Regulation Of Bone Morphogenetic Protein Signaling Research Articles

BMP-binding protein twisted gastrulation is required in mammary gland epithelium for normal ductal elongation and myoepithelial compartmentalization

Bone morphogenetic proteins (BMPs) are involved in embryonic mammary gland (MG) development and can be dysregulated in breast cancer. However, the role BMPs play in the postnatal MG remains virtually unknown. BMPs are potent morphogens that are involved in cell fate determination, proliferation, apoptosis and adult tissue homeostasis. Twisted gastrulation (TWSG1) is a secreted BMP binding protein that modulates BMP ligand availability in the extracellular space. Here we investigate the consequences of TWSG1 deletion on development of the postnatal MG. At puberty, Twsg1 is expressed in the myoepithelium and in a subset of body cells of the terminal end buds. In the mature duct, Twsg1 expression is primarily restricted to the myoepithelial layer. Global deletion of Twsg1 leads to a delay in ductal elongation, reduced secondary branching, enlarged terminal end buds, and occluded lumens. This is associated with an increase in luminal epithelial cell number and a decrease in apoptosis. In the MG, pSMAD1/5/8 level and the expression of BMP target genes are reduced, consistent with a decrease in BMP signaling. GATA-3, which is required for luminal identity, is reduced in Twsg1−/− MGs, which may explain why K14 positive cells, which are normally restricted to the myoepithelial layer, are found within the luminal compartment and shed into the lumen. In summary, regulation of BMP signaling by TWSG1 is required for normal ductal elongation, branching of the ductal tree, lumen formation, and myoepithelial compartmentalization in the postnatal MG.

Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated geneGREM2regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm

SUMMARYAtrial fibrillation (AF) is the most common cardiac arrhythmia and carries a significant risk of stroke and heart failure. The molecular etiologies of AF are poorly understood, leaving patients with limited therapeutic options. AF has been recognized as an inherited disease in almost 30% of patient cases. However, few genetic loci have been identified and the mechanisms linking genetic variants to AF susceptibility remain unclear. By sequencing 193 probands with lone AF, we identified a Q76E variant within the coding sequence of the bone morphogenetic protein (BMP) antagonist gremlin-2 (GREM2) that increases its inhibitory activity. Functional modeling in zebrafish revealed that, through regulation of BMP signaling, GREM2 is required for cardiac laterality and atrial differentiation during embryonic development. GREM2 overactivity results in slower cardiac contraction rates in zebrafish, and induction of previously identified AF candidate genes encoding connexin-40, sarcolipin and atrial natriuretic peptide in differentiated mouse embryonic stem cells. By live heart imaging in zebrafish overexpressing wild-type or variant GREM2, we found abnormal contraction velocity specifically in atrial cardiomyocytes. These results implicate, for the first time, regulators of BMP signaling in human AF, providing mechanistic insights into the pathogenesis of the disease and identifying potential new therapeutic targets.

Autosomal dominant mesomandibular fibro-osseous dysplasia: a self-resolving inherited fibro-osseous lesion of the jaws

A hereditary congenital condition characterized by a fibro-osseous lesion sharing some features with fibrous dysplasia and affecting the middle aspect of the mandible is presented. The condition was initially described as congenital monostotic fibrous dysplasia in two siblings, a male and a female. However, there is sufficient evidence that the disorder is autosomal dominant since it has been encountered in two of four children, both males, of the female propositus and one child, a boy, of the male propositus. All patients presented at birth or right after birth with enlargement of the middle part of the mandible. Radiographs from affected individuals have shown mesomandibular enlargement with irregular trabeculation akin of “ground-glass” appearance. Histologically, samples from all patients revealed woven bone proliferation in a cellular fibroblastic stroma. Interestingly, the originally described siblings, now in their 30s, have no evidence of jaw lesions either radiographically or clinically, thus indicating that the condition is self-limiting or self-resolving. An autosomal dominant mode of inheritance with apparent male predilection is favored. The molecular basis of this condition is currently unknown. However, the location of the lesions in the middle aspect of the mandible suggests dysregulation of Bone Morphogenetic Protein (BMP) signaling since BMPs regulate mandibular morphogenesis in utero, particularly in the medial region as well as postnatal bone remodeling. Immunohistochemical evaluation for a BMP-binding protein Twisted Gastrulation (TWSG1) revealed mosaic pattern of staining, with some cells, including osteoclasts, strongly stained and others exhibiting faint or no staining, thus supporting active regulation of BMP signaling within the lesion. Future investigations will determine if dysregulation of BMP signaling plays a causative role or rather reflects secondary activation of repair mechanisms and/or bone remodeling.

