Biochemical and Biophysical Research Communications 396 (2010) 950–955
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Induced expression of bone morphogenetic protein-6 and Smads signaling
in human monocytes derived dendritic cells during sickle-cell pathology with
orthopedic complications
Kumar Abhishek a,d, Ritesh Kumar b, Ehtesham Arif c, P.K. Patra a, S.B. Choudhary d, Mohammad Sohail e,*
a
Department of Biochemistry, Pandit Jawaharlal Nehru Memorial Medical College, Raipur, Chhattisgarh, India
Department of Biotechnology, Hamdard University, New Delhi, India
School of Medicine, University of Pennsylvania, Philadelphia 19104, United States
d
Department of Botany, Vinoba Bhave University, Hazaribagh, Jharkhand, India
e
Department of Entomology, Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, United States
b
c
a r t i c l e
i n f o
Article history:
Received 23 April 2010
Available online 9 May 2010
Keywords:
Sickle cell disease
BMP6
Smad
Alkaline phosphatase
Apoptosis
a b s t r a c t
BMP-SMAD (bone morphogenetic protein) signaling pathways in association with APT play paramount
roles in osteoblastic differentiation, bone formation and embryonic development of human and animals.
However, the implications of potent components (BMP6, Smad1, Smad2 and APT) of this pathway in SCD
(sickle cell disease) pathology with orthopedic complications (Ortho + SS) are poorly elucidated and substantially unknown. Here, we address the role of BMP6, Smad1, Smad2 and APT mRNA and protein
expression in hMDDCs obtained from Ortho + SS patients, employing RT-PCR, qRT-PCR and immunoblotting. Interestingly, we observed that SCD pathology exhibited significantly up-regulated expression of
those signaling components at the level of mRNA and protein. Furthermore, exogenous BMP6 induced
apoptosis was observed to be significantly associated in Ortho + SS complication and markedly increased
the percentage of cells undergoing apoptosis as compared to healthy group. Interestingly, the non-stimulated cells have shown higher apoptotic nuclei percentage than the stimulated cells in pathological condition. Thus, expression of BMP-SMAD signaling components augments apoptosis and up regulates the
transcription of these genes and it suggests that induction is due to transcriptional regulation. Taken
together, our findings provide evidence that BMP-SMAD signaling components along with APT were over
expressed, mediates apoptosis and may play an important role in the SCD pathology with orthopedic
complications.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
Sickle cell disease (SCD) is an inherited disorder of haemoglobin
(Hb) synthesis affecting many individuals throughout the world. Its
complications are varied: acute chest syndrome, proliferative retinopathy, pulmonary hypertension, renal insufficiency, cerebral vascular accident and musculoskeletal complications [1]. The bone
involvement in SCD ranges from acute manifestations, such as painful vaso-occlusive crisis or osteomyelitis, to more chronic and debilitating complications, such as osteonecrosis, osteoporosis and
osteopenia, impaired growth and chronic infections [2]. The de novo
bone formation is potentially associated with several structurally
related proteins from the transforming growth factor-b superfamily,
particularly bone morphogenetic proteins (BMPs). BMPs constitute
the largest group of proteins within the TGF-b superfamily of growth
and differentiation factors. It is now well documented that BMPs
* Corresponding author. Fax: +1 951 827 3086.
E-mail addresses: soh.khan@gmail.com, drsohail.delhi@gmail.com (M. Sohail).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.05.029
orchestrate a wide range of developmental processes, including
BMP-induced differentiation of osteoblast progenitors and subsequent bone development in adults, skeletal development, neuronal
patterning and limb formation in embryos [3]. At the cellular level,
a heterotetrameric complex composed of type I and II transmembrane serine/threonine kinase receptors initiates downstream cytoplasmic signaling following BMP binding [4]. These BMPRs have
different BMP affinities and form receptor complexes prior to or after
BMP binding, which is thought to regulate BMP signaling (Smaddependent vs. Smad-independent) [5]. Heteromeric receptor complexes comprising type I and II receptors lead to ligand-induced
phosphorylation of type I receptors [4]. Following the activation of
this receptor, receptor kinases phosphorylate the transcription factors Smad 1, 5, 8, which subsequently form heteromeric complexes
with Smad4 and activate the expression of target genes in the nucleus orchestrated by other co-activators [6,7].
