PLoS
Biology 6, e123.
Dubrac, S., Schmuth, M., and Ebner, S. (2010). Atopic dermatitis: the
role of Langerhans cells in disease pathogenesis. Immunology and Cell
Biology 88, 400–409.
Dumortier, A., Durham, A.-D., Di Piazza, M., Vauclair, S., Koch, U.,
Ferrand, G., Ferrero, I., Demehri, S., Song, L.L., Farr, A.G., et al.
(2010). Atopic Dermatitis-Like Disease and Associated Lethal
Myeloproliferative Disorder Arise from Loss of Notch Signaling in the
Murine Skin. PLoS ONE 5, e9258.
Fuchs, E., and Raghavan, S. (2002). Getting under the skin of
epidermal morphogenesis. Nat Rev Genet 3, 199–209.
Ganguli-Indra, G., Liang, X., Hyter, S., Leid, M., Hanifin, J., and Indra,
A.K. (2009). Expression of COUP-TF-interacting protein 2 (CTIP2) in
human atopic dermatitis and allergic contact dermatitis skin.
Experimental Dermatology 18, 994–996.
Golonzhka, O., Leid, M., Indra, G., and Indra, A.K. (2007). Expression
of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during
development and in adulthood. Gene Expr Patterns 7, 754–760.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J.-M., Campbell,
A.L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M., and Indra,
A.K. (2008). Dual Role of COUP-TF-Interacting Protein 2 in Epidermal
Homeostasis and Permeability Barrier Formation. J Investig Dermatol
129, 1459–1470.
Grewe, M., Bruijnzeel-Koomen, C.A., Schöpf, E., Thepen, T.,
Langeveld-Wildschut, A.G., Ruzicka, T., and Krutmann, J. (1998). A
role for Th1 and Th2 cells in the immunopathogenesis of atopic
dermatitis. Immunol. Today 19, 359–361.
Hyter, S., Bajaj, G., Liang, X., Barbacid, M., Ganguli-Indra, G., and
Indra, A.K. (2010). Loss of nuclear receptor RXRα in epidermal
keratinocytes promotes the formation of Cdk4-activated invasive
melanomas. Pigment Cell Melanoma Res 23, 635–648.
Indra, A.K., Dupe, V., Bornert, J.M., Messaddeq, N., Yaniv, M., Mark,
M., Chambon, P., and Metzger, D. (2005a). Temporally controlled
134
targeted somatic mutagenesis in embryonic surface ectoderm and fetal
epidermal keratinocytes unveils two distinct developmental functions of
BRG1 in limb morphogenesis and skin barrier formation. Development
132, 4533–4544.
Indra, A.K., Mohan, W.S., Frontini, M., Scheer, E., Messaddeq, N.,
Metzger, D., and Tora, L. (2005b). TAF10 is required for the
establishment of skin barrier function in foetal, but not in adult mouse
epidermis. Dev Biol 285, 28–37.
Indra, A.K., Warot, X., Brocard, J., Bornert, J.M., Xiao, J.H., Chambon,
P., and Metzger, D. (1999). Temporally-controlled site-specific
mutagenesis in the basal layer of the epidermis: comparison of the
recombinase activity of the tamoxifen-inducible Cre-ER(T) and CreER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327.
Jakasa, I., Koster, E.S., Calkoen, F., McLean, W.H.I., Campbell, L.E.,
Bos, J.D., Verberk, M.M., and Kezic, S. (2010). Skin Barrier Function in
Healthy Subjects and Patients with Atopic Dermatitis in Relation to
Filaggrin Loss-of-Function Mutations. Journal of Investigative
Dermatology 131, 540–542.
Kashyap, M., Rochman, Y., Spolski, R., Samsel, L., and Leonard, W.J.
(2011). Thymic stromal lymphopoietin is produced by dendritic cells. J.
Immunol. 187, 1207–1211.
Kastner, P., Chan, S., Vogel, W.K., Zhang, L.-J., Topark-Ngarm, A.,
Golonzhka, O., Jost, B., Le Gras, S., Gross, M.K., and Leid, M. (2010).
