Target Of BMP Signaling Research Articles

Bone Morphogenetic Protein 13 Has Protumorigenic Effects on Hepatocellular Carcinoma Cells In Vitro.

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrosis and, thus, build the "soil" for hepatocarcinogenesis. Furthermore, HSCs are known to promote the progression of hepatocellular carcinoma (HCC), but the molecular mechanisms are only incompletely understood. Recently, we newly described the expression of bone morphogenetic protein 13 (BMP13) by HSCs in fibrotic liver tissue. In addition, BMP13 has mostly been studied in the context of cartilage and bone repair, but not in liver disease or cancer. Thus, we aimed to analyze the expression and function of BMP13 in HCC. Expression analyses revealed high BMP13-expression in activated human HSCs, but not in human HCC-cell-lines. Furthermore, analysis of human HCC tissues showed a significant correlation between BMP13 and α-smooth muscle actin (α-SMA), and immunofluorescence staining confirmed the co-localization of BMP13 and α-SMA, indicating activated HSCs as the cellular source of BMP13 in HCC. Stimulation of HCC cells with recombinant BMP13 increased the expression of the inhibitors of differentiation 1 (ID1) and 2 (ID2), which are known targets of BMP-signaling and cell-cycle promotors. In line with this, BMP13-stimulation caused an induced SMAD 1/5/9 and extracellular signal-regulated kinase (ERK) phosphorylation, as well as reduced expression of cyclin-dependent kinase inhibitors 1A (CDKN1A) and 2A (CDKN2A). Furthermore, stimulation with recombinant BMP13 led to increased proliferation and colony size formation of HCC cells in clonogenicity assays. The protumorigenic effects of BMP13 on HCC cells were almost completely abrogated by the small molecule dorsomorphin 1 (DMH1), which selectively blocks the intracellular kinase domain of ALK2 and ALK3, indicating that BMP13 acts via these BMP type I receptors on HCC cells. In summary, this study newly identifies stroma-derived BMP13 as a potential new tumor promotor in HCC and indicates this secreted growth-factor as a possible novel therapeutic target in HCC.

Expression of Bone Morphogenetic Protein 14 in Liver Disease and Cancer

The activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, activated HSCs also play an important role in the progression of hepatocellular cancer (HCC). Bone morphogenetic protein 14 (BMP14) is a member of the TGF-β/BMP superfamily. So far, most studies have analyzed BMP14 in the context of bone and cartilage formation and homeostasis. The aim of this study was to assess the expression and function of BMP14 in liver fibrosis and HCC. The BMP14 expression increased during the in vitro activation of primary human HSCs and also in mouse models of liver fibrosis. In human HCC, as well as non-tumorous liver tissues, there was a significant correlation between the expression of BMP14 and alpha-smooth-muscle actin (α-SMA), an established marker for HSC activation. RNAi-mediated BMP14 suppression in activated HSCs resulted in the reduced expression of the transcription factors inhibitor of differentiation 1 (ID1) and ID2, known targets of BMP signaling. Interestingly, α-SMA and collagen expression was also reduced in BMP14-depleted cells, while treatment with recombinant BMP14 induced ID1, ID2, α-SMA and collagen expression. In human HCC cell lines, treatment with recombinant BMP14 induced proliferation, migratory activity and colony formation. In summary, our data indicate activated HSCs as a major cellular source of enhanced BMP14 expression in fibrotic liver disease and HCC, and show that BMP14 exhibits pro-fibrogenic as well as pro-tumorigenic effects. Future analyses will reveal the potential of this soluble growth factor as a therapeutic target or prognostic marker for the progression of fibrosis and HCC in patients with chronic liver disease.

