PIAS3 & STAT5 Interactions Disrupted in Nonobese Diabetic (NOD) Mice
N.S. Belkin
Mentor: S.A. Litherland, PhD
100275 JHMHC, 1600 SW Archer Rd, College of Medicine, University of Florida, Gainesville,
Florida 32610; (352) 392-5169; fax (352) 392-3053
Abstract
The phosphorylation, nuclear localization, and DNA binding capacities of STAT5 isoforms are
dysregulated in monocytes of autoimmune (AI) humans and the monocytes and macrophage of
nonobese diabetic (NOD) mice. After exposure to Granulocyte Macrophage-Colony Stimulating
Factor (GM-CSF), activated STAT5 proteins in these AI human and NOD cells become resistant
to IL-10 suppression and independent of further GM-CSF/Jak2 kinase activities. Furthermore,
the DNA binding capacities of truncated repressor isoforms of STAT5 (77kD, 80kD) is greatly
diminished in AI human and NOD monocytes, while that of activator STAT5 isoforms (9296kD) is prolonged in NOD macrophages. Congenic analysis of STAT5 dysfunction suggests
that persistence of STAT5 phosphorylation in the NOD is linked to the regulation of posttranslational modification/function and not to the stat5a/stat5b genes or their expression. Using
immunoprecipitation (IP) and DNA affinity precipitation (DAP) analyses, we have found that in
healthy, unactivated control mouse macrophages, STAT5 isoforms interact with PIAS3. In
contrast, STAT5 proteins failed to interact with PIAS3 in NOD macrophages. Furthermore, we
find a lack of sumolation and ubiquitination of STAT5 in AI cells; whereas, control cell STAT5
proteins found interacting with PIAS3 are modified with sumo and ubiquitin. These data suggest
that the loss of PIAS3 interactions in NOD macrophages are altering post-activation
modifications of STAT5 mediated or facilitated by PIAS3; and thereby, may contribute to the
dysregulation of STAT5 DNA binding, recycling, and /or degradation.
Keywords: STAT5, cytokine, signal transduction, diabetes, PIAS3
Background
Previous studies in the nonobese diabetic (NOD) mouse have suggested that myeloid
antigen presenting cells (APC) differentiation and function are defective at a point or points
during the immunopathogenesis of its autoimmune diabetes where the determination of specific
cell lineage decisions and activation state are made based on the cytokine microenvironment
(Serreze, Gaskins, & Leiter, 1993),(Clare-Salzler, Brooks, Chai et al, 1992), (Clare-Salzler,
1998). Serreze et al (1993) found that myeloid differentiation in the NOD was impaired by a
lack of responsiveness in bone-marrow derived myeloid cells to macrophage colony stimulating
factor (M-CSF). This non-responsiveness was not linked to any defect in M-CSF expression or
receptor binding, but involved impaired M-CSF-induced intracellular signal transduction. Morin
et al (2003) noted that GM-CSF can skew NOD myeloid differentiation away from macrophage
and myeloid dendritic cell development, leading to an excess of granulocyte production.
Furthermore, both overexpression and knock-out deletion of GM-CSF in mice can lead to
dysregulation of myeloid differentiation and autoimmune disease (Feili-Hariri, & Morel, 2001),
suggesting that GM-CSF influence is a critical point of tightly-controlled temporal and
quantitative regulation in myeloid differentiation and activation.
GM-CSF Activation of STAT5 for Opposing Roles in Transcriptional Regulation
Like many of the cytokines involved in hematopoiesis, GM-CSF uses the Janus kinase
Jak2 to activate the signal transduction /transcriptional regulator proteins, STAT5A and
STAT5B, to mediate its influence on gene regulation (Feili-Hariri, & Morel, 2001),(Dong, Liu,
De Koning et al, 1998), (Liu, Itoh, Arai et al, 1999). STAT5A & B are members of the JakSTAT family of signal transduction proteins responsive to cytokines, hormones and growth
factors and are transcribed by 2 closely related genes on Chromosome 11 in the mouse and
Chromosome 17 in humans (Teglund, McKay, Schuetz et al, 1998),(Novak, Mui, Miyajima et al,
1996). STAT5A and B proteins were first discovered as responding signal transducers for the
hormone, prolactin. They share many functions but diverse in their effects on sexually dimorphic
gene expression and mammary gland development. (Darnell, 1997). Stat5a/stat5b double
knock-out mice are embryonic lethal due to lack of erythropoietin stimulation of red blood cell
differentiation (Copeland, Gilbert, Schindler et al, 1995). Mutations in the stat5a/stat5b genes
which yield only truncated isoforms are viable, but have dysfunctional fertility, mammary gland
development, lactation, and hematopoiesis. The latter has been linked to diminished
responsiveness to IL3 in early progenitor cell lineage regulation, IL2 in T cell development, and
GM-CSF in myeloid differentiation(Liu, Itoh, Arai et al, 1999),(Socolovsky, Hyung-song,
Fleming, 2002).
