BMP9 and BMP10 are critical for postnatal retinal vascular remodeling

ALK1 is a type I receptor of the TGF-β family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.     

BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.The ductus arteriosus (DA) is a large blood vessel whose obstruction is essential for the transition from fetal to neonatal circulation. It is a fetal arterial shunt connecting the pulmonary artery with the aortic arch. During fetal life, the DA directs deoxygenated blood away from the pulmonary circulation and toward the descending aorta, bypassing the nonventilated fetal lungs. After birth, the DA closes spontaneously within 1–3 h in small rodents or within 24–48 h in human newborns (1, 2). Although an open DA is required for fetal survival, the persistence of a patent DA (PDA) after birth is a major cause of morbidity and mortality, particularly in preterm neonates, leading to severe complications, including pulmonary hypertension, right ventricular dysfunction, postnatal infections, and respiratory failure. The incidence of PDA has been estimated to be one in 500 in-term newborns and accounts for the majority of all cases of congenital heart disease in preterm newborns. It is currently believed that DA closure involves a two-step process (3, 4). The first, provisional closure, also called functional closure, occurs at birth and is accomplished by smooth muscle cell contraction and DA constriction. The second step, named anatomical closure, involves a profound remodeling of cells within the former DA lumen and permits permanent closure of the DA. Although several factors have been implicated in DA closure (oxygen tension, prostaglandin E2, laminin, growth hormone, and platelets), the precise molecular and cellular signals that promote the transition from initial constriction to definitive DA closure are not yet fully understood.Bone morphogenetic protein 9 (BMP9) and BMP10 are two members of the BMP family that have been demonstrated to play major roles in vascular development (5). In 2007, it was demonstrated that BMP9 and BMP10 bind with high affinity to the endothelial-specific receptor activin receptor-like kinase 1 (ALK1), a type 1 receptor of the TGFβ family (6) whose mutations are involved in vascular diseases (5). Both BMP9 and BMP10 are present in blood, and their circulating levels are particularly elevated in mice around birth (7, 8), suggesting that they could play a role in pre- and postnatal development. In the present work, we addressed whether BMP9 and BMP10 could be involved in DA closure. For this purpose, we used Bmp9-KO mice and neutralizing anti-BMP10 antibody. Herein, we show that injection of a neutralizing anti-BMP10 antibody into Bmp9-KO pups led to an open DA and identified several targets that may be involved in this closure defect. This work is further supported by human genomic data, based on the definition of a 700-kb minimal critical region in chromosome 2 encoding eight genes, including BMP10, that correlated with the presence of a PDA in two patients. This work thus identifies a previously unidentified signaling pathway, the BMP9/10 pathway, in the anatomical closure of the DA.     

Tmem100, an ALK1 receptor signaling-dependent gene essential for arterial endothelium differentiation and vascular morphogenesis

Members of the transforming growth factor-β superfamily play essential roles in various aspects of embryonic development and physiological organ function. Among them, bone morphogenetic protein (BMP) 9 and BMP10 regulate embryonic vascular development by activating their endothelial receptor ALK1 (activin receptor-like kinase 1, also called Acvrl1). ALK1-mediated intracellular signaling is implicated in the etiologies of human diseases, but their downstream functional proteins are largely unknown. In this study, we identified Tmem100, a gene encoding a previously uncharacterized intracellular transmembrane protein, to be an embryonic endothelium-enriched gene activated by BMP9 and BMP10 through the ALK1 receptor. Tmem100 null mice showed embryonic lethality due to impaired differentiation of arterial endothelium and defects of vascular morphogenesis, which phenocopied most of the vascular abnormalities observed with the Acvrl1/Alk1 deficiency. The activity of Notch- and Akt-mediated signaling, which is essential for vascular development, was down-regulated in Tmem100 null mice. Cre-mediated deletion of Tmem100 in endothelial cells was sufficient to recapitulate the null phenotypes. These data indicated that TMEM100 may play indispensable roles downstream of BMP9/BMP10-ALK1 signaling during endothelial differentiation and vascular morphogenesis.     

Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells

ALK1 is an endothelial-specific type I receptor of the TGFbeta receptor family whose heterozygous mutations cause hereditary hemorrhagic telangiectasia type 2. Although TGFbeta1 and TGFbeta3 have been shown to bind ALK1 under specific experimental conditions, they may not represent the physiological ligands for this receptor. In the present study, we demonstrate that BMP9 induces the phosphorylation of Smad1/5/8 in microvascular endothelial cells, and this phosphorylation lasts over a period of 24 hours. BMP9 also activates the ID1 promoter-derived BMP response element (BRE) in a dose-dependent manner (EC50 = 45 +/- 27 pg/mL), and this activation is abolished by silencing ALK1 expression or addition of ALK1 extracellular domain. Overexpression of endoglin increases the BMP9 response, whereas silencing of both BMPRII and ActRIIA expressions completely abolishes it. BMP10, which is structurally close to BMP9, is also a potent ALK1 ligand. Finally, we demonstrate that BMP9 and BMP10 potently inhibit endothelial cell migration and growth, and stimulate endothelial expression of a panel of genes that was previously reported to be activated by the constitutively active form of ALK1. Taken together, our results suggest that BMP9 and BMP10 are two specific ALK1 ligands that may physiologically trigger the effects of ALK1 on angiogenesis.     

Over-expression of BMP4 inhibits experimental choroidal neovascularization by modulating VEGF and MMP-9

Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.     

Reducing Jagged 1 and 2 levels prevents cerebral arteriovenous malformations in matrix Gla protein deficiency

Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp^−/−) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp^−/− mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs.Cerebral arteriovenous malformations (AVMs) are abnormal vascular networks that form direct connections between arteries and veins, thereby short circuiting the cerebral capillary system. Due to the elevated blood pressure in these abnormal connections, vessels may ultimately rupture and result in hemorrhagic strokes (1–3). The development of cerebral AVMs has been shown to involve bone morphogenetic protein (BMP) and Notch signaling, both essential pathways in vascular formation (4, 5). Mutations in the genes for activin-like kinase receptor 1 (ALK1), a BMP type I receptor, and Endoglin, a coreceptor of ALK1, cause hereditary hemorrhagic telangiectasia (HHT) characterized by the presence of AVMs in multiple organs including the brain (6–9). Enhanced endothelial Notch signaling, as mediated by constitutively active Notch4, also promotes the formation of brain AVMs, whereas normalization of Notch4 activity results in regression of arteriovenous (AV) shunts (10, 11).We have previously shown that BMP-4 and -7 induce expression of ALK1 in endothelial cells (ECs) (12, 13). In turn, activation of ALK1 by its ligand BMP-9 induces expression of matrix Gla protein (MGP), an inhibitor of BMP-2, -4 and -7 (12-15), which is highly expressed in brain, lungs, and kidneys (13, 16). MGP provides negative feedback inhibition for BMP-4 and -7 (13, 17), which limits the ALK1 induction. Gene deletion of MGP in mice results in excess ALK1 activation and severe AVMs in lungs and kidneys (13). However, it has not yet been shown whether AVMs also form in the brain of MGP null (Mgp^−/−) mice.Connections between BMP-9/ALK1 and Notch signaling have been reported. Larrivée et al. reported that BMP-9/ALK1 activation induces the expression of Notch ligands and targets in vascular ECs (18), suggesting that Notch signaling acts on the vasculature downstream of BMP-9/ALK1. Even so, it is not clear how ALK1 signaling affects Notch signaling and vice versa during the formation of AVMs.Here, we report that the formation of cerebral AVMs occurs in Mgp^−/− mice, but is prevented by decreasing the expression of the Notch ligands Jagged 1 and 2, which compensates for the excess ALK1 activation in these mice. The results suggest that maintaining the balance between BMP and Notch signaling is essential to avoid AVMs.     

Bone morphogenetic protein 4 mediates myocardial ischemic injury through JNK-dependent signaling pathway

Bone morphogenetic protein (BMP) signaling regulates embryonic development of many organ systems and defective BMP signaling has been implicated in adult disorders of many of these systems. However, its relevance in cardiac disease has not been reported. Here we demonstrate for the first time that Bmp4 activity promotes cellular apoptosis following ischemia-reperfusion (I/R) injury induced myocardial infarction (MI). Bmp4 heterozygous null mice (Bmp4^+/−) demonstrated reduced infarct size, less myocardial apoptosis and down-regulation of pro-apoptotic proteins relative to wild-type mice following I/R injury. This was associated with reduction in I/R induced BMP4 levels in the left ventricular infarcted region. Furthermore, treatment of neonatal cardiomyocytes with BMP4 resulted in time and dose-dependent increase in cellular apoptosis and activation of the JNK MAP kinase pathway. In contrast, while JNK activation was significantly attenuated in Bmp4^+/− mice and following Smad1 inhibition in myocytes, inhibition of JNK with a specific inhibitory peptide, TAT-JBD_20, blocked BMP4 induced apoptosis. In vivo treatment of mice with Noggin, an endogenous extracellular BMP antagonist, or dorsomorphin, a small molecule inhibitor of BMP signaling, reduced infarct size, and inhibited pro-apoptotic signaling accompanied by an inhibition of Smad1 phosphorylation and JNK activation. These studies identify a novel role for Bmp4 in the pathogenesis of myocardial infarction and illustrate the use of a small molecule inhibitor of BMP signaling for treatment of acute I/R injury.     

