Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension.
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BACKGROUND: Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. METHODS AND RESULTS: Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38(MAPK) phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. CONCLUSION: BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH. (+info)
Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors.
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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. (+info)
ERK signaling is a central regulator for BMP-4 dependent capillary sprouting.
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OBJECTIVE: Bone Morphogenetic Protein-4 (BMP-4) and Extracellular-Signal Regulated Kinases (ERK) play crucial roles in vascular diseases. Here, we demonstrate that BMP-4 not only signals through the classical Smad cascade but also activates ERK phosphorylation as an alternative pathway in human umbilical vein endothelial cells (HUVEC) and that Smad and ERK pathways communicate through signal crosstalk. METHODS: HUVECs were treated with BMP-4 and/or MEK inhibitors. Smad 6 and constitutively active (ca) MEK1 were overexpressed. Loss of function of Smad 4 and Smad 6 was achieved by specific siRNA transfection. Cell lysates were analyzed by western blotting for Smad and ERK phosphorylation. HUVEC spheroids were generated for angiogenesis quantification. RESULTS: Treatment with BMP-4 results in a dose- and time-dependent activation of the MEK-ERK 1/2 pathway in addition to activation of the Smad pathway and is blocked by MEK inhibitors. Quantitative in-gel angiogenesis assays in the presence or absence of MEK inhibitors demonstrate that ERK signals are necessary for BMP-4 induced capillary sprouting. Furthermore sprouting is not blocked by inhibition of the Smad signaling pathway. Overexpression of the inhibitory Smad 6 inhibits ERK phosphorylation and ERK-induced capillary sprouting, whereas loss of function of Smad 4 has no effect. CONCLUSIONS: We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signaling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. ERK is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, stimulation of Smad 6 inhibits ERK activation and thus results in a negative feedback loop to fine-tune BMP signaling in HUVECs. (+info)
Negative regulation of inducible nitric-oxide synthase expression mediated through transforming growth factor-beta-dependent modulation of transcription factor TCF11.
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Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation. (+info)
Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevis.
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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. (+info)
TGF-beta signaling is dynamically regulated during the alveolarization of rodent and human lungs.
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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. (+info)
Etoposide-induced Smad6 expression is required for the G1 to S phase transition of the cell cycle in CMT-93 mouse intestinal epithelial cells.
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The inhibitory Smad6 and Smad7 are responsible for cross-talk between TGF-betabone morphogenic protein (BMP) signaling and other cellular signaling pathways, as well as negative feedback on their own signaling functions. Although inhibitory Smads are induced by various stimuli, little is known about the stimuli that increase Smad6 transcription, in contrast to Smad7. Here we demonstrate that etoposide, which induces double strand breaks during DNA replication, significantly up-regulates the transcription of the Smad6 gene in CMT-93 mouse intestinal cells by increasing specific DNA binding proteins. In addition, endogenous inhibition of the Smad6 gene by RNAi interference led to transient accumulation of G1 phase cells and reduction in incorporation of bromodeoxyuridine (BrdU). These findings strongly suggest that Smad6 plays a distinct role in the signaling of etoposide-induced DNA damage. (+info)
Unexpected activities of Smad7 in Xenopus mesodermal and neural induction.
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