BMP Target Research Articles

Integration of BMP/Wnt signaling to control clonal growth of limbal epithelial progenitor cells by niche cells

Both BMP and Wnt signaling control stem cells in bulge/dermal papilla, intestinal crypt, and bone marrow. To explore their roles in the limbal niche, which govern corneal epithelial homeostasis, we established an in vitro model of sphere growth by reunion between single limbal epithelial progenitor cells (LEPCs) and aggregates of limbal niche cells (LNCs) in 3D Matrigel. Compared to LEPCs alone, spheres formed by LEPC+LNC exhibited higher clonal growth and less corneal epithelial differentiation. Furthermore, pSmad1/5/8 was in the nucleus of LEPCs, but not LNCs, and correlated with upregulation of BMP1, BMP3, BMP4, all three BMP receptors, and BMP target genes. Inactivation of BMP signaling in LNCs was correlated with upregulation of noggin preferentially expressed by LNCs. Additionally, β-catenin was stabilized in the perinuclear cytoplasm in LEPCs and correlated with upregulation of Wnt7A and FZD5 preferentially expressed by LEPCs. Inactivation of Wnt signaling in LNCs was correlated with upregulation of DKK1/2 by LNCs. Addition of XAV939 that expectedly downregulated perinuclear β-catenin in LEPCs led to significant reduction of epithelial clonal growth, but upregulated all three BMP receptors and downregulated LNC-derived noggin, resulting in activation of BMP signaling in LNCs. Addition of noggin that expectedly downregulated nuclear localization of pSmad1/5/8 in LEPCs led to nuclear localization of β-catenin in larger LEPCs but membrane relocation of β-catenin in smaller LEPCs and significant upregulation of DKK1/2. Hence, balancing acts between Wnt signaling and BMP signaling exist not only within LEPCs but also between LEPCs and LNCs to regulate clonal growth of LEPCs.

Postsynaptic glutamate receptors regulate local BMP signaling at theDrosophilaneuromuscular junction

Effective communication between pre- and postsynaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. Here, we report that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlated with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis.

Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

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.

Inverse regulation of target genes at the brink of the BMP morphogen activity gradient

BMP-dependent patterning in the Drosophila melanogaster wing imaginal disc serves as a paradigm to understand how morphogens specify cell fates. The observed profile of the transcriptional response to the graded signal of BMP relies upon two counter-active gradients of pMad and Brinker (Brk). This patterning model is inadequate to explain the expression of target genes, like vestigial and spalt, in lateral regions of the wing disc where BMP signals decline and Brk levels peak. Here, we show that in contrast to the reciprocal repressor gradient mechanism, where Brk represses BMP targets in medial regions, target expression in lateral regions is downregulated by BMP signalling and activated by Brk. Brk induces lateral expression indirectly, apparently through repression of a negative regulator. Our findings provide a model explaining how the expression of an established BMP target is differentially and inversely regulated along the anterior-posterior axis of the wing disc.

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.

Bmp Indicator Mice Reveal Dynamic Regulation of Transcriptional Response

Cellular responses to Bmp ligands are regulated at multiple levels, both extracellularly and intracellularly. Therefore, the presence of these growth factors is not an accurate indicator of Bmp signaling activity. While a common approach to detect Bmp signaling activity is to determine the presence of phosphorylated forms of Smad1, 5 and 8 by immunostaining, this approach is time consuming and not quantitative. In order to provide a simpler readout system to examine the presence of Bmp signaling in developing animals, we developed BRE-gal mouse embryonic stem cells and a transgenic mouse line that specifically respond to Bmp ligand stimulation. Our reporter identifies specific transcriptional responses that are mediated by Smad1 and Smad4 with the Schnurri transcription factor complex binding to a conserved Bmp-Responsive Element (BRE), originally identified among Drosophila, Xenopus and human Bmp targets. Our BRE-gal mES cells specifically respond to Bmp ligands at concentrations as low as 5 ng/ml; and BRE-gal reporter mice, derived from the BRE-gal mES cells, show dynamic activity in many cellular sites, including extraembryonic structures and mammary glands, thereby making this a useful scientific tool.

