BMP Target Genes Research Articles

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.

L3MBTL1: A Polycomb Protein At the Node of Crosstalk Between the BMP4 and Hippo Signaling Pathways in Erythropoiesis.

AbstractAbstract 2296The Hippo signaling pathway, first discovered in Drosophila, is emerging as an important regulator of stem cell behavior. Upon still-unclear upstream stimuli, the hippo pathway kinase cascade phosphorylates and inhibits the function of YAP, a transcription coactivator, by inducing its cytoplasmic retention. While recent evidences indicate that inhibition of YAP affects cell fate decisions, and proliferation, in many tissues, little is known about the relevance of this pathway in hematopoiesis. However, the interaction of YAP with Smad1, identified in flies and human cells (Alarcon C. et al. Cell 2009), prevents smurf-mediated Smad1 degradation, potentially enhancing BMP signaling.Our ongoing studies have indentified crosstalk between the BMP4 and the Hippo pathways in hematopoietic cells, and in induced-pluripotent stem (iPS) cells that we differentiated towards the erythroid lineage. This crosstalk involves the chromatin-binding, Polycomb protein L3MBTL1, which clearly regulate the effects of BMP on the erythroid differentiation of hematopoietic stem/progenitor cells and on fetal globin gene expression.We find that the Lats2 kinase, a core component of the Hippo pathway, physically interacts with L3MBTL1 and that treatment with BMP4 or Erythropoietin decreases the expression of both proteins in various hematopoietic cells, including primary human cord blood-derived CD34+ cells. By altering L3MBTL1 levels in K562 cells, we were able to show that the L3MBTL1-Lats2 interaction enhances Lats-mediated phosphorylation and the cytoplasmic retention of YAP. Furthermore, L3MBTL1-depleted iPS cells have an enhanced smad-mediated transcriptional response; by analyzing the gene expression profile of these cells, we found increased expression of several BMP target genes (such as HHEX and ID genes), suggesting that L3MBTL1 negatively titrates the BMP4 signaling pathway at least in part by affecting YAP phosphorylation and localization. Gene Set Enrichment Analysis confirmed enrichment of many smad-related genes, and yet, these cells presented enhanced smad1/5/8 phosphorylation by WB analysis, indicating that BMP4 signaling is triggered by L3MBTL1 depletion. We also found that hematopoietic differentiation of L3MBTL1-KD iPS cells generates high-fetal globin gene expressing erythroid progeny, suggesting a role for the BMP4 signaling pathway and the targeting of L3MBTL1 in the treatment of hemoglobinopathies.To further evaluate the effect of BMP4 signaling on hematopoietic cells that lack L3MBTL1, we analyzed the stress erythroid response of L3MBTL1 KO mice: while no difference was observed at baseline in the null mice compared to wt littermates, the L3mbtl1 null mice had a more severe anemia, with increased leukocytosis, and thrombocytosis post-hydrazine (PHZ) or Epo. We found a significant increase in the colony-forming ability of the l3mbtl1 null spleen and bone marrow cells, compared to controls, as well as increased spleen size and an expansion of the spleen erythroid compartment. Thus, l3mbtl1 null hematopoietic stem cells are more sensitive to the PHZ-mediated cytokine storm, which includes BMP4. Interestingly, the L3mbtl1 null BM and spleen cells showed diminished expression of Lats2 and phospho-YAP, consistent with our in vitro findings.In conclusion, these investigations have shown that L3MBTL1 not only negatively titrates the BMP4 signaling pathway, but also provides a nodal point for crosstalk between the BMP4 and Hippo signaling pathways in erythropoiesis. Thus, these data provide insights into possible novel treatments for genetic red cell disorders (such as β-thalassemia) and for acquired bone marrow failure syndromes such as Epo-resistant anemia. Disclosures:Levine:Agios Pharmaceuticals: Research Funding.

