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升高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Ca2+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在很大程度上仍然未知。骨形态发生蛋白（BMPs）是转化生长因子-2 （TGF-2）超家族的一个亚群，被认为是哺乳动物发育、细胞增殖、分化和凋亡的关键调节因子。近年来，在家族性肺动脉高压中发现BMP受体II （BMPRII）种系突变等证据，提示BMP信号异常参与肺动脉高压的发病机制。特别是，CH对骨形态发生蛋白4 （bone morphogenetic protein 4, BMP4）的调节被认为是影响CHPH发展的重要因素。我们之前证明，CH升高了肺动脉平滑肌细胞（PASMCs）的基底细胞内[Ca2+] ([Ca2+]i)，这在很大程度上是由于通过存储操作的Ca2+通道（SOCCs）增强了存储操作的钙进入（SOCE），可能由典型瞬时受体电位蛋白（trpc）组成。在我们最近的研究中，我们获得的数据显示BMP4在调节TRPCs表达和Ca2+内流中的作用。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)暴露于CH会增加PASMCs中TRPC1和TRPC6、SOCE和basal [Ca2+]i的mRNA和蛋白表达，并且通过特异性BMP4 siRNA敲低BMP4表达或使用其拮抗剂noggin消耗BMP4，可以减弱CH诱导的这些增加；2) CH增强小鼠肺组织BMP4 mRNA和蛋白的表达；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部分缺陷小鼠中受到损害。这些结果表明，BMP4通过调节TRPC通道参与CH期间PASMCs中Ca2+稳态的调节，要么在HIF-1a的下游起作用，要么与HIF-1a依赖的TRPC上调一致。基于上述发现和我们研究中的其他一些数据，我们假设CH导致PASMCs中[Ca2+]i升高是由于或部分原因是HIF-1依赖性BMP4上调，导致远端PASMCs中TRPCs表达、SOCE和基础[Ca2+]i升高，从而导致CHPH。为了验证这一假设，我们将使用微荧光测量和分子生物学相结合的技术在肺、PA和/或PASMCs中进行实验，以实现以下具体目标：1)确定HIF-1和BMP4在CH期间上调TRPCs表达中的作用；2)确定导致缺氧下TRPCs表达增加的BMP4受体和拮抗剂；3)确定BMP4调控PASMCs中TRPCs表达的信号通路；4)确定哪种TRPC有助于SOCE和基础[Ca2+]i响应CH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.