Work by Piazza et al (2000) suggests that STAT5 isoform changes can act as key
regulatory ‘switches’ for myeloid differentiation and activation. They found that in early
myeloid differentiation stages, IL-3 and GM-CSF can induce signaling through both full-length
STAT5A (94k-96k) and B (94-92k) isoforms, as well as through truncated isoforms (77k & 80k)
that lack the transcriptional activator motif (Bunting, Bradley, Hawley et al, 2002), (Piazza,
Vlens, Lagassee et al, 2000), (Lehtonen, Matikainen, Miettinen et al, 2002), (Lee, Piazza,
Brutsaert et al, 1999). Truncated STAT5 isoforms are not derived from splice variations as seen
in other STAT proteins, but produced post-translationally by the actions of a myeloid-specific
nuclear serine protease (Bunting, Bradley, Hawley et al, 2002), (Lee, Piazza, Brutsaert et al,
1999),(Azam, Lee, Strehlow, 1997). As myeloid cells mature to macrophages and granulocytes,
they down regulate the protease and lose their ability to produce truncated STAT5 isoforms, so
that signaling through M-CSF and G-CSF signals in matured and activated cells act only through
the full-length STAT5 isoforms (Bunting, Bradley, Hawley et al, 2002), (Lee, Piazza, Brutsaert
et al, 1999), (Ilaria, Hawley, & Van Etten, 1999). During cytokine-induced differentiation,
truncated STAT5 isoforms can act as repressors of gene transcription in immature/unactivated
cells, while full-length STAT5 isoforms induced in mature/activated cells act as gene
transcription activators (Bunting, Bradley, Hawley et al, 2002), (Lee, Piazza, Brutsaert et al,
1999).
Regulation of STAT5 Activation & Function
STAT5 proteins are recruited to the GM-CSF receptor and activated by the Jak2
associated with the common c chain of the receptor (Liu, Itoh, Arai et al, 1999),(Al-Shami,
Mahanna, & Naccache,
Figure 1. STAT5-PIAS-3 Interaction in STAT5 Regulatory
Pathways
1998), (Itho, Liu,
Yokota et al, 1998)
(Figure 1). Both
phosphorylated tyrosine
residues on  and c
chains of the GM-CSF
receptor have been
implicated in the
binding of STAT5 proteins through its SH2 domains (Doyle, & Gasson, 1998). Tyrosine
phosphorylation of STAT5 leads to dimerization, mixing STAT5A & B truncated and full-length
isoforms (Darnell, 1997), (Lehtonen, Matikainen, Miettinen et al, 2002), (Azam, Lee, Strehlow
et al, 1997), (McBride, & Reich, 2003). Dimerization allows transport to the nucleus. Once at
the nuclear membrane, phosphorylated STAT5 can be bound by PIAS3 (Nakagawa, &
Yokosawa, 2002), a protein that has or associates with a SUMO-1 ligase protein modification
activity (Nakagawa, & Yokosawa, 2002), (Kotaja, Karvonen, Janne et al, 2002) and can prohibit
STAT5 binding to DNA (Park, Yahashita, Rui et al, 2001).
PIAS3 is thought to interact with SUMO-1, an ubiquitin ligase protein modifier enzyme
(Nakagawa, & Yokosawa, 2002), (Kotaja, Karvonen, Janne et al, 2002) and promote ubiquitin
modification of STAT proteins in preparation for their removal by the proteasome. If there is a
problem with ubiquitination and proteasome function in the NOD, this would affect the
degradation of STAT5 in the cytoplasm. Wang et al (2000) have suggested that
dephosphorylation of STAT5 tyrosines is necessary for its ubiquitination, as is the COOH region
that is lost in truncated STAT5 isoforms. Others have suggested that serine-threonine
phosphorylation is required, possibly by the nuclear kinase, GSK-3, for STAT protein DNA
release and nuclear export, akin to the mechanism seen in N-FAT subcellular translocation (Park,
Yamashita, Rui et al, 2001). If phosphorylated STAT5 is allowed to enter the nucleus, it can
bind somewhat promiscuously to GAS(gamma activating) (Meyer, Jucker, Ostertag et al, 1998)
sequences in promoter and enhancer regions of genes, usually within 15min of the initial ligandreceptor binding (Park, Yamashita, Rui et al, 2001).