TGFβ Signaling Regulates Hepcidin Gene Expression

AbstractAbstract 992 Introduction:Hepcidin regulates iron metabolism by reducing duodenal iron absorption and iron release from macrophages and hepatocytes. In inflammatory states, including infection, neoplasia, and heart failure, cytokines induce hepcidin synthesis leading to the development of anemia of inflammation. The regulation of hepcidin gene expression by bone morphogenetic proteins (BMPs), members of the TGFβ family of growth factors, has been extensively investigated. In contrast, less is known about the regulation of hepcidin gene expression by other stimuli, including TGFβ itself. Although TGFβ expression is increased in inflammatory states, the role of TGFβ in the induction of hepcidin gene expression is controversial. To further elucidate the role TGFβ in iron metabolism, we investigated the regulation of hepcidin gene expression in the hepatoma cell line, HepG2. Methods:HepG2 cells were incubated with TGFβ (0.1, 0.5, 1, 2.5, and 5 ng/ml) for varying durations. RNA was extracted for measurement of levels of mRNAs encoding hepcidin, PAI-1 (a TGFβ-target gene), and Id-1 (a BMP-target gene). Cellular proteins were extracted to measure levels of phosphorylated TGFβ-responsive SMADs (using antibodies directed against phosphorylated SMAD2 or SMAD3) and levels of phosphorylated BMP-responsive SMADs (using antibodies directed to phosphorylated SMADs 1 and 5, SMAD1/5). The mechanisms by which TGFβ regulates hepcidin were investigated by pretreating cells with cycloheximide, an inhibitor of protein synthesis (50 μg/mL); Noggin (250 ng/mL) or LDN-193189 (100 nM), inhibitors of BMP signaling; or SB-431542 (5 μM), an inhibitor of the TGFβ type 1 receptor, Alk5. In additional experiments, HepG2 cells were transfected with an siRNA directed against Alk5, 72 hours before exposure to TGFβ. Results:In HepG2 cells, TGFβ induced hepcidin gene expression in a time- and dose-dependent manner: hepcidin mRNA levels were maximal at 2 hours after stimulation with TGFβ (1 ng/ml) and declined thereafter. Incubation of HepG2 cells increased PAI-1 and Id-1 mRNA levels, although increased PAI-1 mRNA levels persisted for at least 8 hours whereas Id-1 mRNA levels peaked at 2 hours. Cycloheximide did not block the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, or Id-1. TGFβ induced phosphorylation of SMADs 2 and 3, as well as SMAD1/5. Pretreatment of HepG2 cells with LDN-193189 (at concentrations that inhibit all four BMP type I receptors, as well as Alk1 which is a target of both BMPs and TGFβ) did not block the ability of TGFβ to induce hepcidin or Id-1 gene expression or phosphorylation of SMADs 2, 3, or 1/5. Pretreatment with Noggin gave similar results. Inhibition of Alk5 with SB-421542 blocked the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, and Id-1, as well as phosphorylation of SMADs 2, 3, or 1/5. TGFβ-stimulated hepcidin gene expression was inhibited by siRNA-mediated knockdown of Alk5. Conclusion:In HepG2 cells, TGFβ induces hepcidin gene expression via a mechanism which requires Alk5. Although, in addition to phosphorylation of SMADs 2 and 3, TGFβ induces phosphorylation of BMP-responsive SMADs, the failure of cycloheximide to inhibit the induction of hepcidin gene expression by TGFβ suggests that synthesis of BMPs is not required. Moreover, the inability of LDN-193189 to inhibit TGFβ-stimulated hepcidin gene expression suggests against a role for activation of Alk1 by TGFβ. Taken together our findings suggest that TGFβ stimulates hepcidin gene expression via a mechanism that requires Alk5 and may be mediated by signaling either via SMADs 2 and 3 or SMAD1/5. Targeting the regulation of hepcidin gene expression by TGFβ may offer a novel therapeutic approach to the anemia of inflammation. Disclosures:No relevant conflicts of interest to declare.

Abstract 5089: Evaluation of BMP4 and BMP7 induced gene expression changes in breast cancer cells reveals time-dependent transcription patterns and a common synexpression group of genes

