Role of Bone Morphogenetic Protein 4 in Hypoxic Pulmomary Hypertension

描述（由申请人提供）：慢性缺氧（CH）诱导的血管张力持续增加和肺血管重塑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Ca 2+在这些过程中的作用方面取得了进展，但其潜在的分子机制仍然很大程度上未知。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，在哺乳动物的发育、细胞增殖、分化和凋亡过程中起着重要的调节作用。近年来，家族性肺动脉高压中BMP受体II（BMPRII）的胚系突变的发现以及其他相关证据表明，BMP信号异常在肺动脉高压的发病机制中起重要作用。尤其是CH对骨形态发生蛋白4（BMP 4）的调节作用可能是影响CHPH发生的重要因素。我们先前证明CH升高肺动脉平滑肌细胞（PASMCs）中的基础细胞内[Ca 2 +]（[Ca 2 +]i）在很大程度上是由于通过可能由典型瞬时受体电位蛋白（TRPC）组成的钙库操纵的Ca 2+通道（SOCCs）增强的钙库操纵的钙内流（SOCE）。在我们最近的研究中，我们获得的数据显示BMP 4在TRPC表达和Ca 2+内流的调节中的作用。这些数据包括：1）BMP 4处理增加PASMCs中TRPC 1和TRPC 6的表达、SOCE和基础[Ca 2 +]i：2）CH暴露增加PASMCs中TRPC 1和TRPC 6的mRNA和蛋白表达、SOCE和基础[Ca 2 +]i，并且这些CH诱导的增加通过特异性BMP 4 siRNA敲低BMP 4表达或使用其拮抗剂noggin耗竭BMP 4而减弱; 2）CH促进小鼠肺组织BMP 4的mRNA和蛋白表达; 3）HIF-1a过表达促进PASMCs BMP 4的表达，而CH诱导的HIF-1a部分缺陷小鼠肺组织BMP 4表达的增加被削弱。这些结果表明，BMP 4通过调节TRPC通道参与CH期间PASMCs中Ca 2+稳态的调节，其作用在HIF-1a的下游或与HIF-1a依赖的TRPC上调一致。基于上述结果和我们的研究中的一些其他数据，我们推测CH引起的PASMCs [Ca 2 +]i增加是由于或部分由于HIF-1依赖性BMP 4上调，导致远端PASMCs中TRPCs表达增加，SOCE和基础[Ca 2 +]i增加，从而促进CHPH。为了验证这一假设，我们将在肺、PA和/或PASMCs中使用显微荧光测量和分子生物学的组合技术进行实验，以实现以下特定目的：1）确定HIF-1和BMP 4在CH期间上调TRPCs表达中的作用; 2）确定负责缺氧增加TRPCs表达的BMP 4受体和拮抗剂; 3）确定BMP 4调节PASMCs中TRPC表达的信号通路; 4）确定哪种TRPC有助于响应CH的SOCE和基础[Ca 2 +]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.