Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.     

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.     

ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.     

Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis

Molecules associated with the transforming growth factor β (TGF-β) superfamily, such as bone morphogenic proteins (BMPs) and TGF-β, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-β1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.     

Deficient Alk3-mediated BMP signaling causes prenatal omphalocele-like defect

BMP signaling plays important roles in many embryonic developmental processes. Alk3 is one of two BMP type I receptors that transduces BMP signal from the cell surface into cell. Conventional knockout of Alk3 resulted in early embryonic lethality around E7.5-E9.5. In this study, we have generated embryonic mesoderm-specific Alk3 conditional knockout by crossing Dermo1-Cre and floxed Alk3 mice. Abrogation of Alk3-mediated BMP signaling in this mouse resulted in severe defect of secondary ventral body wall formation, replicating the omphalocele phenotype in human. Our finding suggests that Alk3 plays an essential role in the formation of embryonic ventral abdominal wall, and abrogation of BMP signaling activity due to gene mutations in its signaling components could be one of the underlying causes of omphalocele at birth.     

Divergence and convergence of TGF-beta/BMP signaling

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.     

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.     

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.     

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.     

The bone morphogenetic protein type Ib receptor is a major mediator of glial differentiation and cell survival in adult hippocampal progenitor cell culture

Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture.     

Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin

Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor beta (TGF-beta) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-beta superfamily. GDF9 and BMP4 induce gremlin, but TGF-beta does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.     

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

BMP signaling in skeletal development

Development of the vertebrate skeleton, a complex biological event that includes diverse processes such as formation of mesenchymal condensations at the sites of future skeletal elements, osteoblast and chondrocyte differentiation, and three dimensional patterning, is regulated by many growth factors. Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, play a pivotal role in the signaling network and are involved in nearly all processes associated with skeletal morphogenesis. BMP signals are transduced from the plasma membrane receptors to the nucleus through both Smad pathway and non-Smad pathways, and regulated by many extracellular and intercellular proteins that interact with BMPs or components of the BMP signaling pathways. To gain a better understanding of the molecular mechanisms underlying the role of BMP in early skeletal development, it is necessary to elucidate the BMP signaling transduction pathways in chondrocytes and osteoblasts. The major objective of this review was to summarize BMP signaling pathways in the context of craniofacial, axial, and limb development. In particular, this discourse will focus on recent advances of the role of different ligands, receptors, Smads, and BMP regulators in osteoblast and chondrocyte differentiation during embryonic development.     

Cripto binds transforming growth factor beta (TGF-beta) and inhibits TGF-beta signaling

Cripto is a developmental oncoprotein and a member of the epidermal growth factor-Cripto, FRL-1, Cryptic family of extracellular signaling molecules. In addition to having essential functions during embryogenesis, Cripto is highly expressed in tumors and promotes tumorigenesis. During development, Cripto acts as an obligate coreceptor for transforming growth factor beta (TGF-beta) ligands, including nodals, growth and differentiation factor 1 (GDF1), and GDF3. As an oncogene, Cripto is thought to promote tumor growth via mechanisms including activation of mitogenic signaling pathways and antagonism of activin signaling. Here, we provide evidence supporting a novel mechanism in which Cripto inhibits the tumor suppressor function of TGF-beta. Cripto bound TGF-beta and reduced the association of TGF-beta with its type I receptor, TbetaRI. Consistent with its ability to block receptor assembly, Cripto suppressed TGF-beta signaling in multiple cell types and diminished the cytostatic effects of TGF-beta in mammary epithelial cells. Furthermore, targeted disruption of Cripto expression by use of small inhibitory RNA enhanced TGF-beta signaling, indicating that endogenous Cripto plays a role in restraining TGF-beta responses.