Recently, a number of molecular markers have been investigated
in terms of outcome prediction and potential therapeutic targets for
various diseases including SCD pathology with orthopedic
K. Abhishek et al. / Biochemical and Biophysical Research Communications 396 (2010) 950–955
complications [1]. These studies are mainly directed towards BMPs
signaling pathways and dysregulation of BMP signaling has been
linked to various clinical disorders [4]. BMPs are synthesized and secreted from a variety of cell types including dendritic and endothelial cells and play an important role in regulating cell proliferation,
apoptosis, and differentiation [8–10]. So far in human, little experimental data are available regarding the mechanisms underlying the
regulation of BMP signaling in SCD-specific pathology and other
associated disease. Importantly, the precise molecular mechanisms
and functional studies by which BMP signaling regulate during SCD
pathology in human have been largely unknown because of the complexity in the in vivo system. From this viewpoint, we used human
monocytes derived dendritic cells, an in vitro system for study SCD
pathology with orthopedic complications (Ortho + SS) to dissect
the role of BMP-Smad signaling pathways. In the present study, we
have evaluated the components of BMP-Smads, which modulates
the signaling pathway during SCD pathology in association with
APT (alkaline phosphatase) activity. We demonstrate that hMDDCs
transduce signals via activation of BMP6-Smad1/2 in association
with upregulated APT with an evidence of the production of biologically active BMPs by human monocytes-derived DCs. This study further demonstrates that apoptosis in human monocytes-derived DCs
is regulated by BMP6-Smad1/2 signaling, which can also be induced
by exogenous BMP6. These results raise the possibility that hMDDCs
could be under the influence of BMP-Smad signaling during the
development of Ortho + SS. There has been no published study
examining the expression of BMP6, Smad1, Smad2 and APT mRNA
and protein in Ortho + SS. However, to the best of our knowledge,
our investigation is the first report attempting to evaluate the association between expressions of BMP-Smad signaling pathway in
association with APT in hMDDCs during Ortho + SS as compared to
healthy subjects.
2. Materials and methods
2.1. Study population
The consent were taken from the participants and human blood
samples were taken as per protocols approved by the Ethical Review Board of the Pt. J.N.M. Medical College, Raipur, Chhattisgarh,
human ethical guideline as reflected in the guidelines of the medical ethics committee, Ministry of Health, Government of India.
Subjects were taken from Chhattisgarh and Jharkhand state for this
study, which consists of 44 (case) and 44 (control), respectively.
The group was divided in two categories: case and control. In case
group, we have taken sickle cell disease (SS) and sickle cell disease
with orthopedic complication (Ortho + SS) subjects, whereas, in
control group, we have taken sickle cell trait (AS) and healthy
(AA) subjects. The selection criterion for patients was that they
had Ortho + SS and no other malformations. An individual with
any other anomalies, than the above said has not included in study.
To be classified as a case, all the patients were diagnosed through
clinical examinations and their diseased status was checked by
electrophoresis. The outdoor patients and inpatients with no such
complications were considered as control group. The selection criteria for controls had no evidence of any other serious illness, particularly for other hereditary diseases. The control group was
matched to the case group by age, weight, height, haemoglobin,
calcium, uric acid and LDH (lactate dehydrogenase) as shown in
Supplementary Table 1.
2.2. Western blot analysis
Human monocytes-derived DCs (hMDDCs) from patients and
control subjects were plated onto 24-well plates at a cell density
of 1.2 106 cells/ml and after 24 h cells were washed twice with
951
PBS and then lysed with RIPA cell lysis buffer. The cell lysates were
incubated on ice for 20 min and centrifuged at 10,000g for 15 min
at 4 °C. Supernatant containing 20–50 lg of protein was loaded
onto a sodium dodecyl sulfate (SDS)–polyacrylamide gel. After
electrophoresis, proteins were transferred onto nitrocellulose
membrane were incubated with the affinity purified rabbit polyclonal primary antibody against the phosphorylated Smad1, Smad2
(Upstate Biotechnology, Lake Placid, NY), BMP6 and APT (Cell Signalling Technology, Beverly, MA) and detection was done by horseradish peroxidase-conjugated goat anti-rabbit secondary
antibodies and enhanced chemiluminescence (ECL; Amersham
Biosciences, Piscataway, NJ) Western blotting detection reagent
was used to visualize immunoreactive bands and densitometer
evaluation was done by ImageJ software (NIH).