Abstract Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily of growth factors. They are well-known for their key roles in the regulation of various developmental processes and bone formation, but in recent years, evidence has also accumulated of their crucial contribution in tumor biology. In particular, BMP4 and BMP7 have been implicated in breast cancer pathogenesis. However, little is known about BMP target genes in the context of tumor cells. Here, we used whole-genome microarrays to explore the effects of BMP4 and BMP7 treatment on global gene expression levels in seven breast cancer cell lines during a 6-point time series. Several complementary methods, including hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data, were used for data analysis. Both BMP ligands had a strong influence on gene expression levels, although the response to BMP4 treatment was more pronounced as compared to BMP7. As might have been expected, the cellular functions most strongly affected by the stimulation of BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, where the regulation of gene expression and signal transduction was followed by changes in transcripts involved in metabolism and cell proliferation. Hierarchical clustering analysis revealed distinct differences in the response of individual cell lines to BMPs, but also underlined a common synexpression group of genes for both ligands. Remarkably, the majority of the genes within these synexpression groups were shared by the two ligands, and thus these genes are likely to represent the core molecular responses common to BMP4 and BMP7 signaling pathways. Taken together, our data demonstrate that BMP signaling has a substantial effect on gene transcription in breast cancer cells and that the cellular functions affected follow a logical temporal pattern. Our results also reveal components of the cellular transcriptional response that are common to BMP4 and BMP7. Finally, our study provides a list of putative novel BMP target genes relevant in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5089. doi:1538-7445.AM2012-5089

In This Issue

HomeCirculation ResearchVol. 110, No. 4In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published17 Feb 2012https://doi.org/10.1161/RES.0b013e31824d9f4bCirculation Research. 2012;110:523Dll1 Controls Vascular Branching (p 530)Notch ligand Dll1 is an extravascular cue that tells vessels where to branch, report Napp et al.Download figureDownload PowerPointDuring angiogenesis, the branching of new vessels begins when specific cells in the vessel wall become tip cells. The expression of the Notch ligand, Dll4, defines tip cells and prevents neighboring cells from adopting the same fate. Dll4 is induced by the external growth factor VEGF (vascular endothelial growth factor), which then prompts sprouting of the vessel led by the tip cell. But VEGF does not work alone. Napp et al now show that Notch ligand, Dll1, is also essential for tip cell selection. The team studied mice that had a heterozygous deletion of Dll1 and found that in the newborn retina, a site of abundant angiogenesis, there were fewer tip cells and fewer vessel branches. Expression analysis revealed that Dll1 was never found in the developing vessel's endothelial cells themselves but rather was immediately adjacent to the vessels at the sites of tip cell formation. In vitro studies showed that recombinant Dll1 could induce human endothelial cells to upregulate tip cell–specific genes, including Dll4. Understanding the molecular mechanisms of angiogenesis has far-reaching implications in areas such as development, wound healing, and tumor formation.Left-Right Asymmetry in Vascular Cells (p 551)Given a choice of direction, vascular mesenchymal cells turn right and reveal an inherent asymmetry, say Chen et al.Download figureDownload PowerPointAlthough animals might appear to be symmetrical on the outside, their innards reveal a striking left-right asymmetry in both the positioning of organs and the structure of the organs themselves. In the early embryo, the beating of cilia on the surface of certain cells sets up left-right asymmetry by washing molecules in one direction over neighboring cells. This model implies that cells themselves have an inherent left-right asymmetry, but the random orientation of cells in culture makes testing that theory rather difficult. Chen et al noticed that vascular mesenchymal cells (VMCs) form distinct patterns when grown on plates striped with adherent and nonadherent surfaces. Closer inspection revealed that as proliferating VMCs filled the adherent stripes and were forced to venture toward nonadherent stripes, they instinctively turned right, eventually leading to the observed patterns. This preference was dependent on intracellular stress fiber formation and was not unique to VMCs; 1 other cell type, ST2, showed a left turn preference, whereas 2 other cell types showed no preference. These findings could be important not only in studies of development but also in tissue engineering, say the team.Bmp and Myocardial Differentiation (p 578)de Pater et al have determined when and where BMP is needed during heart development.Download figureDownload PowerPointDespite its name, bone morphogenetic protein (BMP) is required for differentiation of a variety of tissues, including the myocardium. In vitro studies of cardiomyocyte differentiation, however, have suggested seemingly contradictory roles for BMP. Pater et al have now set things straight, in the zebrafish at least. They showed that although BMP signaling is present in cardiac progenitor cells in the early fish embryo and is required for induction of cardiomyocytes, it is later detrimental to heart development and is downregulated. The team showed that inhibition of BMP signaling between 10 and 24 hours after fertilization reduced the numbers of atrial and ventricular cardiomyocytes, but inhibition of BMP after 24 hours had no effect. Once cardiomyocyte differentiation was under way, BMP activity in the embryo was normally inhibited by a signaling factor called Smad6. This reduction in BMP activity was essential for proper morphogenesis of the heart, because reactivation led to ectopic expression of BMP-target genes and impaired chamber formation. Understanding precisely when and where BMP is required should help researchers improve their in vitro methods for cardiomyocyte differentiation of embryonic or induced pluripotent stem cells.Written by Ruth Williams Previous Back to top Next FiguresReferencesRelatedDetails February 17, 2012Vol 110, Issue 4 Advertisement Article InformationMetrics © 2012 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31824d9f4b Originally publishedFebruary 17, 2012 PDF download Advertisement

