Related Articles

Background

TGF-β acts as a suppressor of primary tumour initiation but has been implicated as a promoter of the later malignant stages. Here associations with risk of invasive breast cancer are assessed for SNPs tagging seventeen genes in the canonical TGF-β ALK5/SMADs 2&3 and ALK1/SMADs 1&5 signalling pathways: LTBP1, LTBP2, LTBP4, TGFB1, TGFB2, TGFB3, TGFBR1(ALK5), ALK1, TGFBR2, Endoglin, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD72.

Methods

354 tag SNPs (minor allele frequency>0.05) were selected for genotyping in a staged study design using 6,703 cases and 6,840 controls from the SEARCH study. Significant associations were meta-analysed with data from the NCI Polish Breast Cancer Study (PBCS) (1,966 cases and 2,347 controls) and published data from the Breast Cancer Association Consortium (BCAC).

Results

Associations of three SNPs, tagging TGFB1 (rs1982073), TGFBR1 (rs10512263) and TGFBR2 (rs4522809) were detected in SEARCH; however associations became weaker in meta-analyses including data from PBCS and BCAC. Tumour sub-type analyses indicated that the TGFB1 rs1982073 association may be confined to increased risk of developing progesterone receptor negative (PR−) tumours (1.18 (95% CI 1.09-1.28), 4.1×10−5 (P value for heterogeneity of ORs by PR status = 2.3 × 10−4)). There was no evidence for breast cancer risk associations with SNPs in the endothelial-specific pathway utilising ALK1/SMADs 1&5 that promotes angiogenesis.

Conclusion

Common variation in the TGF-β ALK5/SMADs 2&3 signalling pathway, which initiates signalling at the cell surface to inhibit cell proliferation, might be related to risk of specific tumour sub-types.

Impact

The subtype specific associations require very large studies to be confirmed.

Endoglin, an endothelial cell-specific transforming growth factor-β (TGF-β) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-β superfamily signaling in endothelial cells through regulating the balance between two TGF-β-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-β and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-β superfamily signaling and migration in endothelial cells.

Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.

Endoglin is a transforming growth factor β (TGFβ) superfamily auxiliary receptor. We had previously shown that it suppressed prostate cancer (PCa) cell motility, and that its expression was lost during PCa progression. The mechanism by which endoglin inhibits PCa cell motility is unknown. Here we demonstrate that endoglin abrogates TGFβ-mediated cell motility, but does not alter cell surface binding of TGFβ. By measuring Smad-specific phosphorylation and Smad-responsive promoter activity, endoglin was shown to constitutively activate Smad1, with little-to-no effect upon Smad3. Knockdown of Smad1 increased motility and abrogated endoglin's effects. As type I activin receptor-like kinases (ALKs) are necessary for Smad activation, we went on to show that knockdown of ALK2, but not TGFβRI (ALK5), abrogated endoglin-mediated decreases in cell motility and constitutively active ALK2 was sufficient to restore a low-motility phenotype in endoglin deficient cells. These findings provide the first evidence that endoglin decreases PCa cell motility through activation of the ALK2-Smad1 pathway.

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor–like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Bone morphogenetic protein (BMP) signaling is required for endochondral bone formation. However, whether or not the effects of BMPs are mediated via canonical Smad pathways or through noncanonical pathways is unknown. In this study we have determined the role of receptor Smads 1, 5 and 8 in chondrogenesis. Deletion of individual Smads results in viable and fertile mice. Combined loss of Smads 1, 5 and 8, however, results in severe chondrodysplasia. Smad1/5CKO (cartilage-specific knockout) mutant mice are nearly identical to Smad1/5CKO;Smad8-/- mutants, indicating that Smads 1 and 5 have overlapping functions and are more important than Smad8 in cartilage. The Smad1/5CKO phenotype is more severe than that of Smad4CKO mice, challenging the dogma, at least in chondrocytes, that Smad4 is required to mediate Smad signaling through BMP pathways. The chondrodysplasia in Smad1/5CKO mice is accompanied by imbalances in cross-talk between the BMP, FGF and Ihh/PTHrP pathways. We show that Ihh is a direct target of BMP pathways in chondrocytes, and that FGF exerts antagonistic effects on Ihh expression. Finally, we tested whether FGF exerts its antagonistic effects directly through Smad linker phosphorylation. The results support the alternative conclusion that the effects of FGFs on BMP signaling are indirect in vivo.