Abstract 17230: BMP9 Regulates Endothelin-1 Gene Transcription in Cooperation With TGF-β Signaling

Pulmonary arterial hypertension (PAH) is characterized by abnormal pulmonary vasoreactivity and pulmonary angiopathy, leading to progressively increased pulmonary resistance, right heart failure and high mortality. The mechanisms of PAH have not been fully elucidated, although dysregulated bone morphogenetic protein (BMP) signaling due to loss-of-function mutations in the gene encoding the BMP type II receptor (BMPRII) are implicated in heritable PAH. It has been recently reported that BMP9, a BMPRII ligand, regulates transcription of the potent vasoconstrictor and smooth muscle mitogen endothelin-1 (ET-1), an important mediator of PAH. BMP9 functions as a circulating vascular quiescence factor that promotes endothelial survival while inhibiting angiogenesis. We hypothesized that altered BMP signaling in pulmonary vascular endothelium may affect the regulation of ET-1 and thereby contribute to the pathophysiology of PAH. We found that stimulation of cultured human and murine lung microvascular endothelial cells with BMP9 activates both canonical BMP and TGF-β pathways via their respective effector molecules, SMAD1/5/8 and SMAD2/3. BMP9-induced transcription of ET-1 appeared to require coordinated activation of both BMP and TGF-β signaling pathways, based on the finding that ET-1 gene transcription was inhibited by selective small molecule inhibitors of BMP or TGF-β receptor kinases, by recombinant BMP receptor ectodomains, or by siRNA-mediated suppression of BMP or TGF-β type I receptor expression. In addition, functional analysis of the human ET-1 gene promoter revealed the presence of distinct cis -regulatory elements involved in BMP9- versus TGF-β-mediated activation of ET-1 gene transcription. Mutational inactivation of a novel BMP-responsive regulatory element, as well as known TGF-β-responsive elements, abolished ET-1 gene promoter activity in response to BMP9, underscoring further the cooperative nature of BMP and TGF-β signaling in ET-1 regulation. These results suggest that perturbation of BMP signaling may alter the regulation of ET-1 in the endothelium, impact endothelial and vascular homeostasis, and thus contribute to the pathogenesis of PAH.

Distinct bone morphogenetic proteins activate indistinguishable transcriptional responses in nephron epithelia including Notch target genes

Endogenous Bone Morphogenetic Protein (BMP) signaling plays a significant role in the kidney's recovery from acute injury and exogenous administration of BMP7 has therapeutic potential in numerous rodent models of renal injury and disease. However, in the healthy kidney endogenous BMP7 ligand is vigorously counteracted by extracellular antagonists such as USAG1 and CHRDL1. Little is known about the degree of BMP signaling and the ligands driving it in the healthy adult kidney. In this study we characterize basal BMP signaling in the healthy tubular nephron, and show that BMP2 is expressed in proximal nephron epithelial cells. Comparative gene profiling of proximal tubule cell responses to BMP2 and BMP7 does not reveal any qualitative difference, suggesting that identical BMP gene targets may be activated in healthy and injured organs. Interestingly, our gene profiling analysis shows that BMP signaling activates a number of Notch regulated transcription factors, including HEY1. As in other biological systems, HEY1 functions as a negative feedback regulator of BMP2 expression in the proximal tubule. In summary, this work reveals endogenous BMP signaling patterns in the healthy human and mouse kidneys, and identifies novel gene targets, some of which are involved in the complex regulation of BMP signaling in the adult kidney.

Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification

Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.

Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-β and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Application of Small Organic Molecules Reveals Cooperative TGFβ and BMP Regulation of Mesothelial Cell Behaviors

Epicardial development is a process during which epithelial sheet movement, single cell migration, and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement, and single cell migration by Transforming Growth Factor β (TGFβ). With the DM analogue DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.

Bone Morphogenetic Protein Endothelial Cell Precursor–Derived Regulator Regulates Retinal Angiogenesis In Vivo in a Mouse Model of Oxygen-Induced Retinopathy

Bone morphogenetic proteins (BMPs) are potently proangiogenic; however, the mechanisms underlying the regulation of vessel development by BMPs are not fully understood. To assess the significance of BMP endothelial cell precursor-derived regulator (BMPER) in blood vessel formation in vivo, we investigated its role in retinal angiogenesis. In a model of oxygen-induced retinopathy, Bmper mRNA expression and protein levels are downregulated, correlating with the initiation of Sma and Mad related protein phosphorylation in endothelial cells. Moreover, Bmper haploinsufficiency results in an increased rate of retinal revascularization, with retinas from Bmper+/- mice displaying increased numbers of branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Furthermore, although Bmper haploinsufficiency does not alter Bmp expression, it does lead to an increase in BMP signaling, as evidenced by increased phosphorylated Sma and Mad related protein levels in endothelial cells and increased expression of known BMP target genes. These observations provide compelling evidence that BMPER is important in the regulation of BMP signaling and revascularization in the hypoxic retina. These bring forth the possibility of novel therapeutic approaches for pathological angiogenesis based on manipulation of BMP signaling.