Bone morphogenetic protein-3b (BMP-3b) inhibits osteoblast differentiation via Smad2/3 pathway by counteracting Smad1/5/8 signaling

Despite the involvement of BMP-3b (also called GDF-10) in osteogenesis, embryogenesis and adipogenesis, the functional receptors and intracellular signaling of BMP-3b have yet to be elucidated. In the present study, we investigated the cellular mechanism of BMP-3b in osteoblast differentiation using mouse myoblastic C2C12 cells. BMP-3b stimulated activin/TGF-β-responsive promoter activities. The stimulatory actions of BMP-3b on activin/TGF-β-responsive activities were suppressed by co-treatment with BMP-2. BMP-responsive promoter activities stimulated by BMP-2 were significantly inhibited by treatment with BMP-3b. BMP-3b suppressed the expression of osteoblastic markers including Runx2, osteocalcin and type-1 collagen induced by BMP-2, -4, -6 and -7. BMP-2-induced Smad1/5/8 phosphorylation and mRNA levels of the BMP target gene Id-1 were suppressed by co-treatment with BMP-3b, although BMP-3b failed to activate Smad1/5/8 signaling. Of interest, the BMP-3b suppression of BMP-2-induced Id-1 expression was not observed in cells overexpressing Smad4 molecules. On the other hand, BMP-3b directly activated Smad2/3 phosphorylation and activin/TGF-β target gene PAI-1 mRNA expression, while BMP-2 suppressed BMP-3b-induced Smad2/3 signal activation. BMP-2 inhibition of BMP-3b-induced PAI-1 expression was also reversed by overexpression of Smad4. Analysis using inhibitors for BMP–Smad1/5/8 pathways revealed that these BMP-3b effects were mediated via receptors other than ALK-2, -3 and -6. Furthermore, results of inhibitory studies using extracellular domains for BMP receptor constructs showed that the activity of BMP-3b was functionally facilitated by a combination of ALK-4 and ActRIIA. Collectively, BMP-3b plays an inhibitory role in the process of osteoblast differentiation, in which BMP-3b and BMP-2 are mutually antagonistic possibly by competing with the availability of Smad4.

Analysis of BMP4 and BMP7 signaling in breast cancer cells unveils time-dependent transcription patterns and highlights a common synexpression group of genes

BackgroundBone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors. They are known for their roles in regulation of osteogenesis and developmental processes and, in recent years, evidence has accumulated of their crucial functions in tumor biology. BMP4 and BMP7, in particular, have been implicated in breast cancer. However, little is known about BMP target genes in the context of tumor. We explored the effects of BMP4 and BMP7 treatment on global gene transcription in seven breast cancer cell lines during a 6-point time series, using a whole-genome oligo microarray. Data analysis included hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data.ResultsBoth ligands had a strong effect on gene expression, although the response to BMP4 treatment was more pronounced. The cellular functions most strongly affected by BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, with regulation of gene expression and signal transduction leading to changes in metabolism and cell proliferation. Hierarchical clustering revealed distinct differences in the response of individual cell lines to BMPs, but also highlighted a synexpression group of genes for both ligands. Interestingly, the majority of the genes within these synexpression groups were shared by the two ligands, probably representing the core molecular responses common to BMP4 and BMP7 signaling pathways.ConclusionsAll in all, we show that BMP signaling has a remarkable effect on gene transcription in breast cancer cells and that the functions affected follow a logical temporal pattern. Our results also uncover components of the common cellular transcriptional response to BMP4 and BMP7. Most importantly, this study provides a list of potential novel BMP target genes relevant in breast cancer.