Further, detailed methodology related to cell culture, alkaline
phosphatase activity and LDH measurement, semi quantitativePCR, qRT-PCR and apoptosis assay has been given in Supplementary material attached to this article.
3. Results
3.1. Induced expression of BMP6 and associated proteins in SCD with
orthopedic complications
Previous studies have described that BMPs activate the signaling pathways in various pathological state in association with
other components of the pathways [11] and one approach to define
the role of specific BMP in SCD disease is to examine the expression
profile in affected patients for the development of disease through
a defined signal transduction pathway. We found, by Western blot
analysis, relatively high expression of mature BMP-6 protein in
Ortho + SS patients as compared to healthy group as well as marginally higher than SS patients also (Fig. 1A, panel 3). Further, to access the role of sickle cell disease in BMP-SMAD signaling pathway,
we also analyzed the expression of Smad1, Smad2 and APT protein.
We distinctly observed upregulated protein expression in diseased
group as compared to healthy group (Fig. 1A, panel 1, 2 and 4).
However, the levels of BMP6, Smad1, Smad2 and APT protein were
approximately 1.5-fold higher than healthy group as quantified by
NIH imaging software. Interestingly, the difference in protein
expression was found to be statistically significant (Fig. 1B–E)
and our observation of up-regulated expression of proteins in
BMP-SMAD signaling pathway component is in accordance with
our findings observed during assessment of mRNA levels of these
components also. Taken together, our data strongly suggest that
in orthopedic complications during SCD is clearly marked by perturbed and augmented BMP-SMAD signaling and subsequent disease progression.
3.2. SCD with orthopedic complications induces BMP6, Smad and APT
expression in hMDDCs
SCD pathology has been reported to have perturbed role of BMPSMAD signaling and to stimulate the proliferation and differentiation of bone forming cells leading to various orthopedic complications [12]. In order to assess the effect of BMP6, Smad1, Smd2 and
APT expression in Ortho + SS, hMDDCs were cultured as mentioned
in the methodology section and the mRNA expression was determined by semi-quantitative RT-PCR. We observed sustained and
up-regulated expression of the BMP6 in SCD pathology as compared
to healthy group (Supplementary Fig. 1A, panel 3) and it was found
to be 0.5-fold higher than the healthy group, although the difference
was not statistically significant (Supplementary Fig. 1C). Furthermore, SCD pathology induced the expression BMP signaling
component Smad1, Smad2 and APT as compared to healthy group
(Supplementary Fig. 1A panel 1, 2 and 4, respectively) and the
952
K. Abhishek et al. / Biochemical and Biophysical Research Communications 396 (2010) 950–955
Fig. 1. Immunoblot of partially purified protein from control group (AS and AA) and case group (SS, Ortho + SS). (A) Cell lysates were subjected to immunoblot for Smad1,
Smad2, BMP6, APT and b-actin as loading control. The expression level of Smad1, Smad2, BMP6 and APT were expressed in different isolates (B–E). The mean fold were
changed in control and case group relatively and normalized to the level of cellular actin, as assessed by densitometry. The data represents the mean ± SE of two independent
experiments and the statistical significance was calculated using Student’s t-test (N value is 5 for each bar represented).
difference was observed to be significant as shown in Supplementary Fig. 1B, D and E. These data suggest that SCD pathology with
orthopedic complication upregulates the expression of BMP6 and
its signaling component Smad1, Smad2 and APT.
of APT and biochemical level of APT is modulated by SCD
pathology.
3.5. BMP6 induces apoptosis in SCD pathology with orthopedic
complication
3.3. Gene expression analysis by quantitative PCR
Based on the results of semi-quantitative RT-PCR and Western
blotting, we hypothesized that SCD with orthopedic complication
specific pathology modulates the induction of BMP-Smad signaling
pathways in association with APT and differentially expressed in
group of patients during pathology as compared to healthy group.