Brain arteriovenous malformation in hereditary hemorrhagic telangiectasia: Recent advances in cellular and molecular mechanisms.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by vessel dilatation, such as telangiectasia in skin and mucosa and arteriovenous malformations (AVM) in internal organs such as the gastrointestinal tract, lungs, and brain. AVMs are fragile and tortuous vascular anomalies that directly connect arteries and veins, bypassing healthy capillaries. Mutations in transforming growth factor β (TGFβ) signaling pathway components, such as ENG (ENDOGLIN), ACVRL1 (ALK1), and SMAD4 (SMAD4) genes, account for most of HHT cases. 10-20% of HHT patients develop brain AVMs (bAVMs), which can lead to vessel wall rupture and intracranial hemorrhages. Though the main mutations are known, mechanisms leading to AVM formation are unclear, partially due to lack of animal models. Recent mouse models allowed significant advances in our understanding of AVMs. Endothelial-specific deletion of either Acvrl1, Eng or Smad4 is sufficient to induce AVMs, identifying endothelial cells (ECs) as primary targets of BMP signaling to promote vascular integrity. Loss of ALK1/ENG/SMAD4 signaling is associated with NOTCH signaling defects and abnormal arteriovenous EC differentiation. Moreover, cumulative evidence suggests that AVMs originate from venous ECs with defective flow-migration coupling and excessive proliferation. Mutant ECs show an increase of PI3K/AKT signaling and inhibitors of this signaling pathway rescue AVMs in HHT mouse models, revealing new therapeutic avenues. In this review, we will summarize recent advances and current knowledge of mechanisms controlling the pathogenesis of bAVMs, and discuss unresolved questions.

C-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

Essential function and targets of BMP signaling during midbrain neural crest delamination

BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.

Bone morphogenetic protein and blood vessels: new insights into endothelial cell junction regulation.

BMP signaling is an important regulator of vascular development and homeostasis, and perturbations of BMP pathway components are linked to vascular disease. However, until recently BMP's broad requirements in many developmental programs delayed cause-and-effect and mechanistic studies of its vascular role in vivo. This review covers recent findings that illuminate the role of BMP signaling in endothelial cells of blood vessels, and highlights effects of BMP signaling on endothelial cell junctions and vascular barrier function. BMP signaling in endothelial cells of blood vessels is context-dependent, and can either be pro-angiogenic and promote vascular sprouting, or antiangiogenic and promote vascular homeostasis. I discuss how distinct BMP signaling inputs impact blood vessel formation and function, with emphasis on new studies that investigate how BMP signaling affects endothelial cell junctions and vascular permeability. BMP signaling is important but complex in endothelial cells of blood vessels, with multiple distinct inputs leading to opposing cellular behaviors and phenotypic outputs in ways that are poorly understood. Endothelial cell-cell junctions are a target of BMP signaling, and junction stability can be tuned in either direction by BMP inputs. Several human diseases have perturbed junctions linked to BMP signaling changes.

Oleanolic Acid Enhances Mesenchymal Stromal Cell Osteogenic Potential by Inhibition of Notch Signaling

Oleanolic acid (OA), a pentacyclic triterpenoid, has been shown to modulate multiple signaling pathways in a variety of cell linages. But the mechanisms underlying OA-mediated mesenchymal stromal cell (MSC) osteogenic differentiation are not known. In this study, we examined effects of OA on cell viability, osteogenic differentiation in MSCs, and the involvement of Notch and BMP signaling. OA induced bone marrow derived MSC differentiation towards osteoprogenitor cells and inhibited Notch signaling in a dose dependent manner. Constitutive activation of Notch signaling fully blocked OA induced MSC osteogenic differentiation. The expression level of early osteogenic marker genes, ALP, Runx2, and type I collagen, which play a critical role in MSC to osteoblast transition and servers as a downstream target of BMP signaling, was significantly induced by OA. Furthermore, BMP2 mediated MSC osteogenic differentiation was significantly enhance by OA treatment, indicating a synergistic effect between BMP2 and OA. Our results suggest that OA is a promising bioactive agent for bone tissue regeneration, and inhibition of Notch signaling is required for its osteogenic effects on MSCs.