Bcl11b represses a mature T-cell gene expression program in immature
CD4(+)CD8(+) thymocytes. Eur. J. Immunol. 40, 2143–2154.
Lee, H.-C., Headley, M.B., Iseki, M., Ikuta, K., and Ziegler, S.F. (2008).
Cutting edge: Inhibition of NF-kappaB-mediated TSLP expression by
retinoid X receptor. J. Immunol. 181, 5189–5193.
Lee, H.-C., and Ziegler, S.F. (2007). Inducible expression of the
proallergic cytokine thymic stromal lymphopoietin in airway epithelial
cells is controlled by NF B. Proceedings of the National Academy of
Sciences 104, 914–919.
Leung, D.Y., and Bieber, T. (2003). Atopic dermatitis. The Lancet 361,
151–160.
135
Li, M. (2005). Retinoid X receptor ablation in adult mouse keratinocytes
generates an atopic dermatitis triggered by thymic stromal
lymphopoietin. Proceedings of the National Academy of Sciences 102,
14795–14800.
Li, M. (2006). Topical vitamin D3 and low-calcemic analogs induce
thymic stromal lymphopoietin in mouse keratinocytes and trigger an
atopic dermatitis. Proceedings of the National Academy of Sciences
103, 11736–11741.
Liang, X., Bhattacharya, S., Bajaj, G., Guha, G., Wang, Z., Jang, H.-S.,
Leid, M., Indra, A.K., and Ganguli-Indra, G. (2012). Delayed Cutaneous
Wound Healing and Aberrant Expression of Hair Follicle Stem Cell
Markers in Mice Selectively Lacking Ctip2 in Epidermis. PLoS ONE 7,
e29999.
Liu, Y. (2009). Chapter 1 TSLP in Epithelial Cell and Dendritic Cell
Cross Talk. In Advances in Immunology, (Elsevier), pp. 1–25.
Liu, Y.-J. (2006). Thymic stromal lymphopoietin: master switch for
allergic inflammation. J. Exp. Med. 203, 269–273.
Nakayama, T., Hieshima, K., Nagakubo, D., Sato, E., Nakayama, M.,
Kawa, K., and Yoshie, O. (2004). Selective induction of Th2-attracting
chemokines CCL17 and CCL22 in human B cells by latent membrane
protein 1 of Epstein-Barr virus. J. Virol. 78, 1665–1674.
Novak, N., and Leung, D.Y. (2011). Advances in atopic dermatitis.
Current Opinion in Immunology 23, 778–783.
Palmer, C.N.A., Irvine, A.D., Terron-Kwiatkowski, A., Zhao, Y., Liao, H.,
Lee, S.P., Goudie, D.R., Sandilands, A., Campbell, L.E., Smith, F.J.D.,
et al. (2006). Common loss-of-function variants of the epidermal barrier
protein filaggrin are a major predisposing factor for atopic dermatitis.
Nat. Genet. 38, 441–446.
Proksch, E., Brandner, J.M., and Jensen, J.M. (2008). The skin: an
indispensable barrier. Exp Dermatol.
Proksch, E., Feingold, K.R., Man, M.Q., and Elias, P.M. (1991). Barrier
function regulates epidermal DNA synthesis. Journal of Clinical
Investigation 87, 1668–1673.
136
Rang, H.P. (2003). Pharmacology (Edinburgh; New York: Churchill
Livingstone).
Scharschmidt, T.C., and Segre, J.A. (2008). Modeling atopic dermatitis
with increasingly complex mouse models. J. Invest. Dermatol. 128,
1061–1064.
Soumelis, V., Reche, P.A., Kanzler, H., Yuan, W., Edward, G., Homey,
B., Gilliet, M., Ho, S., Antonenko, S., Lauerma, A., et al. (2002). Human
epithelial cells trigger dendritic cell–mediated allergic inflammation by
producing TSLP. Nature Immunology.
Spergel, J. (2003). Atopic dermatitis and the atopic march. Journal of
Allergy and Clinical Immunology 112, S118–S127.
Takai, T. (2012). TSLP expression: cellular sources, triggers, and
regulatory mechanisms. Allergol Int 61, 3–17.
Tokura, Y. (2010). Extrinsic and intrinsic types of atopic dermatitis.
Journal of Dermatological Science 58, 1–7.
Topark-Ngarm, A., Golonzhka, O., Peterson, V.J., Barrett, B., Martinez,
B., Crofoot, K., Filtz, T.M., and Leid, M. (2006). CTIP2 associates with
the NuRD complex on the promoter of p57KIP2, a newly identified
CTIP2 target gene. J Biol Chem 281, 32272–32283.
van Vliet, E., Uhrberg, M., Stein, C., and Gleichmann, E. (1993). MHC
control of IL-4-dependent enhancement of B cell Ia expression and Ig
class switching in mice treated with mercuric chloride. Int. Arch. Allergy
Immunol. 101, 392–401.
Wang, Z., Coleman, D.J., Bajaj, G., Liang, X., Ganguli-Indra, G., and
Indra, A.K. (2011). RXRα ablation in epidermal keratinocytes enhances
UVR-induced DNA damage, apoptosis, and proliferation of
keratinocytes and melanocytes. J. Invest. Dermatol. 131, 177–187.
Yoo, J. (2005). Spontaneous atopic dermatitis in mice expressing an
inducible thymic stromal lymphopoietin transgene specifically in the
skin. Journal of Experimental Medicine 202, 541–549.
Ziegler, S.F. (2010). The role of thymic stromal lymphopoietin (TSLP) in
allergic disorders. Current Opinion in Immunology 22, 795–799.
137
Ziegler, S.F., and Artis, D. (2010). Sensing the outside world: TSLP
regulates barrier immunity. Nature Immunology 11, 289–293.
138
Chapter 4
General Conclusion
139
Skin, the largest body organ, provides both a physical and a chemical
barrier against the outside world (Proksch et al., 2006, 2008; Segre,
2006). The protective skin barrier is a very complicated system formed
by a large number of inter-related components including proteins and
lipids (Fuchs and Raghavan, 2002). The alteration of any of these
components results in altered barrier function(s), which can lead to mild
to lethal phenotype. Therefore, altered skin barrier function is a central
event in various skin diseases (Segre, 2006). Our study revealed a
novel role of transcription factor Ctip2 in skin barrier formation,
maintenance and homeostasis.
The establishment of an effective physical barrier protecting the living
organism from its surrounding environment is the key function of
mammalian skin (Hoffjan and Stemmler, 2007). Most of the transport of
substances across the stratum corneum (SC) takes place through the
lipid bilayer, suggesting its essential role for the barrier function
(Jungersted et al., 2008). The major SC lipids, ceramides, cholesterol
and fatty acids have distinct roles in maintaining normal barrier
functions (Elias and Menon, 1991; Ishikawa et al., 2010). Ceramides as
well as cholesterol were shown to have an influence on (and are
influenced by) the integrity of the SC, while free fatty acids play a major
140
role in the lipid bilayer formation and pH maintenance (Baroni et al.,
2012). We have recently identified Ctip2 as a key regulator of skin lipid
metabolism, which plays an important role in establishment of
epidermal permeability barrier (EPB). Compared with wildtype, uneven
and thinner distribution of neutral lipids was observed in the Ctip2 null
mice at E17.5 (Golonzhka et al., 2008). Furthermore, the ultrastructural
analyses of the epidermis have revealed defects in lipid disc formation,
loosely packed corneocytes, as well as disorganized intercellular
lamellar body membranes in Ctip2 null mice (Golonzhka et al., 2008).
Ablation of Ctip2 leads to altered lipid composition in the developing
mouse epidermis by modulating the expression levels of key enzymes
involved in lipid metabolism. We also demonstrated that Ctip2 was
recruited to the promoter regions of several genes involved in the
ceramide biosynthesis pathways and could directly regulate their
expression.