To test these hypotheses and to further understand the BMP6SMAD signaling pathway, we analyzed expression changes in
SCD patients and healthy subjects. We compared the amount of
BMP6, Smad1, Smad2 and APT mRNA by qRT-PCR and the expression levels were found to be more than 1.5-folds high as those of
healthy subjects, respectively (Fig. 2A–D). Indeed, the transcriptional response analysis by qRT-PCR revealed the differed quantitative expression of the BMP-Smad signaling component in SCD
pathology and found to be statistically significant as compared to
healthy subjects. The above data validates the results of qualitative
estimation by RT-PCR and immunoblot; that SCD pathology promotes the expression of BMP-Smad signaling components and further these results collectively suggest that enhanced expression of
these genes are associated with SCD pathology and orthopedic
complications.
3.4. Induction of alkaline phosphatase activity in cultured hMDDCs
upon SCD pathology with orthopedic complications
Since a high level of alkaline phosphatase activity is considered
a hallmark of the osteogenic phenotype, we evaluated the effect of
alkaline phosphatase activity in hMDDCs. As expected, we also observed significantly elevated alkaline phosphatase activity in SCD
pathology as compared to healthy group of subjects (Supplementary Fig. 2). Our observation of elevated alkaline phosphatase activity in diseased state is in accordance with findings of up-regulated
expression of mRNA and protein level suggesting that expression
We investigated a possible proapoptotic effect of BMP6 treatment on hMDDCs during SCD pathology. To test whether BMP6
can mediate apoptosis in hMDDCs, we first examined the potential
damage to the plasma membrane generated by BMP6 stimulation.
For this purpose, we measured the amount of LDH released into the
medium by lysed cells in response to BMP6 stimulation. As shown
in Supplementary Fig. 3, release of LDH in the medium was observed to be statistically significant in case of patients group of
SCD with orthopedic complications and patients with only SCD
pathology as compared to healthy group (P = 0.005).
It has been demonstrated that doses (10–100 nM) of BMPs and
TGF-b1 inhibited cellular proliferation in various pathological conditions in human [13]. To test whether the antiproliferative effects
of BMP6 also involve mediating apoptosis in Ortho + SS, we determined and compared the percentage of cells undergoing apoptosis
in cells cultured in media with or without BMP6. Under baseline
conditions, 6.46 ± 2.56% of hMDDCs were stained positive for annexin V. After treatment with 100 nM BMP-6 for 48 h, there was
a significant increase in annexin V staining to 34.68 ± 5.46%
(Fig. 3A and B). The annexin V-positive cells exhibited the decreased cell size that is indicative of apoptotic cell shrinkage. Under baseline conditions, 7.98 ± 0.42% of the cells were in a size
range of 50–260 AU; after treatment of the cells with BMP6,
26.23 ± 9.87% of the cells were in the observed size range.
Furthermore, in hMDDCs isolated from these patients and
healthy subjects, the apoptotic nuclei under baseline conditions
were found to be 28.6 ± 0.3% (N = 44) from case group and
5.2 ± 0.4% (N = 44) in control group (P = 0.005) as shown in Fig. 3C.
Interestingly, the exogenous treatment of the cells with BMP6 also
induced apoptosis but somewhat lesser in case of SCD group of
patients (21.5 ± 0.4%) as compared to non-stimulated patients
group, whereas the stimulated cells from healthy group has shown
increased level of apoptotic nuclei (8.5 ± 0.3%) as compared to
K. Abhishek et al. / Biochemical and Biophysical Research Communications 396 (2010) 950–955
953
Fig. 2. Total RNA was isolated from control group (AS and AA) and case group (SS, Ortho + SS) for cDNA synthesis. Differential expression of different group in panel A, B, C and
D represented as Smad1, Smad2, BMP6 and APT, respectively. Fold induction was calculated using the DDCT method of qRT-PCR, relative to b-actin levels. Data is presented as
mean and error bar represent the plus or minus SE from 4 isolates. Results are representative of at least two independent experiments. **P 6 0.05; ***P 6 0.0005 compared
with healthy subjects by one-way analysis using Tukey’s multiple comparison test through Graphpad Prism version 5.0.