BMP activated Smad signaling strongly promotes migration and invasion of hepatocellular carcinoma cells

Several of the different bone morphogenetic proteins (BMPs) are involved in development and progression of specific tumors. For hepatocellular carcinoma (HCC) only BMP4 and BMP6 are described to be important for carcinogenesis. However, up to now neither the influence of other BMPs on tumor progression, nor the responsible signaling pathways to mediate target gene expression in HCC are known.In order to characterize BMP expression pattern in HCC cell lines, we performed RT-PCR analysis and revealed enhanced expression levels of several BMPs (BMP4, 6, 7, 8, 9, 10, 11, 13 and 15) in HCC. Thus, we treated HCC cells with the general BMP inhibitors chordin and noggin to determine the functional relevance of BMP overexpression and observed decreased migration and invasion of HCC cells. A cDNA microarray of noggin treated HCC cells was performed to analyze downstream targets of BMPs mediating these oncogenic functions. Subsequent analysis identified collagen XVI as ‘Smad signaling specific’ and nidogen-2 as ‘MAPK/ERK signaling specific’ BMP-target genes. To examine which signaling pathway is mainly responsible for the oncogenic role of BMPs in HCC, we treated HCC cells with dorsomorphin to determine the influence of BMP activated Smad signaling. Interestingly, also migratory and invasive behavior of dorsomorphin treated HCC cells was diminished.In summary, our findings demonstrate enhanced expression levels of several BMPs in HCC supporting enhanced migratory and invasive phenotype of HCC cells mainly via activation of Smad signaling.

Effects of intermittent parathyroid hormone treatment on osteoprogenitor cells in postmenopausal women

Intermittent parathyroid hormone (PTH) 1-34 treatment stimulates bone formation, but the molecular mechanisms mediating this effect have not been previously studied in humans. Thus, we used magnetic activated cell sorting to isolate hematopoietic lineage negative (lin-)/alkaline phosphatase positive (AP+) osteoprogenitor cells from bone marrow of 20 postmenopausal women treated with PTH (1-34) for 14 days and 19 control subjects. Serum PINP and CTX increased in PTH-treated subjects (by 97% and 30%, respectively, P<0.001). Bone marrow lin-/AP+ cells from PTH-treated subjects showed an increase in the RANKL/OPG mRNA ratio (by 7.5-fold, P=0.011) and in the mRNAs for c-fos (a known PTH-responsive gene, by 42%, P=0.035) and VEGF-C (by 57%, P=0.046). Gene Set Enrichment Analysis (GSEA, testing for changes in pre-specified pathways) demonstrated that PTH had no effect on osteoblast proliferation, apoptosis, or differentiation markers. However, PTH treatment resulted in a significant decrease (GSEA P-value, 0.005) in a panel of BMP target genes in the lin-/AP+ cells. Our findings thus identify several future directions for studying mechanisms of PTH action in humans. First, given the increasing evidence that PTH induces angiogenesis, the role of increased VEGF-C production by bone marrow osteoprogenitor cells in mediating this effect and the anabolic response to PTH warrants further study. Second, while the observed inhibition of BMP target gene expression by PTH is not consistent with the anabolic effects of PTH on bone and requires further validation, these data do generate the hypothesis that an inhibition of BMP signaling by PTH may, over time, limit the availability of mature osteoblasts on bone surfaces and thereby contribute to the observed waning of the anabolic response to PTH.

Direct visualization of Smad1/5/8‐mediated transcriptional activity identifies podocytes and collecting ducts as major targets of BMP signalling in healthy and diseased kidneys

Bone morphogenetic protein 7 (BMP7) is a key determinant of renal response to injury, exhibiting strong protective as well as regenerative potential in a variety of experimental models. In vitro, beneficial effects of stimulation with BMP7 and other BMPs have been observed in many renal cell types. Still, it remains poorly understood which cells in the native kidney actually respond to BMPs in health and disease. Here, we report the use of BRE:gfp mice expressing green fluorescent protein (GFP) under the control of a pSmad1/5/8-specific BMP-responsive element (BRE) to directly visualize the spatiotemporal distribution of transcriptional activity downstream of canonical BMP signalling in healthy kidneys and in two distinct models of kidney disease. BRE-GFP signal coincided with expression of endogenous BMP target genes but, surprisingly, it was much more restricted than expected from the widespread distribution of pSmad1/5/8, a classical component of canonical BMP signal tranduction. BRE-GFP was mainly present in podocytes and collecting duct cells, and both glomerular and medullary BRE-GFP decreased following ischaemia-reperfusion injury as well as following unilateral ureteric obstruction, together with decreased BMP7, pSmad1/5/8 and BMP target gene expression. Remarkably, however, BRE-GFP was increased in injured proximal tubules in association with up-regulation of BMP receptors ALK2 and ALK3. Thus, native BMP transcriptional activity is much more restricted than previously suggested based on pSmad1/5/8 detection alone, and its response to injury varies according to cell type and nephron segment.