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb^th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.     

TGF‐β & BMP Receptors Endoglin and ALK1: Overview of their Functional Role and Status as Antiangiogenic Targets

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF‐β superfamily, TGF‐β1, TGF‐β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell–cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1‐mediated cells signaling.     

Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions

The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.Ligands of the TGF-β superfamily, the largest family of secreted proteins in mammals, are synthesized as dimers and bind transmembrane type 1 and type 2 serine-threonine kinase receptors to activate downstream signaling cascades (e.g., the SMADs) in many developmental, physiological, and pathophysiological processes (1, 2). Growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) are key oocyte-secreted members of the TGF-β superfamily and can regulate female fertility in several mammals (2, 3). Although GDF9 and BMP15 are closely related paralogs, they have been shown in vitro to signal through divergent SMAD2/3 and SMAD1/5/8 pathways, respectively (4–6).By studying gene knockouts and mutant models, putative roles of GDF9 and BMP15 in female reproduction have been described in mice, sheep, and humans. Our group previously discovered that Gdf9-null female mice are sterile (7), and Gdf9^+/−Bmp15^−/− double-mutant mice had more severe fertility defects than subfertile Bmp15^−/− mice (8, 9). BMP15 or GDF9 heterozygous mutant sheep have increased litter size, whereas homozygous mutants are sterile and phenocopy Gdf9^−/− mice (10, 11). In humans, mutations in GDF9 and BMP15 have been associated with premature ovarian failure and dizygotic twinning (12–14). These data suggest synergistic functions of the two gene products and potential species-specific differences in the bioactivity of these proteins. Although an in vitro study has detected the GDF9:BMP15 heterodimer by immunoprecipitation (15), and cooperative effects of the two homodimers were studied by other groups (16–18), the functions of GDF9:BMP15 heterodimers in any species remain largely unknown.In the present study, we demonstrate that GDF9:BMP15 heterodimers are the most bioactive ligands in the regulation of cumulus expansion genes. These heterodimers signal through a unique BMP receptor type 2 (BMPR2)-ALK4/5/7-ALK6 receptor complex to induce the phosphorylation of SMAD2/3 in human and mouse granulosa cells. Our findings open up prospects for the understanding of the synergistic roles of GDF9 and BMP15 proteins in ovarian functions and have important implications for improving female reproductive productivity in mammals.     

Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin

Hemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.      

Iron overload induces BMP6 expression in the liver but not in the duodenum

Background

The bone morphogenetic protein BMP6 regulates hepcidin production by the liver. However, it is not yet known whether BMP6 derives from the liver itself or from other sources such as the small intestine, as has been recently suggested. This study was aimed at investigating the source of BMP6 further.

Design and Methods

We used three different strains of mice (C57BL/6, DBA/2, and 129/Sv) with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene. We examined Bmp6 expression at both the mRNA (by quantitative PCR) and protein (by immunohistochemistry and Western blotting analyses) levels.

Results

We showed that iron overload induces Bmp6 mRNA expression in the liver but not in the duodenum of these mice. Bmp6 is also detected by immunohistochemistry in liver tissue sections of mice with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene, but not in liver tissue sections from iron-loaded Bmp6-deficient mice. Bmp6 in the duodenum was below immunodetection threshold, thus confirming quantitative PCR data. Lack of specificity of available antibodies together with slight heterogeneity between 129 substrains may account for the differences with previously published data.

Conclusions

Our data strongly support the importance of liver BMP6 for regulation of iron metabolism. Indeed, they demonstrate that intestinal Bmp6 expression is modulated by iron neither at the mRNA nor at the protein level.     

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice

The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. CONCLUSION: Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.     

Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1^−/− and Sulf-2^−/− mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf^−/− mice. Articular cartilage and cultured chondrocytes from Sulf^−/− mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.     

BMP9 induces EphrinB2 expression in endothelial cells through an Alk1-BMPRII/ActRII-ID1/ID3-dependent pathway: implications for hereditary hemorrhagic telangiectasia type II

ALK1 (ACVRL1) is a member of the TGFβ receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for hereditary hemorrhagic telangiectasia type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations. Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1-restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.