Fig. 3. Apoptotic effect of BMP-6 on hMDDCs. (A) Representative cell nuclei stained with DAPI and annexin-V in a control cell and a cell treated with 100 nM of BMP-6. Apoptotic
cell was stained positively with annexin V (green). (B) hMDDCs stained with annexin V-FITC and propidium iodide (PI) were analyzed by flow cytometry. A scatter plot of PI
(y-axis) vs. annexin V fluorescein (x-axis) indicates the alive cells (bottom-left quadrant), necrotic cells (top-left quadrant), end-stage apoptotic cells (top-right quadrant), and early
apoptotic cells (bottom-right quadrant). (C) SCD pathology with orthopedic complications-mediated apoptotic effects as compared to healthy subjects in hMDDCs. (D) BMP-6
induced hMDDCs apoptosis; N = 18 fields of cells from three cover slips for each data bar; data are plotted as mean ± SE of two independent experiments. ***P = 0.001 vs. control
cells (100 nM BMP-6 treatment). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
non-stimulated group of healthy subjects as shown in Fig. 3D. These
results demonstrate that BMP6 also induces apoptosis through BMP
mediated apoptotic pathway in hMDDCs in SCD pathology in addition to up-regulated expression of BMP-SMAD signaling pathway
in diseased state as compared to healthy subject.
4. Discussion
Among BMP family members, BMP6 has been extensively studied and demonstrated to play a crucial role in inducing osteoblast
differentiation and bone formation during embryonic skeletal
development and bone remodeling [14]. In recent years, BMP-6
has generated considerable attention in various pathological conditions like, cancer biology and bone related genetic disorder
[15]. Bone cells respond to changes in the microenvironment by
altering gene expression profile. Typically, this involves the expression of genes required for adaptation to the new conditions. BMPSmad signaling among several molecular pathways has been
shown to mediate pathology sensing at the cellular and molecular
levels [14]. Thus, in order to elucidate the role of Smads/BMPs in
954
K. Abhishek et al. / Biochemical and Biophysical Research Communications 396 (2010) 950–955
SCD pathology with orthopedic complications, it is very important
to understand the signaling mechanisms of those components
in vitro for future therapeutic intervention. The human monocytes-derived DCs provide an in vitro system and we took advantage to explore the effect of BMP-Smad signaling and underlying
molecular mechanism involved in SCD pathology.
In this study, we used RT-PCR and immunoblotting to examine
the expression of BMP6, Smad1, Smad2 and APT in human monocytes-derived DCs samples from sickle cells patients for the first
time. Our observations are in agreement with the results of earlier
studies in various pathological conditions [16]. Our results showed
that these signaling components for osteoblast development [3]
are significantly up-regulated expression assessed by real time
quantification in hMDDCs along with the alkaline phosphatase
activity in SCD pathology as compared to healthy subjects. These
data suggests that the downstream signals of BMP6 and Smads
are triggered during Ortho + SS and can be explained in several
ways; firstly the kinetics of SMAD phosphorylation in association
with BMP ligands and receptors are much faster and more transient in pathological condition than healthy state. Second, there
may be cascade effect through the BMP-SMAD signaling pathway
that is able to amplify the impairment step by step in pathological
conditions. We found significant differences in protein levels of
those Smads when monitored by Western blot. These data suggests
that cultured hMDDCs should be able to form active heterotetrameric receptor complexes capable of mediating BMPs signaling
during pathology. It is well established that canonical BMPs signaling is mediated by BMP receptors activation followed by nuclear
translocation of Smads protein complexes, which in turn modulate
gene expression. The intracellular Smad proteins are signaling molecules that act in transduction of signaling pathways triggered by
members of TGF-b family and subsequently play a modulating role
in BMP signaling [17]. It has been shown that Smad1 directly interacts with Hoxc8 to activate the transcription of osteopontin gene
[18], which is a marker gene for osteoblast and chondrocyte differentiation. It is well established that BMP6 transmit signal trough
phosphorylation of Smads, which in turn modulate gene expression. With this perception, we tried to analyze the Smad expression
at the level of mRNA during Ortho + SS. As expected, when we analyze the Smads mRNA in hMDDCs cultures we observed the upregulated expression of Smad1 and Smad2 in SCD pathology. This
up regulation is in agreement with the results of earlier studies
as an indicator of activation of the BMP6 signaling cascade
[19,16]. Altogether these results also demonstrate that the BMP6
induced apoptotic process requires a functional Smads pathway.