HMGB factors are required for posterior digit development through integrating signaling pathway activities

The chromatin factors Hmgb1 and Hmgb2 have critical roles in cellular processes, including transcription and DNA modification. To identify the function of Hmgb genes in embryonic development, we generated double mutants of Hmgb1;Hmgb2 in mice. While double null embryos arrest at E9.5, Hmgb1(-/-) ; Hmgb2(+/-) embryos exhibit a loss of digit5, the most posterior digit, in the forelimb. We show that Hmgb1(-/-) ; Hmgb2(+/-) forelimbs have a reduced level of Shh signaling, as well as a clear downregulation of Wnt and BMP target genes in the posterior region. Moreover, we demonstrate that hmgb1 and hmgb2 in zebrafish embryos enhance Wnt signaling in a variety of tissues, and that double knockdown embryos have reduced Wnt signaling and shh expression in pectoral fin buds. Our data show that Hmgb1 and Hmgb2 function redundantly to enhance Wnt signaling in embryos, and further suggest that integrating Wnt, Shh, and BMP signaling regulates the development of digit5 in forelimbs.

Real-time PCR analysis of mechanical strain and BMPs in human periosteal cells: an in vitro model of entheseal stress

Background and objectivesEnthesitis with new cartilage and bone formation, which leads to joint ankylosis, is a hallmark of spondyloarthritis (SpA). Ankylosis is thought to occur initially in entheseal sites, however,...

Dorsal–Ventral patterning: Crescent is a dorsally secreted Frizzled-related protein that competitively inhibits Tolloid proteases

In Xenopus, dorsal–ventral (D–V) patterning can self-regulate after embryo bisection. This is mediated by an extracellular network of proteins secreted by the dorsal and ventral centers of the gastrula. Different proteins of similar activity can be secreted at these two poles, but under opposite transcriptional control. Here we show that Crescent, a dorsal protein, can compensate for the loss of Sizzled, a ventral protein. Crescent is a secreted Frizzled-Related Protein (sFRP) known to regulate Wnt8 and Wnt11 activity. We now find that Crescent also regulates the BMP pathway. Crescent expression was increased by the BMP antagonist Chordin and repressed by BMP4, while the opposite was true for Sizzled. Crescent knock-down increased the expression of BMP target genes, and synergized with Sizzled morpholinos. Thus, Crescent loss-of-function is compensated by increased expression of its ventral counterpart Sizzled. Crescent overexpression dorsalized whole embryos but not ventral half-embryos, indicating that Crescent requires a dorsal component to exert its anti-BMP activity. Crescent protein lost its dorsalizing activity in Chordin-depleted embryos. When co-injected, Crescent and Chordin proteins greatly synergized in the dorsalization of Xenopus embryos. The molecular mechanism of these phenotypes is explained by the ability of Crescent to inhibit Tolloid metalloproteinases, which normally degrade Chordin. Enzyme kinetic studies showed that Crescent was a competitive inhibitor of Tolloid activity, which bound to Tolloid/BMP1 with a KD of 11 nM. In sum, Crescent is a new component of the D–V pathway, which functions as the dorsal counterpart of Sizzled, through the regulation of chordinases of the Tolloid family.

Bone Morphogenetic Protein Type II Receptor Mutations Causing Protein Misfolding in Heritable Pulmonary Arterial Hypertension

More than 70% of cases of heritable pulmonary arterial hypertension are due to heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor (BMPR-II), a receptor for the transforming growth factor-β/BMP superfamily. Among the many mutations identified, some involve substitution of cysteine residues in the ligand-binding domain or the kinase domains of BMPR-II. These mutants are characterized by retention within the endoplasmic reticulum. This retention causes loss of function in terms of phosphorylation of downstream Smad1, Smad5, and Smad8 and the transcription of BMP target genes. The retention has a dominant negative effect on BMP signaling because it also impairs trafficking of the associated type I receptor. Studies suggest a more severe phenotype in patients with this class of mutation. We have shown that trafficking of cysteine-substituted mutants can be partially restored in the presence of chemical chaperones. Restoration of cell surface expression of ligand-binding domain mutants leads to partial rescue of BMP signaling and suggests that small-molecule pharmacological chaperones may be a therapeutic option in these patients.