Microarray meta-analysis identifies evolutionarily conserved BMP signaling targets in developing long bones

In vertebrates, BMP signaling has been demonstrated to be sufficient for bone formation in several tissue contexts. This suggests that genes necessary for bone formation are expressed in a BMP signaling dependent manner. However, till date no gene has been reported to be expressed in a BMP signaling dependent manner in bone. Our aim was to identify such genes. On searching the literature we found that several microarray experiments have been conducted where the transcriptome of osteogenic cells in absence and presence of BMP signaling activation have been compared. However, till date, there is no evidence to suggest that any of the genes found to be upregulated in presence of BMP signaling in these microarray analyses is indeed a target of BMP signaling in bone. We wanted to utilize this publicly available information to identify candidate BMP signaling target genes in vivo. We performed a meta-analysis of six such comparable microarray datasets. This analysis and subsequent experiments led to the identification of five targets of BMP signaling in bone that are conserved both in mouse and chick. Of these Lox, Klf10 and Gpr97 are likely to be direct transcriptional targets of BMP signaling pathway. Dpysl3, is a novel BMP signaling target identified in our study. Our data demonstrate that Dpysl3 is important for osteogenic differentiation of mesenchymal cells and is involved in cell secretion. We have demonstrated that the expression of Dpysl3 is co-operatively regulated by BMP signaling and Runx2. Based on our experimental data, in silico analysis of the putative promoter-enhancer regions of Bmp target genes and existing literature, we hypothesize that BMP signaling collaborates with multiple signaling pathways to regulate the expression of a unique set of genes involved in endochondral ossification.

MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting

Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Id proteins are critical downstream effectors of BMP signaling in human pulmonary arterial smooth muscle cells

Bone morphogenetic protein type II receptor (BMPR-II) mutations are responsible for over 70% of cases of heritable pulmonary arterial hypertension (PAH). Loss of BMP signaling promotes pulmonary vascular remodeling via modulation of pulmonary artery smooth muscle cell (PASMC) proliferation. Id proteins (Id1-4) are major downstream transcriptional targets of BMP signaling. However, the impact of BMPR-II mutation on the expression of the range of Id proteins and the contribution of individual Id proteins to abnormal PASMC function remain unclear. Human PASMCs were used to determine the expression of Id proteins (Id1-4) by real-time PCR and immunoblotting. The BMP responses in control cells were compared with PASMCs harboring BMPR-II mutations and cells in which BMPR-II was knocked down by siRNA transfection. Id3 expression in pulmonary vessels was also investigated in BMPR-II mutant mice and in patients with heritable PAH. BMP4 and BMP6, but not BMP9, induced mRNA expression of Id1, Id2, and Id3. The BMP-stimulated induction of Id1 and Id3 was markedly reduced in BMPR-II mutant PASMCs and in control PASMCs following siRNA silencing of BMPR-II. Pulmonary arteries in BMPR-II mutant mice and patients with heritable PAH demonstrated reduced levels of Id3 compared with control subjects. Lentiviral overexpression of Id3 reduced cell cycle progression and inhibited proliferation of PASMCs. Lipopolysaccharide further reduced Id3 expression in mutant PASMCs. In conclusion, Id proteins, and particularly Id1 and Id3, are critical downstream effectors of BMP signaling in PASMCs. Loss of BMPR-II function reduces the induction of Id genes in PASMCs, Id1, and Id3 regulate the proliferation of PASMCs via cell cycle inhibition, an effect that may be exacerbated by inflammatory stimuli.

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.

BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis

Expression of bone morphogenetic protein 4 (BMP4) in adipocytes of white adipose tissue (WAT) produces "white adipocytes" with characteristics of brown fat and leads to a reduction of adiposity and its metabolic complications. Although BMP4 is known to induce commitment of pluripotent stem cells to the adipocyte lineage by producing cells that possess the characteristics of preadipocytes, its effects on the mature white adipocyte phenotype and function were unknown. Forced expression of a BMP4 transgene in white adipocytes of mice gives rise to reduced WAT mass and white adipocyte size along with an increased number of a white adipocyte cell types with brown adipocyte characteristics comparable to those of beige or brite adipocytes. These changes correlate closely with increased energy expenditure, improved insulin sensitivity, and protection against diet-induced obesity and diabetes. Conversely, BMP4-deficient mice exhibit enlarged white adipocyte morphology and impaired insulin sensitivity. We identify peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) as the target of BMP signaling required for these brown fat-like changes in WAT. This effect of BMP4 on WAT appears to extend to human adipose tissue, because the level of expression of BMP4 in WAT correlates inversely with body mass index. These findings provide a genetic and metabolic basis for BMP4's role in altering insulin sensitivity by affecting WAT development.

Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts

Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts

BMP signaling regulates sympathetic nervous system development through Smad4-dependent and -independent pathways

Induction of the sympathetic nervous system (SNS) from its neural crest (NC) precursors is dependent on BMP signaling from the dorsal aorta. To determine the roles of BMP signaling and the pathways involved in SNS development, we conditionally knocked out components of the BMP pathways. To determine if BMP signaling is a cell-autonomous requirement of SNS development, the Alk3 (BMP receptor IA) was deleted in the NC lineage. The loss of Alk3 does not prevent NC cell migration, but the cells die immediately after reaching the dorsal aorta. The paired homeodomain factor Phox2b, known to be essential for survival of SNS precursors, is downregulated, suggesting that Phox2b is a target of BMP signaling. To determine if Alk3 signals through the canonical BMP pathway, Smad4 was deleted in the NC lineage. Loss of Smad4 does not affect neurogenesis and ganglia formation; however, proliferation and noradrenergic differentiation are reduced. Analysis of transcription factors regulating SNS development shows that the basic helix-loop-helix factor Ascl1 is downregulated by loss of Smad4 and that Ascl1 regulates SNS proliferation but not noradrenergic differentiation. To determine if the BMP-activated Tak1 (Map3k7) pathway plays a role in SNS development, Tak1 was deleted in the NC lineage. We show that Tak1 is not involved in SNS development. Taken together, our results suggest multiple roles for BMP signaling during SNS development. The Smad4-independent pathway acts through the activation of Phox2b to regulate survival of SNS precursors, whereas the Smad4-dependent pathway controls noradrenergic differentiation and regulates proliferation by maintaining Ascl1 expression.

Antagonism of BMP4 Signaling Disrupts Smooth Muscle Investment of the Ureter and Ureteropelvic Junction

Congenital ureteropelvic junction obstruction has been associated with aberrant ureteral smooth muscle organization. Recent evidence has shown that BMP4 may be involved in ureteral morphogenesis. We determined whether the disruption of BMP4 signaling results in abnormal smooth muscle investment of the ureter and ureteropelvic junction. We used a Cre mediated Bmp4 knockout system to conditionally excise the Bmp4 gene in developing mouse embryos. Kidney rudiments were isolated from embryos at varying gestational ages from WT and conditional knockout mice. Metanephric kidney explants were cultured in the presence or absence of the BMP antagonist Noggin. Agarose beads pre-incubated with Gremlin, another BMP antagonist, were used for localized disruption of BMP signaling. Frozen sections and whole metanephric explants were then analyzed by immunofluorescence. Bmp4 gene excision resulted in a dose dependent loss of ureteral smooth muscle. Antagonism of BMP signaling inhibited ureteral smooth muscle investment in a dose dependent manner and was paralleled by a dose dependent decrease in the immediate downstream targets of BMP signaling, phosphorylated Smad1, 5 and 8. Localized antagonism of BMP resulted in the focal disruption of ureteral smooth muscle investment. We report that decreased BMP signaling, whether by the loss of BMP4 in vivo or direct antagonism in vitro, results in a gradual reduction of the normal, well organized coat of smooth muscle surrounding the ureter. Our results also suggest that this occurs via a direct Smad dependent pathway. This raises the possibility that abnormalities in BMP4 signaling may have a role in the development of congenital ureteropelvic junction obstruction.

An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney

BackgroundBone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney.ResultsMouse strains reporting on BMP pathway activation were generated by transgenically expressing β-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips.ConclusionTransgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.

Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.

