Developmental SMAD6 loss leads to blood vessel hemorrhage and disrupted endothelial cell junctions.

The BMP pathway regulates developmental processes including angiogenesis, yet its signaling outputs are complex and context-dependent. Recently, we showed that SMAD6, an intracellular BMP inhibitor expressed in endothelial cells, decreases vessel sprouting and branching both in vitro and in zebrafish. Genetic deletion of SMAD6 in mice results in poorly characterized cardiovascular defects and lethality. Here, we analyzed the effects of SMAD6 loss on vascular function during murine development. SMAD6 was expressed in a subset of blood vessels throughout development, primarily in arteries, while expression outside of the vasculature was largely confined to developing cardiac valves with no obvious embryonic phenotype. Mice deficient in SMAD6 died during late gestation and early stages of postnatal development, and this lethality was associated with vessel hemorrhage. Mice that survived past birth had increased branching and sprouting of developing postnatal retinal vessels and disorganized tight and adherens junctions. In vitro, knockdown of SMAD6 led to abnormal endothelial cell adherens junctions and increased VE-cadherin endocytosis, indicative of activated endothelium. Thus, SMAD6 is essential for proper blood vessel function during murine development, where it appears to stabilize endothelial junctions to prevent hemorrhage and aberrant angiogenesis.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis.

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Semaphorin 7A contributes to West Nile virus pathogenesis through TGF-ß1/Smad6 signaling.

Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood-brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF-ß1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A-blocking therapies for WNV and possibly other flaviviral infections.

Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord.

The roof plate resident BMPs have sequential functions in the developing spinal cord, establishing cell fate and orienting axonal trajectories. These activities are, however, restricted to the dI1-dI3 neurons in the most dorsal region of the spinal cord. What limits the extent of the action of the BMPs to these neurons? To address this question, we have examined both the distribution of the inhibitory Smads (I-Smads), Smad6 and Smad7 in the spinal cord and the consequence of ectopically expressing the I-Smads in chicken embryos. Our studies suggest that the I-Smads function in vivo to restrict the action of BMP signaling in the dorsal spinal cord. Moreover, the I-Smads have distinct roles in regulating the diverse activities of the BMPs. Thus, the ectopic expression of Smad7 suppresses the dI1 and dI3 neural fates and concomitantly increases the number of dI4-dI6 spinal neurons. In contrast, Smad6 most potently functions to block dI1 axon outgrowth. Taken together, these experiments suggest that the I-Smads have distinct roles in spatially limiting the response of cells to 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.

Inhibitory Smad proteins promote the differentiation of mouse embryonic stem cells into ependymal-like ciliated cells.

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.

Increased expression of Smad6 deteriorates murine acute experimental pancreatitis in two models.

BACKGROUND: Smad6 is implicated in the inhibition of bone morphogenetic protein signalling. However, the function of Smad6 in the pancreas remains obscure. METHODS: To elucidate the unknown function of Smad6, we developed transgenic mice selectively expressing Smad6 in pancreatic acinar cells using a plasmid construct coding rat elastase 1 enhancer/promoter. RESULTS: Smad6 transgenic mice had no specific distinguishing phenotype such as body weight, pancreatic wet weight and concentrations of pancreatic protein. However, Smad6 transgenic mice reacted to hyperstimulation by caerulein injection or a diet containing 0.5% ethionine. Maximal amylase release stimulated by CCK-8 was significantly decreased in Smad6 transgenic mice acini, and trypsin activities in transgenic mice acini were significantly increased after stimulation of CCK-8. There was no difference in effect of CCK-8 stimulation on the subsequent increase in intracellular free Ca2+ concentration ([Ca2+](i)) between wild-type and transgenic mice acini. These findings suggest that reduced pancreatic enzyme secretion was caused by the disorder of its downstream signal transduction pathways in acinar cells. The amino acid sequence at the N-terminus of Smad6 was similar to that of synaptosome-associated protein (SNAP) 25 interacting protein, which plays an important role in regulating exocytosis of pancreatic enzymes in acinar cells. Pancreatic SNAP25 protein levels in transgenic mice were decreased after caerulein-induced pancreatitis. CONCLUSIONS: These results suggest that elevated expression of Smad6 inhibits normal function of SNAP25-interacting protein and SNAP25, reduces amylase secretion in acinar cells, and increases the susceptibility of acinar cells to the onset of pancreatitis.

Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.

Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-beta), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.

TGF-beta signaling is dynamically regulated during the alveolarization of rodent and human lungs.

Although transforming growth factor-beta (TGF-beta) signaling negatively regulates branching morphogenesis in early lung development, few studies to date have addressed the role of this family of growth factors during late lung development. We describe here that the expression, tissue localization, and activity of components of the TGF-beta signaling machinery are dynamically regulated during late lung development in the mouse and human. Pronounced changes in the expression and localization of the TGF-beta receptors Acvrl1, Tgfbr1, Tgfbr2, Tgfbr3, and endoglin, and the intracellular messengers Smad2, Smad3, Smad4, Smad6, and Smad7 were noted as mouse and human lungs progressed through the canalicular, saccular, and alveolar stages of development. TGF-beta signaling, assessed by phosphorylation of Smad2, was detected in the vascular and airway smooth muscle, as well as the alveolar and airway epithelium throughout late lung development. These data suggest that active TGF-beta signaling is required for normal late lung development.

Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevis.

The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct.

A critical look at growth factors and epithelial-to-mesenchymal transition in the adult kidney. Interrelationships between growth factors that regulate EMT in the adult kidney.

In the adult kidney, the cellular phenotypes are maintained by a strict balance of growth factors. Epithelial-to-mesenchymal transition (EMT) is a program whereby injured epithelial cells that function as ion and fluid transporters become matrix remodelling mesenchymal cells. This process requires either transcriptional repression of genes that maintain the epithelial phenotype and transcriptional activation, or relieved repression of genes needed for functional myofibroblasts. The transcriptional regulators are controlled by several integrated signalling pathways which are triggered by growth factors. Emerging evidence indicates that the growth factors TGFbeta/CTGF and BMP-7/HGF are the main determinants that maintain the two cellular phenotypes. Both TGFbeta and BMP-7 counteract the activity of each other by cross-inducing their respective inhibitory Smads. Both growth factors may also induce the expression of other factors that can change the cellular environment and enhance their function. Chronic kidney diseases (regardless of the aetiology of the disease) are associated with increased TGFbeta and CTGF expression levels which, in turn, have an inverse effect on the activity level of BMP-7 and HGF, leading to an EMT of injured tubular epithelial cells and a progression of the disease. A detailed understanding of the complex interrelationship between these growth factors may lead to the development of novel drugs.

OAZ regulates bone morphogenetic protein signaling through Smad6 activation.

The intensity and duration of activation of a signal transduction system are important determinants of the specificity of the cellular response to the stimulus. It is unclear how different cells can generate a signal of varying intensity and duration in response to the same cytokine. We investigated the role of the transcriptional activator and Smad1/4 cofactor OAZ in regulating bone morphogenetic protein (BMP) signaling. We demonstrate that upon BMP4 stimulation, an OAZ-Smad1/4 complex binds to and activates the gene encoding Smad6, a specific inhibitor of the BMP pathway. Removal of endogenous OAZ from pluripotent embryonal carcinoma cells prevents the induction of Smad6 by BMP4 and extends the period of detection of phosphorylated Smad1 after BMP stimulation. Conversely, in cells that do not normally express OAZ, such as myoblasts and smooth muscle cells, forced OAZ expression leads to faster and higher Smad6 induction in response to BMP4, decrease of Smad1 phosphorylation, and attenuation of BMP-mediated responses. Our results demonstrate that OAZ can alter the intensity and duration of the BMP stimulus through Smad6 and indicate that the tissue-specific expression of OAZ is a critical determinant of the cellular response to the BMP signal.

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors.

Embryonic ectoderm is fated to become either neural or epidermal, depending on patterning processes that occur before and during gastrulation. It has been stated that epidermal commitment proceeds from a bone morphogenetic protein-4 (BMP-4)-dependent inhibition of dorsal ectoderm neuralization. We recently demonstrated that murine embryonic stem (ES) cells treated with BMP-4 undergo effective keratinocyte commitment and epidermogenesis. Focusing on the precise role of BMP-4 in the early choice between neural and epidermal commitment, we show here that BMP-4 treatment of ES cells leads to a dramatic apoptotic death of Sox-1+ neural precursors with concomitant epidermal engagement. In addition, neutralization of the Smad pathway prevents both the BMP-4 apoptotic process and the inhibition of neural differentiation. Our results suggest that, in mammals, BMP-4, as an active inducer of epidermal commitment, interferes with the survival of neural precursors through induction of their apoptotic cell death.

The Smad6-histone deacetylase 3 complex silences the transcriptional activity of the glucocorticoid receptor: potential clinical implications.

Glucocorticoids play pivotal roles in the maintenance of homeostasis but, when dysregulated, may also have deleterious effects. Smad6, one of the transforming growth factor beta (TGFbeta) family downstream transcription factors, interacts with the N-terminal domain of the glucocorticoid receptor (GR) through its Mad homology 2 domain and suppresses GR-mediated transcriptional activity in vitro. Adenovirus-mediated Smad6 overexpression inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of phosphoenolpyruvate carboxykinase, a well known rate-limiting enzyme of liver gluconeogenesis. Smad6 suppresses GR-induced transactivation by attracting histone deacetylase 3 to DNA-bound GR and by antagonizing acetylation of histone H3 and H4 induced by p160 histone acetyltransferase. These results indicate that Smad6 regulates glucocorticoid actions as a corepressor of the GR. From our results and known cross-talks between glucocorticoids and TGFbeta family molecules, it appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anticatabolic and pro-wound healing properties of the TGFbeta family of proteins.