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

Absence of Canonical Smad Signaling in Ureteral and Bladder Mesenchyme Causes Ureteropelvic Junction Obstruction

Obstruction of the ureteropelvic junction (UPJ) is a common congenital anomaly frequently associated with ureteral defects. To study the molecular mechanisms that modulate ureteral development, we inactivated Smad4, the common Smad critical for transcriptional responses to TGF-β and Bmp signaling, in the ureteral and bladder mesenchyme during embryogenesis. Loss of canonical Smad signaling in these tissues caused bilateral UPJ obstruction and severe hydronephrosis beginning at embryonic day 17.5. Despite a reduction in quantity of ureteral smooth muscle, differentiation proceeded without Smad4, producing a less severe phenotype than Bmp4 mutants; this finding suggests that at least some Bmp4 functions in ureteral smooth muscle may be Smad-independent. The absence of canonical Smad signaling in the ureteral mesenchyme, but not in the urothelium itself, led to urothelial disorganization, highlighting the importance of mesenchymal support for epithelial development. Transcript profiling revealed altered expression in known Bmp targets, smooth muscle-specific genes, and extracellular matrix-related genes in mutant ureters before the onset of hydronephrosis. Expression of the Bmp target Id2 was significantly lower in Smad4 mutants, consistent with the observation that Id2 mutants develop UPJ obstruction. In summary, Smad4 deficiency reduces the number and contractility of ureteral smooth muscle cells, leading to abnormal pyeloureteral peristalsis and functional obstruction. The subsequent bending and luminal constriction of the ureter at the UPJ marks the transition from a functional obstruction to a more intractable physical obstruction, suggesting that early intervention for this disease may prevent more irreversible damage to the urinary tract.

Inverse regulation of target genes at the brink of the BMP morphogen activity gradient

BMP-dependent patterning in the Drosophila melanogaster wing imaginal disc serves as a paradigm to understand how morphogens specify cell fates. The observed profile of the transcriptional response to the graded signal of BMP relies upon two counter-active gradients of pMad and Brinker (Brk). This patterning model is inadequate to explain the expression of target genes, like vestigial and spalt, in lateral regions of the wing disc where BMP signals decline and Brk levels peak. Here, we show that in contrast to the reciprocal repressor gradient mechanism, where Brk represses BMP targets in medial regions, target expression in lateral regions is downregulated by BMP signalling and activated by Brk. Brk induces lateral expression indirectly, apparently through repression of a negative regulator. Our findings provide a model explaining how the expression of an established BMP target is differentially and inversely regulated along the anterior-posterior axis of the wing disc.

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.

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.

Abstract 1984: Activated BMP signalling modulates multicellular spheroid formation and reattachment of ascites-derived human epithelial ovarian cancer cells in an in vitro model of metastasis

Abstract Epithelial ovarian cancer (EOC) comprises close to 90% of ovarian tumours, and over 75% of cases are not discovered until advanced metastatic stages. Therefore, there remains a critical need to identify the mechanisms controlling late-stage spread of disease and develop more effective treatment regimes. EOC cells have the ability to form multicellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Primary human EOC cells derived from patient ascites secrete and are responsive to bone morphogenetic proteins (e.g. BMP2 and BMP4). BMPs can function in an autocrine fashion to enhance the malignant characteristics displayed by EOC cells in adherent monolayer culture. Herein, we demonstrate that EOC cells with enhanced BMP signalling form smaller, more loosely-aggregated spheroids than control cells when grown in suspension culture. Upon reattachment to standard tissue culture plastic, EOC spheroids readily attach and cells disperse from the attached spheroid; EOC spheroids with enhanced BMP signalling have an increased cell dispersion rate when compared with controls. Activated BMP signalling in EOC spheroids induces Smad1/5/8 phosphorylation, increases ID1 and ID3 proto-oncogene expression, and promotes epithelial-mesenchymal transition as evidenced by increased Snail and Slug and reduced E-cadherin markers. To identify additional target genes mediating the phenotypic changes in EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. From this study, 102 genes were significantly upregulated and 52 downregulated in primary human EOC cells and spheroids with enhanced BMP signalling. A number of these genes have been previously identified as BMP targets or members of the BMP signalling pathway, several of which are involved in negative feedback regulation (e.g. noggin, Smad6 and Smad7), as well as several genes encoding proteins associated with cellular adhesion. In addition, using the online Connectivity Map resource (www.broadinstitute.org/cmap), we revealed that the PI3K-Akt-mTOR pathway may be impacted by an activated BMP pathway in EOC cells. Indeed, BMP signalling induces phosphorylation of Akt in both adherent primary human EOC cells and multicellular spheroids. Thus, we are currently investigating whether the PI3K-Akt pathway and the targets identified by microarray analysis are required for the BMP signalling effects on EOC spheroid formation and reattachment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1984. doi:10.1158/1538-7445.AM2011-1984

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.