Activation of bone morphogenetic protein/Smad signaling in bronchial epithelial cells during airway inflammation

Bone morphogenetic proteins (BMPs) are pleiotropic secreted proteins, structurally related to transforming growth factor (TGF)-beta and activins. BMPs play pivotal roles in the regulation of embryonic lung development and branching of airways and have recently been considered to influence inflammatory processes in adults due to their chemotactic activity on fibroblasts, myocytes, and inflammatory cells. In this study, we have investigated the possible involvement of BMPs in a model of experimental allergic-airway inflammation in situ using antibodies that detect activated Smad proteins, and have monitored the modulation of BMP ligands during the inflammatory response. Inflamed bronchial epithelial cells and a few scattered alveolar cells expressed levels of phosphorylated Smad1 (pSmad1/5), indicative of active BMP/Smad signaling. This was in contrast to healthy epithelium, which was devoid of immunoreactivity. A mechanistic explanation for increased pSmad1/5 staining during inflammation was provided by the upregulated expression of all the BMP type I receptors, i.e., activin receptor-like kinase (ALK)2, ALK3, and ALK6, in the inflamed bronchial epithelial cells. Furthermore, the mRNA and protein profiles for BMP ligands were significantly altered during airway inflammation with induction of BMP2, BMP4, and BMP6, and downregulation of BMP5 and BMP7. Collectively, our data demonstrate for the first time active BMP/Smad signaling during airway inflammation in bronchial epithelial cells and thus raise the possibility that BMPs could play a determining role in respiratory pathophysiology.     

Regulation of bone morphogenetic protein signalling in human pulmonary vascular development

The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH.     

Prenatal lung epithelial cell-specific abrogation of Alk3-bone morphogenetic protein signaling causes neonatal respiratory distress by disrupting distal airway formation

Bone morphogenetic proteins (BMPs) play important roles in regulating lung development and function although the endogenous regulatory effects of BMP signaling are still controversial. We found that BMP type I receptor Alk3 is expressed predominantly in airway epithelial cells during development. The function of Alk3 in lung development was determined using an inducible knockout mouse model by crossing epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre and floxed-Alk3 mice. Abrogation of Alk3 in mouse lung epithelia from either early lung organogenesis or late gestation resulted in similar neonatal respiratory distress phenotypes accompanied by collapsed lungs. Early-induction of Alk3 knockout in lung epithelial cells caused retardation of early lung branching morphogenesis, reduced cell proliferation, and differentiation. However, late gestation induction of the knockout caused changes in cell proliferation and survival, as shown by altered cell biology, reduced expression of peripheral epithelial markers (Clara cell-specific protein, surfactant protein C, and aquaporin-5), and lack of surfactant secretion. Furthermore, canonical Wnt signaling was perturbed, possibly through reduced Wnt inhibitory factor-1 expression in Alk3-knockout lungs. Therefore, our data suggest that deficiency of appropriate BMP signaling in lung epithelial cells results in prenatal lung malformation, neonatal atelectasis, and respiratory failure.     

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.     

Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor beta (TGFbeta) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.     

BMP4 induces HO-1 via a Smad-independent, p38MAPK-dependent pathway in pulmonary artery myocytes

Bone morphogenetic proteins (BMPs) are multifunctional cytokines, which play a key role in vascular development and remodeling. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been shown to be protective against vascular and lung injury. In a microarray study, we identified HO-1 as a major target of BMP4 signaling in human pulmonary artery smooth muscle cells (PASMCs), and confirmed the induction of HO-1 mRNA and protein by RT-PCR and Western blotting, respectively. Immunoblotting demonstrated that incubation of PASMCs with BMP4 rapidly phosphorylated Smad1/5 and activated the mitogen-activated protein kinases, p38(MAPK) and ERK1/2, in PASMCs, but not JNK. Using pathway selective inhibitors, the induction of HO-1 mRNA and protein was shown to be dependent on activation of p38(MAPK). Induction was independent of Smad1/5 signaling, since HO-1 mRNA and protein induction was intact in PASMCs harboring mutations in the kinase domain of BMP type II receptor, with disrupted Smad signaling. In addition, adenoviral transfection of kinase-deficient BMPR-II also failed to inhibit BMP4-induced HO-1 expression. In functional studies, the HO-1 inhibitor, ZnPP-IX, partly reversed the growth-inhibitory effects of BMP4, and overexpression of HO-1 in PASMCs inhibited serum-stimulated [3H]-thymidine incorporation. Taken together, these findings show that HO-1 is an important Smad-independent target of BMP signaling in vascular smooth muscle. Inhibition of HO-1 function or expression will further increase the proproliferative capacity of BMPR-II-deficient PASMCs and may thus represent a potential "second hit" necessary for disease manifestation.     

Dynamic analysis of BMP‐responsive smad activity in live zebrafish embryos

Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor‐specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP‐responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling. Developmental Dynamics 240:682–694, 2011. © 2011 Wiley‐Liss, Inc.     

Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas.

The eight mammalian Smad proteins mediate cellular signaling from members of the transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and activin families. Smads 1, 5, and 8 transmit signals from BMPs, while Smads 2 and 3 transmit signals from TGF-betas and activin. Smad 4 is a common mediator of both pathways, while Smads 6 and 7 inhibit signaling. Signal transduction involves translocation of Smad complexes to the nucleus and subsequent gene activation. Little is known about the expression of endogenous Smad proteins during development. We identified commercially available Smad antibodies that specifically recognize a unique Smad protein and are suitable for immunohistochemistry. Here we compare the localization of Smads 1, 2, 3, 4, 5, and 6 in tissues of the 15-day gestation mouse embryo. Immunoreactive Smad proteins are seen in many tissues with differences in the localization being dependent upon the cell type. All tissues express Smad 4 and at least one each of the BMP-specific and TGF-beta-specific Smads, while expression of Smad 6 is more restricted. Differences are observed in the nuclear versus cytoplasmic localization among the Smads in different cell types or tissues, suggesting selective activation of Smads during this stage of development.     