TGF-β is the primary inducer of extracellular matrix (ECM) proteins in scleroderma (systemic sclerosis, SSc). Previous studies indicate that in a subset of SSc fibroblasts TGF-β signaling is activated via elevated levels of activin receptor-like kinase (ALK) 1 and phosphorylated Smad1 (pSmad1). The goal of this study was to determine the role of endoglin/ALK1 in TGF-β/Smad1 signaling in SSc fibroblasts. In SSc fibroblasts, increased levels of endoglin correlated with high levels of pSmad1, collagen, and connective tissue growth factor (CCN2). Endoglin depletion via siRNA in SSc fibroblasts inhibited pSmad1 but did not affect pSmad2/3. Following endoglin depletion mRNA and protein levels of collagen and CCN2 were significantly decreased in SSc fibroblasts but remained unchanged in normal fibroblasts. ALK1 was expressed at similar levels in SSc and normal fibroblasts. Depletion of ALK1 resulted in inhibition of pSmad1 and a moderate but significant reduction of mRNA and protein levels of collagen and CCN2 in SSc fibroblasts. Furthermore, constitutively high levels of endoglin were found in complexes with ALK1 in SSc fibroblasts. Overexpression of constitutively active ALK1 (caALK1) in normal and SSc fibroblasts led to a moderate increase of collagen and CCN2. However, caALK1 potently induced endothelin 1 (ET-1) mRNA and protein levels in SSc fibroblasts. Additional experiments demonstrated that endoglin and ALK1 mediate TGF-β induction of ET-1 in SSc and normal fibroblasts. In conclusion, this study has revealed an important profibrotic role of endoglin in SSc fibroblasts. The endoglin/ALK1/Smad1 pathway could be a therapeutic target in patients with SSc if appropriately blocked.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

Hepcidin, a key regulator of iron metabolism, is activated by bone morphogenetic proteins (BMPs). Mice pair-fed with regular and ethanol-containing L. De Carli diets were employed to study the effect of alcohol on BMP signaling and hepcidin transcription in the liver. Alcohol induced steatosis and TGF-beta expression. Liver BMP2, but not BMP4 or BMP6, expression was significantly elevated. Despite increased BMP expression, the BMP receptor, and transcription factors, Smad1 and Smad5, were not activated. In contrast, alcohol stimulated Smad2 phosphorylation. However, Smad4 DNA-binding activity and the binding of Smad4 to hepcidin promoter were attenuated. In summary, alcohol stimulates TGF-beta and BMP2 expression, and Smad2 phosphorylation but inhibits BMP receptor, and Smad1 and Smad5 activation. Smad signaling pathway in the liver may therefore be involved in the regulation of hepcidin transcription and iron metabolism by alcohol. These findings may help to further understand the mechanisms of alcohol and iron-induced liver injury.

Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes we characterized TGFβ/BMP receptor and Smad expression in immortalised ovarian surface epithelial cells (IOSE) and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signalling in ovarian cancer. Using siRNA, ligand trap, inhibitor and ligand stimulation approaches we demonstrate that BMP9 acts as a proliferative factor for IOSE and ovarian cancer cell lines, signalling predominantly via an ALK2/Smad1/Smad4 pathway rather than via ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signalling which is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that approximately 25% of epithelial ovarian cancers express BMP9 whereas normal human OSE specimens do not. Our data indicate that BMP9 signalling via ALK2 may be a novel therapeutic target in ovarian cancer.

Osteoarthritis (OA) is a disease of articular cartilage, with aging as the main risk factor. In OA, changes in chondrocytes lead to the autolytic destruction of cartilage. Transforming growth factor-β has recently been demonstrated to signal not only via activin receptor-like kinase 5 (ALK5)-induced Smad2/3 phosphorylation, but also via ALK1-induced Smad1/5/8 phosphorylation in articular cartilage. In aging cartilage and experimental OA, the ratio ALK1/ALK5 has been found to be increased, and the expression of ALK1 is correlated with matrix metalloproteinase-13 expression. The age-dependent shift towards Smad1/5/8 signalling might trigger the differentiation of articular chondrocytes with an autolytic phenotype.

Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.

Summary

Objective

Chondrocytes exhibit specific responses to BMPs and TGF-βs. The bioactivity of these growth factors is regulated by numerous mediators. In our previous study, Smad1 was found to interact with the cytoplasmic domain of the hyaluronan receptor CD44. The purpose of this study was to determine the ability of hyaluronan in the pericellular matrix to modulate the chondrocyte responses to BMP-7 or TGF-β1.

Experimental design

Nuclear translocation of Smad1, Smad2 and Smad4 was studied in bovine articular chondrocytes in response to BMP-7 and TGF-β1. The effects of matrix disruption by hyaluronidase treatment and the initiation of matrix repair by the addition of hyaluronan on the nuclear translocation of Smad proteins, Smad1 phosphorylation and luciferase expression by a CD44 reporter construct in response to BMP-7 were also studied.