If not blocked by PIAS-3 or other regulatory mechanisms, STAT5 will enter the nucleus
and act to regulate gene expression. The intact COOH terminus trans-activation domain of fulllength STAT5 isoforms may bind the acetylase, CBP/gp300, thought to allow for histone
acetylation and opening of silent DNA to activate transcription (O’Shea, Kanno, Chen et al,
2005). In contrast, the N terminus of nuclear truncated or full-length STAT5-containing dimers
can bind the SMRT/N-CoR complex which promotes DNA and histone deacetylation, allowing
for subsequent methylation and DNA silencing, thus repressing transcription (O’Shea, Kanno,
Chen et al, 2005). Dephosphorylation releases STAT5 dimer formation and allows recycling of
the monomeric STAT5 to the cytoplasm, where it is eventually ubiquitinated and degraded by
the proteasome (Park, Yamashita, Rui et al, 2001), (Wang, Moriggl, Starvopodis et al, 2000).
Myeloid Cell GM-CSF and STAT5 Dysfunction in Autoimmune Type 1 Diabetes (T1D)
We have found that unactivated monocytes from people at-risk for or with Type 1
Diabetes (T1D) and unactivated macrophages from the nonobese diabetic (NOD) mouse have
exceptionally high GM-CSF production and responsiveness in vitro (Litherland, Xie, Grebe et al,
2004), (Litherland, Xie, Grebe et al, 2005). We have also found that PGS2/COX2, an early
response gene for inflammation responsive to GM-CSF in monocyte/macrophage activation
(Yamaoka, Otsuka, Nirio et al, 1998), is aberrantly expressed in unactivated monocytes of atrisk/T1D humans (Litherland, She, Schatz et al, 2003), (Litherland, Xie, Hutson et al, 1999) and
in unactivated NOD macrophages (Litherland, Xie, Grebe et al, 2004), (Litherland, Grebe,
Belkin et al, 2005). In addition, we found high levels of phosphorylated STAT5 in these
unactivated myeloid cells. Moreover, a brief (15min) exposure to GM-CSF induces prolonged
truncated STAT5 activation with diminished DNA binding capacity, and prolonged full-length
STAT5 activation with enhanced DNA binding capacity. In longer term exposure, the STAT5
activation in these cells becomes independent of GM-CSF activation and resistant to
dephosphorylation, even in the presence of AG 490, a potent inhibitor of Jak2/3 activity
(Litherland, Xie, Grebe et al, 2004), (Litherland, Grebe, Belkin et al, 2005). However, the
resistance of autoimmune myeloid cell STAT5 to suppression by IL-10 requires at least a brief
prior exposure to GM-CSF (Litherland, Xie, Grebe et al, 2005). Prolonged STAT5
phosphorylation, and its cell type- and isoform-specific alterations in DNA binding, and aberrant
subcellular translocation, along with the GM-CSF-inducible resistance to IL-10, suggest that
STAT5 regulation is dysfunctional in autoimmune myeloid cells.
Our preliminary data suggest that truncated STAT5 in healthy control monocytes is
readily dephosphorylated removed from the nucleus and the cytoplasm, while these STAT5
isoforms in autoimmune monocytes is not. We also see normal serine-threonine phosphorylation
on autoimmune monocyte and NOD macrophage STAT5, and similar GSK-3 levels in controls
and autoimmune myeloid cells. These findings point to PIAS3 as a strong candidate for a central
role regulating STAT5 in the DNA binding, subcellular localization, and the degradation in
monocytes. Therefore, we examined PIAS3 expression and function in NOD macrophages to
see if it is involved in STAT5 aberrant DNA binding, altered subcellular localization and/or
resistance to degradation by the proteasome.
Methods & Materials:
In accordance with the IACUC-approved protocols B083 and D574, peritoneal
macrophages were collected from 8-12 week old female C57BL/6 and NOD mice by post
mortem peritoneal lavage with RPMI (Mediatech, Herndon, VA) +10% FCS (Mediatech) + 1%
PSA (antibiotic/antimycotic mix, Cellgro, Mediatech) and adherence purified prior to culture for
24 hours at 37C/5%CO2 in the same media supplemented with GM-CSF (1000U/mL, Biosource,
Camarillo, CA), anti-GM-CSF (2g/mL, Pierce, Rockford, IL), Jak Inhibitor/AG 490 (100M in
DMSO, CalBiochem, San Diego, CA) or DMSO (volumetrically equivalent to Jak Inhibitor,
Sigma, St. Louis, MO). After 24 hours, culture supernatants were removed and centrifuged at
600xg for 5 minutes at 25˚C to remove cells and frozen at –70˚C for later GM-CSF and PGE2
production analysis by ELISA(BD Biosciences, San Diego, CA and Amersham) (Litherland,
Xie, Hutson et al, 1999), (Litherland, Grebe, Belkin et al, 2005). Cells collected from these
cultures were analyzed by flow cytometry and deconvolution microscopy as previously
described [30]. Extracts were made from adherent cells in situ using STAT5 lysis buffer
[(10mM HEPES pH 7.3 (Gibco, Carlsbad, CA), 1mM dithiothreitol (Sigma), 2mM EDTA
(Sigma), 400mMKCl (Sigma), 0.1% Triton X-100 (Sigma), 10% glycerol (Sigma); with 5g/ml
each of aprotinin, leupeptin, pepstatin, (Sigma) and pefabloc (Roche Molecular
Biochemicals/Boehringer-Mannheim, Indianapolis, IN)] and stored at –70˚C for later proteinDNA binding analysis.
DNA Affinity Precipitation(DAP):
Protein extracts (from 5 x106 cells) Peritoneal macrophage extracts were diluted in Catch &
release lysis (CRLW, UpState Biotech, Charlottesville, VA) buffer (500 μl). Labeled binding
substrates at 16000 fmol of FITC labeled DNA and 2 μg anti-FITC antibody were added for each
sample and incubated for 30 minutes at 25˚C. Negative controls were run without DNA added.
Positive control reactions were set up in parallel using a STAT5 positive human macrophage
line, U937(ATCC). Antibody Capture Affinity Ligand (10μl/reaction, Upstate) was added to
each sample prior to centrifugation in spin columns. Flow through of the column was used as
“unbound” portions of the samples and that in the immunoprecipitated as “bound” portions.
Immunoprecipitation:
Protein Assay (BioRad, Hercules, CA) was preformed on each sample and two 10μg aliquots of
protein were isolated from each sample. One aliquot was prepared in Leammili buffer (BioRad)
and called “total extract”. Anti-STAT5 antibody (2μg, Santa Cruz, Santa Cruz, CA) was added
to the other aliquot, and incubated rocking overnight at 4˚C. Positive (U937 or Jurkat cells) and
negative (no extract) controls were run in parallel. After incubation, 20μL Protein G agarose
beads (UpState) were added and allowed to incubate 45 minutes rocking at 25˚C. The beads
were isolated via centrifugation (600xg, 5min, 25˚C) and washed with 1X PBS (Cellgro). The
beads were resuspended in 1x Leammili buffer, and boiled for 3min. Beads were removed by
centrifugation and the supernatant was resolved on 7.5% SDS PAGE gels (BioRad).
Electrophoresis and Western Blot:
Samples were prepared in Leammili buffer (BioRad) and concentrated when need to 30μL using
a 10K cutoff concentrator column (Amicon, Millipore, Billerica, MA). The Samples were run on
a 7.5% SDS-PAGE gel (BioRad) at 200V for 1 hour at 25˚C. Samples were transferred to
Hybond-P membranes (Amersham, Piscataway, NJ) at 100V for 45 minutes at 25˚C (chilled
buffer to prevent heating). Membranes were washed in 0.25% Tween20 (Sigma) in 1X PBS
(Cellgro) and blocked with 0.5% Amersham block in 1X PBS. The membranes were then
probed with anti-STAT5A/B (Santa Cruz, Santa Cruz, CA) followed with the anti-rabbit Ig-HRP
secondary (UpState or Amersham) and visualized using ECL Plus detection reagents
(Amersham). The membranes were stripped using ECL plus stripping buffer following
Amersham recommended protocol. The probing and stripping steps were repeated with antiSMRTe (UpState), anti-PIAS3 (Santa Cruz), anti-STAT5A/B-Phosphorylated (UpState), antiSUMO (VLI Research, Malvern, PA) and/or anti-Ubiquitin (VLI Research), using species
appropriate secondary anti-Ig-HRP conjugates (UpState or Amersham) to visualize specific
bands.