小鼠乳腺增生组织中BMP信号通路的改变

目的:探明骨形态发生蛋白(BMP)信号分子在乳腺增生组织中的表达模式和BMP通路的激活情况。方法:8周龄的未孕雌性C57BL/C小鼠按体重随机分为2组:对照组和激素组,每组15只。激素组先以2.5mg/kg的戊酸雌二醇灌胃25 d,再腹腔注射黄体酮(4 mg/kg)5 d诱导乳腺增生;对照组予以等体积的PBS。给药结束后收集乳腺组织,观察乳腺的形态,real-time PCR检测BMP信号分子的表达水平,并用Western blot检测Smad1/5/9的磷酸化水平。结果:与对照组比较,在乳腺增生组织中,BMP信号通路配体BMP2、4、5、6、7、9、13和14,受体BMPR1A及拮抗剂Chrdl1和Twsg1的表达异常(P0.05);而BMP3、BMP12、BMPR1B、BMPR2、Chrd、Chrdl2和Noggin等分子的表达则无明显改变;Western blot结果显示乳腺增生组织的Smad1/5/9的磷酸化水平升高(P0.05)。结论:多个BMP信号分子在乳腺增生组织中异常表达,致使BMP通路异常激活。BMP信号通路可能成为治疗乳腺增生的新靶点。     

Smad signaling in the rat model of monocrotaline pulmonary hypertension

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.     

BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2

Spermatogonial stem cells (SSCs) capable of self‐renewal and differentiation are the foundation for spermatogenesis. Although several factors that govern these processes have been investigated, the underlying molecular mechanisms have not been fully elucidated. Here, we investigated the role of BMP4 in mouse SSC differentiation, and found that SSCs cultured in the presence of BMP4 underwent differentiation, characterized by downregulation of SSC self‐renewal markers, Plzf, and upregulation of SSC differentiation marker, c‐kit. Smad1/5/8 proteins were phosphorylated during BMP4‐induced differentiation. The effects of BMP4 on SSCs were blocked by BMP4 inhibitor (Dorsomorphin). The activation of BMP4/Smad signaling pathway in SSCs increased the expression of Sohlh2, which is involved in the early differentiation of spermatogonia. Knockdown sohlh2 expression by RNA interference abolished the effect of BMP4 on SSC differentiation and the upregulation of c‐kit expression. Overall, our results suggest that BMP4 plays an important role during the early differentiation of SSCs via upregulation of sohlh2. Anat Rec, 297:749–757, 2014. © 2014 Wiley Periodicals, Inc.     

Inhibition of BMP2-induced, TAK1 kinase-mediated neurite outgrowth by Smad6 and Smad7

BACKGROUND: BMP2 is known to play a wide variety of roles, including some in the development of the nervous system. This cytokine has been reported to induce neurite outgrowth in rat pheochromocytoma PC12 cells via the activation of a p38 MAP kinase, although its regulatory mechanism remains largely to be elucidated. RESULTS: BMP2-induced neurite outgrowth in PC12 cells was inhibited by the introduction of a kinase-negative form of a MAP kinase kinase kinase, TAK1, an upstream regulatory kinase for p38 kinase. Following BMP2 stimulation, the expression of Smad6 and Smad7, inhibitory Smad species that are known to inhibit the BMP2-restricted Smad species, Smad1, Smad5 and Smad8, was up-regulated. Unexpectedly, over-expression of either Smad6 or Smad7 in PC12 cells repressed the BMP2-induced neurite outgrowth and severely impeded the p38 kinase pathway. Both of these inhibitory Smads were found to interact physically with TAK1-binding protein, a molecule required for TAK1 activation. CONCLUSIONS: This study demonstrates that BMP2-induced neurite outgrowth in PC12 cells involves activation of the TAK1-p38 kinase pathway which is inhibited by Smad6 and Smad7.     

过表达BMP9对人肺鳞状细胞癌细胞NCI-H520迁移和侵袭的影响

目的:探讨骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对人肺鳞状细胞癌细胞NCIH520迁移和侵袭的影响及其机制。方法:采用RT-PCR和Western blot法分别检测NCI-H520细胞和正常人支气管上皮(HBE)细胞中BMP9的mRNA和蛋白表达水平;用Ad BMP9感染NCI-H520细胞,RT-PCR和Western blot法验证BMP9的变化;应用划痕愈合实验检测各组细胞的迁移能力,Transwell小室检测迁移和侵袭能力,并用RTPCR和Western blot法检测迁移相关因子基质金属蛋白酶2(MMP2)的mRNA和蛋白水平。Western blot法检测BMP-Smad经典通路中Smad1/5的磷酸化水平(p-Smad1/5)。用BMP特异性拮抗剂Ad Noggin与Ad BMP9共处理NCI-H520细胞,检测p-Smad1/5及细胞迁移能力的变化。结果:BMP9在NCI-H520细胞中的mRNA和蛋白水平均比在HBE细胞中低;Ad BMP9感染的NCI-H520细胞中BMP9的mRNA和蛋白水平均明显升高,细胞迁移和侵袭能力均明显下降,MMP2的mRNA和蛋白水平下降,且p-Smad1/5的表达水平明显上调。在NCI-H520细胞中,Noggin能有效逆转BMP9导致的p-Smad1/5升高和细胞迁移抑制能力。结论:外源性BMP9转入肺鳞癌细胞NCI-H520可明显抑制其迁移侵袭的能力,该抑制作用可能与BMP-Smad信号通路的激活有关。     

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development.

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian-specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers.