Role of Bone Morphogenetic Protein 4 in Hypoxic Pulmomary Hypertension

描述(申请人提供)：慢性缺氧(CH)引起的血管张力持续增加和肺血管重构在慢性缺氧性肺动脉高压(CHPH)的发病机制中起关键作用。尽管在探索钙离子在这些过程中的作用方面已经取得了进展，但潜在的分子机制仍然很大程度上是未知的。骨形态发生蛋白(BMPs)是转化生长因子-2(TGF-2)超家族的一个亚类，是哺乳动物发育、细胞增殖、分化和凋亡的重要调节因子。近年来，随着BMP受体II(BMPRII)胚系突变在家族性肺动脉高压中的发现及相关证据的发现，提示BMP信号异常在肺动脉高压的发病机制中具有重要意义。特别是CH对骨形态发生蛋白4(BMP4)的超调节作用被认为是影响CHPH发生的重要因素。我们先前的研究表明，CH升高肺动脉平滑肌细胞(PASMC)的基础细胞内[Ca~(2+)]i在很大程度上是由于通过可能由典型的瞬时受体潜能蛋白(TRPC)组成的钙通道(SOCC)促进钙内流(SOCE)。在我们最近的研究中，我们获得了BMP4在调节TRPC表达和钙内流中的作用的数据。这些数据包括：1)BMP4处理增加了PASMCs中TRPC1和TRPC6的表达、SOCE和基础[Ca2+]i；2)CH增加了PASMCs中TRPC1和TRPC6、SOCE和基础[Ca2+]i的mRNA和蛋白表达，而通过特异性BMP4 siRNA抑制BMP4的表达或用其拮抗剂noggin去除BMP4的表达可减弱CH诱导的这些增加；2)CH促进小鼠肺组织BMP4的mRNA和蛋白表达；3)HIF-1a过表达增加了PASMCs中BMP4的表达，而CH诱导的BMP4表达的增加在HIF-1a部分缺陷的小鼠中被减弱。这些结果表明，BMP4通过调节TRPC通道参与CHH时PASMCs内钙稳态的调节，既可作用于HIF-1a下游，也可与HIF-1a依赖的TRPC上调协同作用。根据上述发现和我们研究中的其他一些数据，我们推测，CH引起的PASMCs[Ca~(2+)]i升高是由于或部分由于HIF-1依赖的BMP4上调，从而导致PASMCs远端TRPC表达、SOCE和基础[Ca~(2+)]i增加，从而参与CHPH的发生。为了验证这一假说，我们将利用显微荧光测量和分子生物学相结合的技术在肺、PA和/或PASMCs上进行实验，以完成以下特定目标：1)确定HIF-1和BMP4在CHI过程中TRPC表达上调中的作用；2)确定BMP4受体和拮抗剂(S)，它们与缺氧性TRPC表达增加有关；3)确定BMP4调节PASMCs TRPC表达的信号通路；4)确定哪些TRPC参与了CHI后SOCE和基础[钙]i的增加。

DESCRIPTION (provided by applicant): Chronic hypoxia (CH)-induced sustained increases in vascular tone and pulmonary vascular remodeling play key roles in the pathogenesis of chronic hypoxic pulmonary hypertension (CHPH). Despite progresses have been made on exploring the role of Ca2+ in these processes, the underlying molecular mechanisms, however, remain largely unknown. Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor-2 (TGF-2) superfamily, are known as critical regulators in mammalian development, cell proliferation, differentiation and apoptosis. Recently, the identification of germline mutation of BMP receptor II (BMPRII) in familial pulmonary hypertension and other associated group of evidence indicate the implication of abnormal BMP signaling in the pathogenesis of pulmonary hypertension. In particular, ug-regulation of bone morphogenetic protein 4 (BMP4) by CH was suggested to be an important factor that influences the development of CHPH. We previously demonstrated that CH elevated basal intracellular [Ca2+] ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) due in large part to enhanced store-operated calcium entry (SOCE) through store-operated Ca2+ channels (SOCCs) likely composed of canonical transient receptor potential proteins (TRPCs). In our recent studies, we obtained data showing a role of BMP4 in regulation of TRPCs expression and Ca2+ influx. These data include: 1) BMP4 treatment increased TRPC1 and TRPC6 expression, SOCE and basal [Ca2+]i in PASMCs; 2) Exposure to CH increased mRNA and protein expression of TRPC1 and TRPC6, SOCE and basal [Ca2+]i in PASMCs, and these CH-induced increases were attenuated by knockdown of BMP4 expression via specific BMP4 siRNA, or BMP4 depletion using its antagonist noggin; 2) CH enhanced both mRNA and protein expressions of BMP4 in mouse lung; 3) Overexpression of HIF-1a increased BMP4 expression in PASMCs, and the CH-induced increases of BMP4 expression were impaired in HIF-1a partially deficient mice. These results suggest that BMP4 participate in the regulation of Ca2+ homeostasis in PASMCs during CH via modulation of TRPC channels, acting either in downstream of HIF-1a or in concert with HIF-1a dependent up-regulation of TRPCs. On the basis of the above findings and some other data in our studies, we hypothesize that the increased [Ca2+]i in PASMCs caused by CH is due to or partially due to HIF-1 dependent upregulation of BMP4, which leads to increases in TRPCs expression, SOCE and basal [Ca2+]i in distal PASMCs, thereby contributing to CHPH. To test this hypothesis, we will perform experiments in lung, PA and/or PASMCs using the combined techniques of microfluorescence measurements and molecular biology to accomplish the following specific aims: 1) Determine the roles of HIF-1 and BMP4 in up-regulation of TRPCs expression during CH; 2) Determine BMP4 receptors and antagonist(s) that are responsible for hypoxic increases of TRPCs expression; 3) Determine the signaling pathway through which BMP4 regulates TRPCs expression in PASMCs; 4) Determine which TRPC contributes to the increases of SOCE and basal [Ca2+]i in response to CH.