Besides stratum corneum, other important components contribute the
formation of physical barrier, such as the nucleated epidermis, in
particular the cell–cell junctions and associated cytoskeletal proteins
(Proksch et al., 2008). Ctip2 null mice skin has fewer corneocytes,
which are smaller, more fragile and less resistant to sonication
141
compared to wildtype skin. The linkage of Ctip2 to reduced expression
of serine proteases such as channel-activating serine protease 1
(Cap1) and kallikrein-related peptidase (Klk5, Klk7 and Klk14) as well
as tight junction proteins (Occludin, tight junction protein 1 and 2) in
skin barrier deficiency remains to be addressed (Furuse et al., 2002;
Segre, 2003; Leyvraz et al., 2005; Hachem et al., 2006).
In addition to the physical barrier, the integrity of the skin is also
maintained by the human immune system, which is equipped with a
variety of tools that contribute to the establishment of an efficient
defense system against various challenges including pathogenic
infections. The immune defense system is composed of humoral
(antibody mediated) and cellular (T-cell mediated) constituents
(Proksch et al., 2008; Baroni et al., 2012). The wide range of invading
microorganisms is fended by several immunological facets, including
immediate, nonspecific mechanisms (innate immunity), and delayed,
stimulus-specific responses (adaptive immunity) (Elias, 2007; Jung and
Stingl, 2008; De Benedetto et al., 2012).
Our data demonstrated an important role of Ctip2 in skin immune
defense system. We demonstrated that keratinocytic ablation of Ctip2
142
leads to atopic dermatitis (AD)-like skin inflammation, characterized by
alopecia, pruritus and scaling, as well as extensive infiltration of T
lymphocytes and immune cells. We also observed increased
expression of T-helper 2 (Th2)-type cytokines and chemokines in the
mutant skin, as well as systemic immune responses that share
similarity with human AD patients. Furthermore, we discovered that
TSLP expression was significantly upregulated in the mutant epidermis
as early as postnatal day 1 and ChIP assay revealed that TSLP is likely
a direct transcriptional target of Ctip2 in epidermal keratinocytes.
These results suggest that upregulation of TSLP expression in the
Ctip2ep-/- epidermis could be due to a derepression of gene transcription
in absence of Ctip2.
Besides
these
diverse
chemical/biochemical
strategies,
defense
system,
the
skin
mainly
provides
a
composed
of
antimicrobial peptides (AMPs), also named host defense peptides,
which kill a wide spectrum of bacteria, viruses and fungi, and some
transformed or cancerous cells (Braff et al., 2005; Lai and Gallo, 2009;
Borkowski and Gallo, 2011). AMPs build up an innate epithelial
chemical shield with a rapid and direct first-line defense system termed
as the anti-microbial barrier. AMPs have the ability to enhance immunity
143
by modulating the inflammatory response and activating cellular and
adaptive immunity (Schauber and Gallo, 2009; Gallo and Nakatsuji,
2011). Reduced expression of AMPs such as cathelicidin and βdefensin has been observed in Ctip2 null skin epidermis at embryonic
stage E18.5 (unpublished data), indicating potential role of Ctip2 in
mediating formation of skin chemical/ anti-microbial barrier. The
molecular mechanisms underlying Ctip2 mediated regulation of AMP
expression need to be identified.
Thus, Ctip2 could be involved in a diverse range of events in skin
barrier formation, maintenance and homeostasis. Our study suggests
that Ctip2 is a bi-functional transcription regulator that can either
activate or repress gene expression in a cell and context specific
manner.
During
sphingolipid
biosynthesis,
Ctip2
activate
the
transcription of Lass2 and Gba2 genes by binding to their promoter
regions; however, in the fulfillment of immunological barrier functions,
keratinocytic Ctip2 repress the expression of TSLP gene in a direct
pattern by interacting with TSLP’s promoter region. The roles of Ctip2 in
other aspects of skin function such as hair cycling and cancer
pathogenesis are currently being pursued by Indra group.