Fibrodysplasia Ossificans Progressiva (FOP), a Disorder of Ectopic Osteogenesis, Misregulates Cell Surface Expression and Trafficking of BMPRIA

FOP is a disorder in which skeletal muscle is progressively replaced with bone. FOP lymphocytes, a model system for exploring the BMP pathway in these patients, exhibit a defect in BMPRIA internalization and increased activation of downstream signaling, suggesting that altered BMP receptor trafficking underlies ectopic bone formation in this disease. Fibrodysplasia ossificans progressiva (FOP) is a severely disabling disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the genetic defect and pathophysiology of this condition remain enigmatic, BMP4 mRNA and protein are overexpressed, and mRNAs for a subset of secreted BMP antagonists are not synthesized at appropriate levels in cultured lymphocytes from FOP patients. These data suggest involvement of altered BMP signaling in the disease. In this study, we investigate whether the abnormality is associated with defective BMP receptor function in lymphocytes. Cell surface proteins were quantified by fluorescence-activated cell sorting (FACS). Protein phosphorylation was assayed by immunoprecipitation and immunoblotting. Protein synthesis and degradation were examined by [35S]methionine labeling and pulse-chase assays. mRNA was detected by RT-PCR. FOP lymphocytes expressed 6-fold higher levels of BMP receptor type IA (BMPRIA) on the cell surface compared with control cells and displayed a marked reduction in ligand-stimulated internalization and degradation of BMPRIA. Moreover, in control cells, BMP4 treatment increased BMPRIA phosphorylation, whereas BMPRIA showed ligand-insensitive constitutive phosphorylation in FOP cells. Our data additionally support that the p38 mitogen-activated protein kinase (MAPK) signaling pathway is a major BMP signaling pathway in these cell lines and that expression of inhibitor of DNA binding and differentiation 1 (ID-1), a transcriptional target of BMP signaling, is enhanced in FOP cells. These data extend our previous observations of misregulated BMP4 signaling in FOP lymphocytes and show that cell surface overabundance and constitutive phosphorylation of BMPRIA are associated with a defect in receptor internalization. Altered BMP receptor trafficking may play a significant role in FOP pathogenesis.

Destabilization of ΔNp63α by Nedd4-Mediated Ubiquitination Ubc9-Mediated Sumoylation, and Its Implications on Dorsoventral Patterning of the Zebrafish Embryo

We have recently identified the p53-related ΔNp63α gene as a transcriptional target ofBmp signaling that encodes a transcriptional repressor blocking neural developmentin the zebrafish ectoderm. However, in contrast to Bmps, the neural-repressing effectof forced ΔNp63α expression is restricted to the presumptive forebrain, whileposterior regions of the brain are not affected. Here, we show that this is due toinstability of ΔNp63α protein on the dorsal side of the embryo. In a yeast-two-hybridscreen, we isolated two ΔNp63α-modifying enzymes, the SUMO-conjugating enzymeUbc9 and the ubiquitin ligase Nedd4. The proteins bind to distinct sites in the Cterminalregion of ΔNp63α, which are absent in the shorter and more stable ΔNp63αisoform. Similarly, mutant versions of ΔNp63α unable to bind Nedd4 or Ubc9 arestabilized. ΔNp63α is sumoylated and ubiquitinated both in HEK293 cells and inzebrafish embryos, and Nedd4 promotes ubiquitination and instability of ?Np63?protein, with lysine residue 637 serving as a potential alternative sumoylation andubiquitination site that is crucial forΔNp63α destabilization. In zebrafish, ubc9.1 andnedd4 show restricted expression on the dorsal side of the embryo, where ΔNp63αinstability can be overcome upon blockage of endogenous Nedd4 activity, or uponinjection of mutant versions of ΔNp63α that are unable to bind Nedd4 or Ubc9. Thisresults in a more widespread neural repression, affecting the entire Bmp-sensitiveneuroectoderm. In sum, our data indicate that ΔNp63α is ubiquitinated in a Nedd4-and sumoylated in a Ubc9-dependent fashion, and that these modifications canregulate ΔNp63α stability in the zebrafish ectoderm.