Hence suggesting that Smad mediated BMP signaling was indispensable and could be a relevant signaling pathway in the present
study. We were able to show that SCD pathology induces intracellular signaling in hMDDCs via BMP-Smad pathway in association
with APT under standard cell culture condition, hMDDCs displayed
the increase in alkaline phosphatase activity in SCD pathology. The
relevance of APT activity as a surrogate marker for BMP-Smads
expression has been demonstrated in various human derived cell
culture [20]. Thus increased APT activity would probably be useful
marker for assessing the status of the pathological condition in SCD
with orthopedic complications, as during osteogenesis ascorbic
acid and phosphate are required in high demand due to collagen
I synthesis and hydroxyapatite crystal formation [21].
Apoptosis is a highly regulated cellular process that maintains
tissue homeostasis and programmed cell death has long been recognized as an important mechanism of normal development as
compared to pathological conditions. However, uncontrolled apoptosis is marked by the disease progression. Therefore, understanding the mechanisms regulating cell growth and apoptosis can help
explain the molecular pathogenesis of Ortho + SS. Several lines of
evidence suggest that the activity of BMPs is associated with devel-
opmentally regulated apoptosis [22]. However, the mechanism of
BMP6 mediated cell death remained unexplored in SCD pathology.
Here, we demonstrate that exogenous BMP6, through Smad signaling pathway, induces the apoptotic cell death of hMDDCs in SCD
patients as compared to healthy subjects. Further we also examined the non-stimulated apoptosis in hMDDCs from SCD patients
and remarkably observed significantly higher apoptotic nuclei as
compared to BMP6 induced apoptosis. Based on these differences
in hMDDCs susceptibility for apoptosis, it is concluded that endogenous BMPs mediated killing are pronounced and SCD pathology
sensitizes the apoptosis process in hMDDCs as compared to
healthy subjects. These results suggest that, an additional mechanism involved in the antiproliferative effect of BMPs is to induce
apoptosis in normal human MDDCs when the cells are exposed
to high doses of BMPs as compared to patients, and secondly the
marginally reduced apoptosis in hMDDCs from SCD patients may
contribute to the initiation and/or progression of orthopedic complications in these patients. These results further suggest that
Ortho + SS is caused by different mechanisms in patients; a unique
mechanism involving the BMP signaling pathway responsible for
normal apoptosis in hMDDCs is induced in SCD patients but not
in healthy subjects and most likely proapoptotic effect of BMP6
may be due to an increase in the nuclear translocation of Smad
proteins, indicating the involvement of a canonical pathway.
Although, our observation of induced apoptosis is in accordance
with earlier evidence that BMPs participate in regulating cell
death. It has been shown that BMP-2, BMP-4, BMP-6 and BMP-7
are triggering signals for various programmed cell death [23].
However, the mechanism by which BMPs trigger apoptosis and
the intracellular signaling pathway operating between BMPs remains largely unknown. Collectively, our results suggest a close
association in SCD pathology with orthopedic complication and
expression of induced BMP signals activation in association with
Smads and APT. The authors conclude that BMP-6 may contribute
in the pathology of SCD with orthopedic complications.
5. Conclusion
This is the first report on the expression of BMP-Smad signaling
components along with APT in hMDDCs during Ortho + SS. We
found that BMP6, Smad1, Smad2 and APT mRNA and protein
expression were significantly higher in pathological condition. Further, exogenous BMP6 induces apoptosis in SCD pathology with
orthopedic complication. Taken together, our data significantly
shows correlation between transcriptional and translational regulation of BMP6 with SCD pathology and characteristically marked
by the augmented BMP signaling by a canonical Smad-dependent
signaling cascade during progression of disease. However, longterm follow-up of the patients is needed to determine the possible
functional role of increased expression of BMP6 and other signaling
components.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.05.029.
References
[1] K.J. Gordon, G.C. Blobe, Role of transforming growth factor-beta superfamily
signaling pathways in human disease, Biochim. Biophys. Acta 1782 (2008)
197–228.
[2] A. Almeida, I. Roberts, Bone involvement in sickle cell disease, Br. J. Haematol.
129 (2005) 482–490.
[3] M. Wan, X. Cao, BMP signaling in skeletal development, Biochem. Biophys. Res.
Commun. 328 (2005) 651–657.
K. Abhishek et al. / Biochemical and Biophysical Research Communications 396 (2010) 950–955
[4] R. Derynck, Y.E. Zhang, Smad-dependent and Smad-independent pathways in
TGF-beta family signaling, Nature 425 (2003) 577–584.