Results

The disruption of the hyaluronan-dependent pericellular matrix of chondrocytes resulted in diminished nuclear translocation of endogenous Smad1 and Smad4 in response to BMP-7; however, the nuclear translocation of Smad2 and Smad4 in these matrix-depleted chondrocytes in response to TGF-β1 was not diminished. Incubation of the matrix-depleted chondrocytes with exogenous hyaluronan restored Smad1 and Smad4 nuclear translocation and increased pCD44(499)-Luc luciferase expression in response to BMP-7. Both exogenous hyaluronan and matrix re-growth enhanced by HAS2 transfection restored Smad1 phosphorylation.

Conclusions

Disruption of hyaluronan-CD44 interactions has little effect on the TGF-β responses; however, re-establishing CD44-hyaluronan ligation promotes a robust cellular response to BMP-7 by articular chondrocytes. Thus, changes in cell-hyaluronan interactions may serve as a mechanism to modulate cellular responsiveness to BMP-7.

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, “BMP-specific” Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output.

TGFβ and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1, is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGFβ pathways.

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated ‘gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have reexamined this issue in Xenopus and chick embryos using the powerful and general TGFβ inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not acount for Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFβ pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFβ-pathways should be interpreted with considerable caution.

We have previously shown that keratinocyte-specific deletion of Smad4, a TGFβ/Activin/BMP signaling mediator, results in a progressive alopecia. To further assess the molecular mechanisms of Smad4 loss-mediated alopecia, we examined expression levels of key molecules associated with hair follicle differentiation in Smad4-deleted skin. Among them, Desmoglein 4 (Dsg4) was down-regulated in Smad4-deleted skin prior to the onset of hair follicle abnormalities with gradual depletion coinciding with hair follicle degeneration. Chromatin immunoprecipitation (ChIP) assay showed that Smad4, together with the BMP mediators Smad1 and Smad5, but not the TGFβ/Activin mediators Smad2 or Smad3, bound to the Smad Binding Element (SBE) of the Dsg4 promoter. A Dsg4 reporter assay revealed that Smad4 was required for the maximal transactivation of Dsg4 in cooperation with Smad1 and Smad5. Mutating the SBE of the Dsg4 promoter abrogated Smad4 transactivation of Dsg4. Furthermore, BMP ligands, but not ligands of TGFβ and Activin, induced endogenous Dsg4 expression. Our data demonstrate that in the presence of Smad4, BMP signaling participated in transcriptional regulation of Dsg4. Thus, Smad4 loss-associated Dsg4 depletion contributed, at least in part, to hair follicles degeneration in Smad4 deficient skin.

Introduction

Rheumatoid arthritis is a chronic systemic autoimmune disease affecting peripheral joints and leading to loss of joint function. The severity and outcome of disease are dependent on the balance between inflammatory/destructive and homeostatic or repair pathways. Increasing evidence suggests a role for bone morphogenetic protein (BMP) signaling in joint homeostasis and disease.

Methods

Activation of BMP signaling in collagen-induced arthritis as a model of rheumatoid arthritis was studied by immunohistochemistry and Western blot for phosphorylated SMAD1/5 at different time points. Expression of different BMP ligands and noggin, a BMP antagonist, was determined on synovium and cartilage extracts of arthritic knees, at different time points, with quantitative polymerase chain reaction. At the protein level, BMP2 and BMP7 were studied with immunohistochemistry. Finally, the effect of anti-tumor necrosis factor-alpha (TNFα) treatment on the expression of BMP2, BMP7, and growth and differentiation factor-5 (GDF5) in synovium and cartilage of arthritic knees was investigated.

Results

A time-dependent activation of the BMP signaling pathway in collagen-induced arthritis was demonstrated with a dynamic and characteristic expression pattern of different BMP subfamily members in synovium and cartilage of arthritic knees. As severity increases, the activation of BMP signaling becomes more prominent in the invasive pannus tissue. BMP2 is present in cartilage and the hyperplastic lining layer. BMP7 is found in the sublining zone and inflammatory infiltrate. Treatment with etanercept slowed down progression of disease, but no change in expression of GDF5, BMP2, and BMP7 in synovium was found; in the cartilage, however, blocking of TNFα increased the expression of BMP7.

Conclusions

BMP signaling is dynamically activated in collagen-induced arthritis and is partly TNFα-independent. TNFα blocking increased the expression of BMP7 in the articular cartilage, possibly enhancing anabolic mechanisms. Different types of source and target cells are recognized. These data further support a role for BMP signaling in arthritis.