144
Ctip1 and Ctip2 are highly related proteins which both interact to
COUP-TF family members and have similar sequence-specific DNA
binding activities, it is highly possible that they have some overlapping
functions in vivo (Avram et al., 2000, 2002). Ctip1 has been identified
as a myeloid or B cell proto-oncogene in mouse and human, and it’s
required for the normal lymphoid development as well as human fatal
hemoglobin expression ( Nakamura et al., 2000; Satterwhite, 2001; Uda
et al., 2008; Liu et al., 2003; Sankaran et al., 2008). We have shown
that the severity of the epidermal barrier defects in Ctip2 null mice is
reduced at later timepoints of development (E18.5 compared to E17.5),
which could be due to the compensatory mechanisms mediated by
Ctip1 (Golonzhka et al., 2008). A modest increase of Ctip1 expression
was observed in Ctip2 null mice at E18.5; however, the role of Ctip1 in
epidermal barrier development and maintenance is still unclear
(Golonzhka et al., 2008). Future investigation about the expression
pattern
and
distribution
of
Ctip1
in
mouse
epidermis
during
development, as well as its role in cutaneous barrier formation will be
performed utilizing Ctip1-null mice.
In conclusion, our data suggested the critical role of Ctip2 in regulating
epidermal permeability barrier formation and maintenance. On one
145
hand, in embryonic mouse skin, Ctip2 is required for the establishment
of
functional
biosynthesis
epidermal
and
barrier
metabolism,
by
controlling
keratinocyte
epidermal
lipid
proliferation
and
differentiation, as well as the formation of structurally intact components
of skin barrier such as lipid discs, cornified envelopes, and intercellular
lamellar bodies. On the other hand, Ctip2 is crucial in adult mice
epidermal barrier homeostasis. Loss of keratinocytic Ctip2 leads to
dysregulated skin barrier characterized with AD-like phenotypes, such
as epidermal hyperproliferation, immune cell infiltration, Th2-type skin
inflammation, as well as systemic immunological syndrome. This study
identifies a new mediator, Ctip2, transcriptionally regulating mouse
epidermal barrier formation and homeostasis in both cell-autonomous
and non cell-autonomous manner.
146
Bibliography
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D.J., Ishmael, J.E.,
and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger
proteins implicated in transcriptional repression mediated by chicken
ovalbumin upstream promoter transcription factor (COUP-TF) orphan
nuclear receptors. J Biol Chem 275, 10315–10322.
Avram, D., Fields, A., Senawong, T., Topark-Ngarm, A., and Leid, M.
(2002). COUP-TF (chicken ovalbumin upstream promoter transcription
factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding
protein. Biochem J 368, 555–563.
Baroni, A., Buommino, E., De Gregorio, V., Ruocco, E., Ruocco, V.,
and Wolf, R. (2012). Structure and function of the epidermis related to
barrier properties. Clinics in Dermatology 30, 257–262.
De Benedetto, A., Kubo, A., and Beck, L.A. (2012). Skin Barrier
Disruption: A Requirement for Allergen Sensitization? Journal of
Investigative Dermatology 132, 949–963.
Borkowski, A.W., and Gallo, R.L. (2011). The Coordinated Response of
the Physical and Antimicrobial Peptide Barriers of the Skin. J Investig
Dermatol 131, 285–287.
Braff, M.H., Bardan, A., Nizet, V., and Gallo, R.L. (2005). Cutaneous
defense mechanisms by antimicrobial peptides. J. Invest. Dermatol
125, 9–13.
Elias, P.M. (2007). The skin barrier as an innate immune element.
Seminars in Immunopathology 29, 3–14.
Elias, P.M., and Menon, G.K. (1991). Structural and lipid biochemical
correlates of the epidermal permeability barrier. Adv. Lipid Res 24, 1–
26.