Results:
The GM-CSF production, PGE2 production, and STAT5 phosphorylation in NOD and
C57BL/6 macrophages used in this study reiterated our previous findings (Litherland, Xie, Grebe
et al, 2004), (Litherland, Xie, Grebe et al, 2005), (Litherland, Grebe, Belkin et al, 2005); namely
that NOD macrophages had significantly higher GM-CSF and PGE2 production without
stimulation (Table 1) and significantly higher STAT5 production (Figure 2 & Table 1) by flow
cytometric analysis and deconvolution microscopy (Figure 2b & Litherland, Xie, Grebe et al,
2005).
Table 1. GM-CSF and PGE2 production by NOD and C57BL/6 Macrophages in Treatments
analysis/treatment
media
GM-CSF
Anti-GM-CSF
Jak Inhibitor,
AG490
DMSO
0
226
>1000
>1000
54+/- 76
86+/-122
48+/- 68
42+/- 50
0
107+/- 45
29
113
43+/-3
41+/-21
40+/- 18
54+/- 30
30+/1 19
73+/- 38
33+/- 8
66+/- 23
pg/ml GM-CSF
C57BL/6
NOD
pg/ml PGE2
C57BL/6
NOD
A.
B.
Deconvolution
Analysis of
Unstimulated
Peritoneal
Macrophages
Flow Cytometric Analysis-Mouse Macrophages
DMSO
*p=0.0370
**p=0.0287
ns
AntiGM-CSF
AG 490
ns
80
70
NOD
60
50
40
30
20
10
0
NOD
C57BL/6
media/DMSO
NOD
C57BL/6
GM-CSF
NOD
C57BL/6
anti-GM-CSF
NOD
C57BL/6
B6.NODC11
%STAT5Ptyr+/CD11b+ cells
90
GM-CSF 15’
DAPI
STAT5A/B-FITC
STAT5-PTYR-PE
C57BL/6
100
bg
AG490
Figure 2. A. Flow cytometric and B. deconvolution microscopic analysis of STAT5 phosphorylation. P values are from
pair-wise Mann-Whitney U test analysis.
Using DNA affinity
precipitation (DAP) analyses, we
found that without stimulation neither
NOD nor C57BL/6 macrophage
STAT5 interacts with PIAS3 (0 lanes,
Figure 3a&b). As we described
Figure 4. IP-Western blot analysis of STAT5-PIAS3 interactions
and subsequent STAT5 protein modification (ubiquitination &
sumolation). Blots probed with antibodies to PIAS3 top panel,
anti-SUMO(2nd panel), anti-ubiquitin (UBI, 3rd panel), and
phosphotyrosine specific STAT5 (STAT5Pyr, last panel). SH=
no DNA sham, += positive control; 0= medium only; G=
1000U/ml GM-CSF; A=anti-GM-CSF: J= AG490; D= DMSO.
previously, only in NOD macrophage
does full-length STAT5 bind DNA
without stimulation (Figure 3b, 0 & D
lanes). Under GM-CSF stimulation
conditions, both NOD and C57BL/6
macrophage STAT5 isoforms not
bound to DNA interact with PIAS3
(Figure 2a GU lane). However,
DNA-bound STAT5 in controls also
bound PIAS3 with GM-CSF
Figure 3. DAP analysis of STAT5 in C57BL/6(a) and NOD(b)
macrophages.
Upper panels are blots probed with anti-PIAS3; lower panels are
same blots probed with anti-STAT5 (full-length or truncated
isoforms bands run were indicated. SH= no DNA sham, +=
positive control; 0= medium only; G= 1000U/ml GM-CSF;
A=anti-GM-CSF: J= AG490; D= DMSO; B=protein from lysates
bound to DNA; U= protein from lysates not bound to DNA
stimulation and when anti-GM-CSF
was added (Figure 3a, GB and AB
lanes). In contrast, STAT5 proteins
failed to interact with PIAS3 in NOD
macrophages treated with anti-GM-CSF (Figure 3b, GU lane). Jak Inhibition with AG490
disrupted C57BL/6 STAT5 binding and interactions with PIAS3 when DNA bound, but this
treatment now allows DNA-bound STAT5 in NOD extracts to interact with PIAS3 (Figure
3a&b, J lanes). The higher STAT5 DNA binding seen the NOD DMSO/ media treatments
(Figure 3b; 0 & D lanes) may be in part due to the fact that unactivated GM-CSF production is
markedly higher in the NOD (Table 1) (Litherland, Xie, Grebe et al, 2004), (Litherland, Xie,
Grebe et al, 2005), (Litherland, Grebe, Belkin, 2005). We conclude from these data, that PIAS3STAT5 interactions are impaired in the NOD at least in part by activation of Jak kinase.