P38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

Coordination of dorsoventral axis formation by multiple heparan sulfate proteoglycans

Heparan sulfate proteoglycans are essential mediators of multiple cell‐cell signaling pathways during embryonic development. To better understand how glypicans (GPCs) ‐ a membrane anchored subfamily of heparan sulfate core proteins ‐ function in zebrafish development, we used antisense morpholinos to individually and specifically knock down the expression of all ten GPC genes in zebrafish. Knockdown of GPC2, GPC6a, or GPC6b uniquely resulted in strong dorsoventral phenotypes between gastrulation and 24hpf. To better understand the molecular pathways in which GPCs are involved, we used a candidate gene approach to study changes in transcription of BMP, Wnt and FGF target genes because these cell‐cell signaling pathways are known to regulate dorsoventral patterning. We identified gene expression patterns that were different between each GPC knockdown, with GPC2 having a distinct affect on FGF target genes. These results were confirmed using several complimentary approaches, suggesting that GPC2, GPC6a, and GPC6b regulate distinct steps in dorsoventral patterning, an observation that adds a new dimension to our understanding of how proteoglycans work together to mediate early development.

Schnurri functions in an evolutionarily conserved mechanism for BMP target gene regulation

Schnurri functions in an evolutionarily conserved mechanism for BMP target gene regulation

Novel crosstalk to BMP signalling: cGMP-dependent kinase I modulates BMP receptor and Smad activity

Integration of multiple signals into the canonical BMP/Smad pathway poses a big challenge during the course of embryogenesis and tissue homeostasis. Here, we show that cyclic guanosine 3',5'-monophosphate (cGMP)-dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism enhancing BMP signalling. cGKI, a key mediator of vasodilation and hypertension diseases, interacts with and phosphorylates the BMP type II receptor (BMPRII). In response to BMP-2, cGKI then dissociates from the receptors, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI binds with Smad1 and the general transcription factor TFII-I to promoters of BMP target genes such as Id1 to enhance transcriptional activation. Accordingly, cGKI has a dual function in BMP signalling: (1) it modulates BMP receptor/Smad activity at the plasma membrane and (2) after redistribution to the nucleus, it further regulates transcription as a nuclear co-factor for Smads. Consequently, cellular defects caused by mutations in BMPRII, found in pulmonary arterial hypertension patients, were compensated through cGKI, supporting the positive action of cGKI on BMP-induced Smad signalling downstream of the receptors.

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

TGF-β1 mediated Smad1/Smad5 signalling modulates transdifferentiation of hepatic stellate cells

Aims: The main cellular source of extracellular matrix (ECM) in the fibrotic liver are myofibroblast like cells (MFB). MFB originate from activated hepatic stellate cells (HSC) through a process called „transdifferentiation“. Both transdifferentiation and the fibrotic response of activated HSC are regulated by TGF-β1/ALK5/Smad3 signalling. In addition an involvement of TGF-β1/ALK1/Smad1 in these processes has been postulated [1]. Methods and Results: During transdifferentiation of primary HSC into MFB, Smad1/Smad5 proteins are highly phosphorylated at their C-termini. Inhibition of Smad1/Smad5-signalling using a specific compound (Compound C; [2]) causes complete block of transdifferentiation. Smad1/Smad5 are activated by TGF-β1-mediated phosphorylation in a time- and concentration-dependent manner in quiescent and activated HSC – and similar to Smad3– not in MFB. In contrast to other cell types this activation is independent of ALK5. In addition, we found a strong activation of the „classical“ BMP-target gene, i.e. Runx2, by TGF-β1 in HSC. In line with these findings, Runx2 has been shown previously to regulate TIMP-1 expression in HSC [3]. Conclusions: We show here for the first time that Smad1/Smad5-regulated signalling is critical for activation/transdifferentiation of HSC. This may be at least in part mediated by TGF-β1 in a fashion independent of ALK5. Moreover, we conclude that TGF-β1/Smad1/Smad5 regulate ECM homeostasis through induction of Runx2.