Fuchs, E., and Raghavan, S. (2002). Getting under the skin of
epidermal morphogenesis. Nat Rev Genet 3, 199–209.
Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani,
Y., Noda, T., Kubo, A., and Tsukita, S. (2002). Claudin-based tight
junctions are crucial for the mammalian epidermal barrier: a lesson from
claudin-1-deficient mice. J. Cell Biol. 156, 1099–1111.
147
Gallo, R.L., and Nakatsuji, T. (2011). Microbial Symbiosis with the
Innate Immune Defense System of the Skin. Journal of Investigative
Dermatology 131, 1974–1980.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J.-M., Campbell,
A.L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M., and Indra,
A.K. (2008). Dual Role of COUP-TF-Interacting Protein 2 in Epidermal
Homeostasis and Permeability Barrier Formation. J Investig Dermatol
129, 1459–1470.
Hachem, J.-P., Houben, E., Crumrine, D., Man, M.-Q., Schurer, N.,
Roelandt, T., Choi, E.H., Uchida, Y., Brown, B.E., Feingold, K.R., et al.
(2006). Serine Protease Signaling of Epidermal Permeability Barrier
Homeostasis. Journal of Investigative Dermatology 126, 2074–2086.
Hoffjan, S., and Stemmler, S. (2007). On the role of the epidermal
differentiation complex in ichthyosis vulgaris, atopic dermatitis and
psoriasis. British Journal of Dermatology 157, 441–449.
Ishikawa, J., Narita, H., Kondo, N., Hotta, M., Takagi, Y., Masukawa, Y.,
Kitahara, T., Takema, Y., Koyano, S., Yamazaki, S., et al. (2010).
Changes in the Ceramide Profile of Atopic Dermatitis Patients. Journal
of Investigative Dermatology 130, 2511–2514.
Jung, T., and Stingl, G. (2008). Atopic dermatitis: Therapeutic concepts
evolving from new pathophysiologic insights. Journal of Allergy and
Clinical Immunology 122, 1074–1081.
Jungersted, J.M., Hellgren, L.I., Jemec, G.B.E., and Agner, T. (2008).
Lipids and skin barrier function – a clinical perspective. Contact
Dermatitis 58, 255–262.
Lai, Y., and Gallo, R.L. (2009). AMPed up immunity: how antimicrobial
peptides have multiple roles in immune defense. Trends in Immunology
30, 131–141.
Leyvraz, C., Charles, R.-P., Rubera, I., Guitard, M., Rotman, S.,
Breiden, B., Sandhoff, K., and Hummler, E. (2005). The epidermal
barrier function is dependent on the serine protease CAP1/Prss8. J.
Cell Biol. 170, 487–496.
Liu, P., Keller, J.R., Ortiz, M., Tessarollo, L., Rachel, R.A., Nakamura,
T., Jenkins, N.A., and Copeland, N.G. (2003). Bcl11a is essential for
normal lymphoid development. Nature Immunology 4, 525–532.
148
Proksch, E., Brandner, J.M., and Jensen, J.M. (2008). The skin: an
indispensable barrier. Exp Dermatol.
Proksch, E., Fölster-Holst, R., and Jensen, J.-M. (2006). Skin barrier
function, epidermal proliferation and differentiation in eczema. Journal
of Dermatological Science 43, 159–169.
Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel,
B., Mikkola, H.K.A., Hirschhorn, J.N., Cantor, A.B., and Orkin, S.H.
(2008). Human Fetal Hemoglobin Expression Is Regulated by the
Developmental Stage-Specific Repressor BCL11A. Science 322, 1839–
1842.
Schauber, J., and Gallo, R.L. (2009). Antimicrobial peptides and the
skin immune defense system. J. Allergy Clin. Immunol. 124, R13–18.
Segre, J. (2003). Complex redundancy to build a simple epidermal
permeability barrier. Curr. Opin. Cell Biol. 15, 776–782.
Segre, J.A. (2006). Epidermal barrier formation and recovery in skin
disorders. J Clin Invest 116, 1150–1158.