The mechanistic relationship between the widely used monocrotaline model of primary pulmonary hypertension and altered TGFβ family signaling due to genetic defects in the Bone Morphogenetic Protein type II receptor in affected humans has not been investigated. In this study we use fluorescent microscopy to demonstrate nuclear translocation of Smad 4 in human pulmonary arterial endothelial cell (HPAEC) cultures treated with monocrotaline pyrrole (MCTP), Bone Morphogenetic Protein (BMP) and TGFβ. While MCTP induced transient nuclear accumulation of phosphorylated Smad 1 (P-Smad 1) and phosphylated Smad 2 (P-Smad 2), only expression of P-Smad 1 was significantly altered in western blots. P-Smad 1 expression significantly increased 30 minutes following treatment with MCTP correlating with P-Smad 1 and Smad 4 nuclear translocation. Although a modest, but significant decrease in P-Smad 1 expression occurred 1 hr after treatment, expression was significantly increased at 72 hr. Evaluation of components of the signal and response pathway at 72 hours showed decreased expression of the BMP type II receptor (BMPrII), no change in TGFβ Activin Receptor-like Kinase 1 (Alk 1), no change in Smad 4 but increase in the inhibitory Smad 6, decrease in the alternate BMP signaling pathway p38MAPK but no change in the psmad1 response element ID 1. Our results suggest transient activation of Smad signaling pathways in initial MCTP endothelial cell toxicity, and a persistent dysregulation of BMP signaling. Electron microscopy of cell membrane caveoli revealed a dramatic decrease in these structures after 72 hrs. Loss of these structural elements, noted for their sequestration and inhibition of receptor activity, may contribute to prolonged alterations in BMP signaling.

Background

Bone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway.

Methodology/Principal Findings

Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression.

Conclusions/Significance

Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.

The TGF-β/BMP signaling cascades control a wide range of developmental and physiological functions in vertebrates and invertebrates. In Drosophila melanogaster, members of this pathway can be divided into a Bone Morphogenic Protein (BMP) and an Activin-ß (Act-ß) branch, where Decapentaplegic (Dpp), a member of the BMP family has been most intensively studied. They differ in ligands, receptors and transmitting proteins, but also share some components, such as the Co-Smad Medea (Med). The essential role of Med is to form a complex with one of the two activating Smads, mothers against decapentaplegic (Mad) or dSmad, and to translocate together to the nucleus where they can function as transcriptional regulators of downstream target genes. This signaling cascade underlies different mechanisms of negative regulation, which can be exerted by inhibitory Smads, such as daughters against decapentaplegic (dad), but also by the Ski-Sno family. In this work we identified and functionally analyzed a new member of the Ski/Sno-family, fussel (fuss), the Drosophila homolog of the human functional suppressing element 15 (fussel-15). fuss codes for two differentially spliced transcripts with a neuronal expression pattern. The proteins are characterized by a Ski-Sno and a SAND homology domain. Overexpression studies and genetic interaction experiments clearly reveal an interaction of fuss with members of the BMP pathway, leading to a strong repression of BMP-signaling. The protein interacts directly with Medea and seems to reprogram the Smad pathway through its influence upon the formation of functional Mad/Medea complexes. This leads amongst others to a repression of downstream target genes of the Dpp pathway, such as optomotor blind (omb). Taken together we could show that fuss exerts a pivotal role as an antagonist of BMP signaling in Drosophila melanogaster.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homeostasis, hypertrophy and repair. Here, we have examined the role of Bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor-1A (BMPR-1A) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, or disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature differentiation. BMP signalling acted to inhibit the up-regulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded differentiation. Finally, interference of normal BMP signalling after muscle damage in vivo resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

In vivo cells receive simultaneous signals from multiple extracellular ligands and must integrate and interpret them to respond appropriately. Here we investigate the interplay between pathways downstream of two transforming growth factor β (TGF-β) superfamily members, bone morphogenetic protein (BMP) and TGF-β. We show that in multiple cell lines, TGF-β potently inhibits BMP-induced transcription at the level of both BMP-responsive reporter genes and endogenous BMP target genes. This inhibitory effect requires the TGF-β type I receptor ALK5 and is independent of new protein synthesis. Strikingly, we show that Smad3 is required for TGF-β's inhibitory effects, whereas Smad2 is not. We go on to demonstrate that TGF-β induces the formation of complexes comprising phosphorylated Smad1/5 and Smad3, which bind to BMP-responsive elements in vitro and in vivo and mediate TGF-β-induced transcriptional repression. Furthermore, loss of Smad3 confers on TGF-β the ability to induce transcription via BMP-responsive elements. Our results therefore suggest that not only is Smad3 important for mediating TGF-β's inhibitory effects on BMP signaling but it also plays a critical role in restricting the transcriptional output in response to TGF-β.

BACKGROUND AND AIMS

Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)-SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently demonstrated to be impaired in Hfe knockout (Hfe−/−) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe−/− mice.

METHODS

The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice (Hfe Tg) compared with wildtype (WT) mice. Hfe−/− and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters.

RESULTS

Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, while anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe−/− mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites.

CONCLUSIONS

HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.