[5] A. Hartung, K. Bitton-Worms, M.H. Rechtman, et al., Different routes of bone
morphogenic protein (BMP) receptor endocytosis influence BMP signaling,
Mol. Cell Biol. 26 (2006) 7791–7805.
[6] D. Wotton, R.S. Lo, S. Lee, J. Massague, A smad transcriptional corepressor, Cell
97 (1999) 29–39.
[7] J. Massague, J. Seoane, D. Wotton, Smad transcription factors, Genes Dev. 19
(2005) 2783–2810.
[8] T. Nakaoka, K. Gonda, T. Ogita, et al., Inhibition of rat vascular smooth muscle
proliferation in vitro and in vivo by bone morphogenetic protein-2, J. Clin.
Invest. 100 (1997) 2824–2832.
[9] Y. Yamamura, X. Hua, S. Bergelson, H.F. Lodish, Critical role of SMADs and AP-1
complex in transforming growth factor beta-dependent apoptosis, J. Biol.
Chem. 275 (2000) 36295–36302.
[10] R.J. Wordinger, R. Agarwal, M. Talati, et al., Expression of bone morphogenetic
protein (BMP), BMP receptors, and BMP associated proteins in human
trabecular meshwork and optic nerve head cells and tissues, Mol. Vis. 8
(2002) 241–250.
[11] M.H. Lee, Y.J. Kim, W.J. Yoon, et al., Dlx5 specifically regulates Runx2 type II
expression by binding to homeodomain-response elements in the Runx2 distal
promoter, J. Biol. Chem. 280 (2005) 35579–35587.
[12] A.D. Gheldere, R. Ndjoko, P.L. Docquier, et al., Orthopaedic complications
associated with sickle-cell disease, Acta Orthop. Belg. 72 (2006) 741–747.
[13] N.W. Morrell, X. Yang, P.D. Upton, et al., Altered growth responses of
pulmonary artery smooth muscle cells from patients with primary
pulmonary hypertension to transforming growth factor-beta(1) and bone
morphogenetic proteins, Circulation 104 (2001) 790–795.
955
[14] M. Zhao, S.E. Harris, D. Horn, et al., Bone morphogenetic protein receptor
signaling is necessary for normal murine postnatal bone formation, J. Cell. Biol.
157 (2002) 1049–1060.
[15] J.M. Wozney, V. Rosen, A.J. Celeste, et al., Novel regulators of bone formation:
molecular clones and activities, Science 242 (1988) 1528–1534.
[16] B. Kulterer, G. Friedl, A. Jandrositz, et al., Gene expression profiling of human
mesenchymal stem cells derived from bone marrow during expansion and
osteoblast differentiation, BMC Genomics 8 (2007) 70.
[17] K. Gambaro, E. Aberdam, T. Virolle, et al., BMP-4 induces a Smad-dependent
apoptotic cell death of mouse embryonic stem cell-derived neural precursors,
Cell Death Differ. 13 (2006) 1075–1087.
[18] X. Yang, L. Chen, X. Xu, et al., TGF-beta/Smad3 signals repress chondrocyte
hypertrophic differentiation and are required for maintaining articular
cartilage, J. Cell. Biol. 151 (2001) 35–46.
[19] T. Arikawa, K. Omura, I. Morita, Regulation of bone morphogenetic protein-2
expression by endogenous prostaglandin E2 in human mesenchymal stem
cells, J. Cell. Physiol. 200 (2004) 400–406.
[20] M.S. Friedman, M.W. Long, K.D. Hankenson, Osteogenic differentiation of
human mesenchymal stem cells is regulated by bone morphogenetic protein6, J. Cell Biochem. 98 (2006) 538–554.
[21] N. Jaiswal, S.E. Haynesworth, A.I. Caplan, S.P. Bruder, Osteogenic differentiation
of purified, culture-expanded human mesenchymal stem cells in vitro, J. Cell
Biochem. 64 (1997) 295–312.
[22] J.E. Meredith Jr., B. Fazeli, M.A. Schwartz, The extracellular matrix as a cell
survival factor, Mol. Biol. Cell. 4 (1993) 953–961.
[23] T. Alliston, T.C. Ko, Y. Cao, et al., Repression of bone morphogenetic protein and
activin-inducible transcription by Evi-1, J. Biol. Chem. 280 (2005) 24227–
24237.