Immunoprecipitation with anti-STAT5 antibodies analysis again showed a lack of
STAT5-PIAS3 interactions in unstimulated NOD macrophages, while such interactions were
detected in the C57BL/6 controls (Figure 4). Furthermore, re-probing of these IP blots showed
no sumolation of STAT5 in NOD macrophages; whereas, C57BL/6 STAT5 proteins interacting
with PIAS3 were modified with sumo (Figure 4). Ubiquitination was only inhibited in the
AG490 treated cells. These data suggest that the failure of NOD STAT5 to interact with PIAS3
blocks its ability to sumolate it. Probing of the blots with antibodies specific for tyrosine
phosphorylated STAT5 suggests that NOD STAT5 proteins remain phosphorylated in all
treatments (Figure 4). This supports the theory that PIAS3 sumolation of STAT5 may be
inhibited by its persistent phosphorylation.
Discussion:
In our recent studies, we found that STAT5 signal transduction proteins in unactivated
autoimmune myeloid cells remain persistently phosphorylated, and do not bind DNA in
truncated gene expression suppressor isoforms, while exhibiting enhanced DNA binding
capacity in full-length activator isoforms (Litherland, Xie, Grebe et al, 2005). Persistence of
GM-CSF-induced STAT5 signaling is resistant to IL-10 suppression and may delay or prohibit
progression of bone marrow precursor cells in myeloid cell differentiation. Dysregulation of
STAT5 signaling in autoimmune myeloid cells from humans and nonobese diabetic (NOD) mice
may also to link their GM-CSF overproduction with the GM-CSF inducible IL-10 resistance of
the inducible prostaglandin synthase/cyclooxygenase, PGS2/COX2 (Litherland, Xie, Grebe et al,
2004), (Litherland, Xie, Grebe et al, 2005). Aberrant PGS2/COX2 activity in these cells leads to
overproduction of the pro-inflammatory prostanoid, PGE2 (Litherland, Xie, Grebe et al, 2004),
(Litherland, Xie, Grebe et al, 2005), (Yamaoka, Otsuka, Niiro et al, 1998), (Litherland, She,
Schatz et al, 2003), (Litherland, Xie, Hutson et al, 1999), (Litherland, Grebe, Belkin et al, 2005).
Thus, STAT5 may play a pivotal role in maintaining the delicate balance of cytokine production
and signaling needed for chromatin dynamic changes in myeloid cell 1) differentiation and
activation, and 2) regulation of inflammation.
Analyzing cells from congenic B6.NODC11 (Yui, Muralidharan, Moreno-Altamirano et
al, 1996) and NOD.LC11(DR3) (McDuffie, 2000) recombinant mouse strains, we have shown
that autoimmune myeloid cell STAT5 phenotypes are associated with a small region on
Chromosome 11 which contains both the GM-CSF gene, csf2, and the idd4.3 diabetes
susceptibility locus, but not the stat5a/stat5b genes (Litherland, Grebe, Belkin et al, 2005).
Congenic replacement this csf2/idd4.3 containing region contributes approximate 70% of the
diabetes resistance seen in the NOD.LC11(DR3) congenic mouse strain. Our preliminary data in
human monocytes show STAT5 dysfunction is common phenotype found in multiple
autoimmune disease conditions, including Type 1 diabetes(T1D), Hashimotos thyroiditis(HD),
and Graves Disease(GD) Litherland, Xie, Grebe et al, 2005), suggesting analysis of myeloid cell
STAT5 may serve as diagnostic/prognostic indicators of autoimmune susceptibility and as
potential targets for prevention and/or therapeutic intervention of autoimmune disease.
Using IP and DAP analyses, we have found that in healthy, unactivated control mouse
macrophages, STAT5 isoforms interact with PIAS3. In contrast, STAT5 proteins failed to
interact with PIAS3 in NOD macrophages. Furthermore, we find a lack of sumolation and
ubiquitination of STAT5 in AI cells; whereas, control cell STAT5 proteins found interacting
with PIAS3 are modified with sumo and ubiquitin. These data suggest that the loss of PIAS3
interactions in NOD macrophages are altering post-activation modifications of STAT5 mediated
or facilitated by PIAS3; and thereby, may contribute to the dysregulation of STAT5 DNA
binding, recycling, and /or degradation.
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