Smad6 is essential to limit BMP signaling during cartilage development

Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP-mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6-/- mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6-/- mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6-deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation.

Novel Contact-dependent Bone Morphogenetic Protein (BMP) Signaling Mediated by Heparan Sulfate Proteoglycans

We previously proposed a model that DALLY, a Drosophila glypican, acts as a trans co-receptor to regulate BMP signaling in the germ line stem cell niche. To investigate the molecular mechanisms of contact-dependent BMP signaling, we developed novel in vitro assay systems to monitor trans signaling using Drosophila S2 cells. Using immunoblot-based as well as single-cell assay systems, we present evidence that Drosophila glypicans indeed enhance BMP signaling in trans in a contact-dependent manner in vitro. Our analysis showed that heparan sulfate modification is required for the trans co-receptor activity of DALLY. Two BMP-like molecules, Decapentaplegic (DPP) and Glass bottom boat, can mediate trans signaling through a heparan sulfate proteoglycan co-receptor in S2 cells. The in vitro systems reflect the molecular characteristics of heparan sulfate proteoglycan functions observed previously in vivo, such as ligand specificity and biphasic activity dependent on the ligand dosage. In addition, experiments using a DALLY-coated surface suggested that DALLY regulates DPP signaling in trans by its effect on the stability of DPP protein on the surface of the contacting cells. Our findings provide the molecular foundation for novel contact-dependent signaling, which defines the physical space of the stem cell niche in vivo.

Abstract 2200: CCN6/WISP3 exerts its tumor suppressor function through regulation of BMP signaling by direct binding to BMP4 in the extracellular environment

Abstract CCN6 is a secreted protein with tumor suppressor function in the most aggressive form of locally advanced breast cancer, inflammatory breast cancer. CCN6 was reported to be lost in 80% cases of inflammatory breast cancer and 19% cases of stage matched non inflammatory breast cancer. It was shown that CCN6 knockdown (KD) in benign breast cells triggered an epithelial to mesenchymal transition with E-cadherin downregulation (Am J Pathol 172(4):893-904, 2008) recently shown to be associated with acquisition of growth factor independent signaling (Cancer Res 70 (8):3340-3350, 2010). The molecular mechanism by which CCN6 regulates extracellular signaling to suppress breast cancer invasion is not known. As a member of the CCN family of matricellular proteins, named after connective tissue growth factor, cysteine-rich 61, and nephroblastoma overexpressed, CCN6 shares the highly conserved protein motifs of the CCN family, which in some cases have been reported to bind to bone morphogenetic protein (BMP) and transforming growth factor beta (TGFβ) (J Cell Sci 119(23):4803-4810, 2006). We hypothesized that the invasive phenotype of CCN6 KD cells may be dependent upon cytoskeletal alterations through modulation of growth factor signaling pathways like TGFβ/BMP. The invasive phenotype of lentivirus mediated control and CCN6 KD cells was assessed by growth on matrigel for 15 days following published protocol. The effect on TGFβ/BMP signaling was examined by a pathway specific PCR array for TGFβ/BMP (SA Biosciences), immunoblotting and immunocytochemistry. To ascertain the tumor suppressor effect of CCN6, we examined if recombinant human CCN6 (rhCCN6) could revert the invasive phenotype by treatment with 500 ng/ml rhCCN6 (PeproTech). Similarly small molecule inhibitors were used to inhibit activated P38 kinase (5 μM SB203580), TGFβ type I receptor I kinase (TβRI) (400 nM SB431542) and TGFβ activated kinase1 (TAK1) (0.3 μM 5Z-7-oxozeaenol). The specific binding of CCN6 with BMP4 was confirmed by co-immunoprecipitation, crosslinking, competitive binding and surface plasmon resonance. We found that CCN6 loss in benign breast cells triggers a highly invasive phenotype with loss of α6 integrin, which was rescued by treatment with rhCCN6. CCN6 loss in HME cells antagonized BMP4-mediated Smad1/5 activation and enhanced the effect of BMP4 on the non-canonical pathway to activate TAK1 and P38 through activation of TβRI. Small molecule inhibition of activated P38 or TAK1 or TβRI, or of BMP4 by blocking antibody reverted the invasive phenotype. CCN6 protein had the opposite effect, potentiation of BMP4-mediated Smad1/5 signaling by direct binding to BMP4. Conclusively, by direct binding to BMP4 CCN6 regulates BMP4 mediated Smad and alternate P38 signaling. Thus, CCN6 in the extracellular space may act as a master switch and regulator of BMP4 to suppress invasion in breast cancer. 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 2200. doi:10.1158/1538-7445.AM2011-2200

A mathematical model of bi‐stability and cell‐competition reproduces autoregulation of stem cell number in the Drosophila germarium

Complex organisms maintain stable populations of stem cells to remain healthy and support somatic tissues. In the germline stem cells (GSC) of the Drosophila ovary, Bone Morphogenetic Protein (BMP) signaling regulates the decision between stem cell self renewal or differentiation. We have elucidated key players in the intracellular network that regulates the response to BMPs, however the interaction between intra- and extracellular regulation of BMP signaling remains unknown. To investigate that the proposed system is sufficient to autoregulate population, we developed two mathematical models, considering both intra- and extracellular interactions: a local model for a single cell receiving BMP signaling and a 3D extracellular model of the germarium. As a function of several regulatory parameters, the proposed intracellular feedback mechanism exhibits bistabilty in response to BMP signaling, making cells refractory to additional signal. By combining intra- and extracellular regulation in the 3D multi-cell model, we find that a model of cell-mediated competition for limiting amounts of ligand reproduces autoregulation of stem cell number in the niche. Competition, combined with the bistable intracellular system, constrains the stem cell population, causing differentiation of extraneous GSCs and repopulating if GSCs are lost. This work was supported by the Showalter Trust.

Lissencephaly-1 controls germline stem cell self-renewal through modulating bone morphogenetic protein signaling and niche adhesion

In the Drosophila ovary, bone morphogenetic protein (BMP) signaling activated by the niche promotes germline stem cell (GSC) self-renewal and proliferation, whereas E-cadherin-mediated cell adhesion anchors GSCs in the niche for their continuous self-renewal. Here we show that Lissencephaly-1 (Lis1) regulates BMP signaling and E-cadherin-mediated adhesion between GSCs and their niche and thereby controls GSC self-renewal. Lis1 mutant GSCs are lost faster than control GSCs because of differentiation but not because of cell death, indicating that Lis1 controls GSC self-renewal. The Lis1 mutant GSCs exhibit reduced BMP signaling activity, and Lis1 interacts genetically with the BMP pathway components in the regulation of GSC maintenance. Mechanistically, Lis1 binds directly to and stabilizes the SMAD protein Mothers against decapentaplegic (Mad), facilitates its phosphorylation, and thereby regulates BMP signaling. Finally, the Lis1 mutant GSCs accumulate less E-cadherin in the stem cell-niche junction than do their wild-type counterparts. Germline-specific expression of an activated BMP receptor thickveins (Tkv) or E-cadherin can partially rescue the loss phenotype of Lis1 mutant GSCs. Therefore, this study has revealed a role of Lis1 in the control of Drosophila ovarian GSC self-renewal, at least partly by regulating niche signal transduction and niche adhesion. It has been known that Lis1 controls neural precursor/stem cell proliferation in the developing mammalian brain; this study further suggests that Lis1, which is widely expressed in adult mammalian tissues, could regulate adult tissue stem cells through modulating niche signaling and adhesion.

Neogenin Regulation of BMP-Induced Canonical Smad Signaling and Endochondral Bone Formation

Neogenin has been identified as a receptor for the neuronal axon guidance cues netrins and RGMs (repulsive guidance molecules). Here we provide evidence for neogenin in regulating endochondral bone development and BMP (bone morphogenetic protein) signaling. Neogenin-deficient mice were impaired in digit/limb development and endochondral ossification. BMP2 induction of Smad1/5/8 phosphorylation and Runx2 expression, but not noncanonical p38 MAPK activation, was reduced in chondrocytes from neogenin mutant mice. BMP receptor association with membrane microdomains, which is necessary for BMP signaling to Smad, but not p38 MAPK, was diminished in neogenin-deficient chondrocytes. Furthermore, RGMs appear to mediate neogenin interaction with BMP receptors in chondrocytes. Taken together, our results indicate that neogenin promotes chondrogenesis in vitro and in vivo, revealing an unexpected mechanism underlying neogenin regulation of BMP signaling.

LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche

The microenvironment of growth factors in the subependymal zone (SEZ) of the adult brain provides the instructive milieu for neurogenesis to proceed in this germinal niche. In particular, tight regulation of bone morphogenetic protein (BMP) signaling is essential to balance proliferative and non-proliferative cell fate specification. However, the regulatory pathways that control BMP signaling in the SEZ are still poorly defined. We demonstrate that LRP2, a clearance receptor for BMP4 is specifically expressed in ependymal cells of the lateral ventricles in the adult brain. Intriguingly, expression is restricted to the ependyma that faces the stem cell niche. Expression is not seen in ependyma elsewhere in the lateral ventricles or in the dentate gyrus, the second major neurogenic zone of the adult brain. We further show that lack of LRP2 expression in adult mice results in impaired proliferation of neural precursor cells in the SEZ resulting in decreased numbers of neuroblasts reaching the olfactory bulb. Reduced neurogenesis coincides with increased BMP4 expression and enhanced activation of downstream mediators phospho-SMAD1/5/8 and ID3 in the stem cell niche. Our findings suggest a novel mechanism whereby LRP2-mediated catabolism of BMP4 in the ependyma modulates the microenvironment of the SEZ and enables adult neurogenesis to proceed.

Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling.

Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP–like receptor signaling. SMA-10 acts genetically in a BMP–like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche

The microenvironment of growth factors in the subependymal zone (SEZ) of the adult brain provides the instructive milieu for neurogenesis to proceed in this germinal niche. In particular, tight regulation of bone morphogenetic protein (BMP) signaling is essential to balance proliferative and non-proliferative cell fate specification. However, the regulatory pathways that control BMP signaling in the SEZ are still poorly defined. We demonstrate that LRP2, a clearance receptor for BMP4 is specifically expressed in ependymal cells of the lateral ventricles in the adult brain. Intriguingly, expression is restricted to the ependyma that faces the stem cell niche. Expression is not seen in ependyma elsewhere in the lateral ventricles or in the dentate gyrus, the second major neurogenic zone of the adult brain. We further show that lack of LRP2 expression in adult mice results in impaired proliferation of neural precursor cells in the SEZ resulting in decreased numbers of neuroblasts reaching the olfactory bulb. Reduced neurogenesis coincides with increased BMP4 expression and enhanced activation of downstream mediators phospho-SMAD1/5/8 and ID3 in the stem cell niche. Our findings suggest a novel mechanism whereby LRP2-mediated catabolism of BMP4 in the ependyma modulates the microenvironment of the SEZ and enables adult neurogenesis to proceed.

Cleavage of HJV is independent of its GPI‐anchor

Mutations in hemojuvelin (HJV) lead to the iron overload disorder juvenile hemochromatosis. HJV is a GPI‐linked protein that increases expression of the iron regulatory peptide hormone, hepcidin. Hepcidin negatively regulates iron uptake from the intestine. GPI‐linked HJV potentiates bone morphogenetic protein (BMP) signaling to upregulate hepcidin expression. HJV can also be cleaved by the proteases furin and matriptase‐2, releasing it from its GPI‐anchor and allowing it to be secreted from cells. In contrast to GPI‐linked HJV, secreted HJV negatively regulates BMP signaling and decreases hepcidin expression. To investigate the role of the GPI‐anchor in cleavage and secretion of HJV, we created chimeric proteins with the ectodomain of HJV and the transmembrane and cytoplasmic domains of either low‐density lipoprotein receptor (LDLR) or furin in lieu of the GPI‐anchor. These chimeric proteins appear to traffic similarly to GPI‐HJV, and are cleaved by furin and secreted from cells. Truncated forms of HJV that have neither a transmembrane or GPI‐anchor, are also cleaved by furin and are secreted from cells, but appear to have different kinetics of post‐translational processing than membrane anchored HJV. These studies indicate that the GPI‐anchor is not required for cleavage and secretion of HJV. This work is funded by NIH‐DK080765.

Regulation of Neural Specification from Human Embryonic Stem Cells by BMP and FGF

Inhibition of bone morphogenetic protein (BMP) signaling is required for vertebrate neural induction, and fibroblast growth factors (FGFs) may affect neural induction through phosphorylation at the linker region of Smad1, thus regulating BMP signaling. Here we show that human embryonic stem cells efficiently convert to neuroepithelial cells in the absence of BMP antagonists, or even when exposed to high concentrations of exogenous BMP4. Molecular and functional analyses revealed multiple levels of endogenous BMP signaling inhibition that may account for the efficient neural differentiation. Blocking FGF signaling inhibited neural induction, but did not alter the phosphorylation of the linker region of Smad1, suggesting that FGF enhances human neural specification independently of BMP signaling.