Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

发育期肺损伤常导致外周肺血管平滑肌增生 细胞(SMC)和肺血管的早熟肌化，从而限制肺血流和 导致肺动脉高压。旨在增加肺cGMP水平的药物，如吸入剂 一氧化氮气体，已被观察到可减少异常的肺动脉SMC增殖并改善 动物模型中损伤的新生儿肺发育。然而，这些疗法的保护作用是 不完整。 这项拨款提案的长期目标是通过以下方式进一步表征分子机制 其中cGMP对肺动脉SMC(PASMC)的分化和增殖有调节作用。环状GMP 主要通过刺激cGMP依赖的蛋白激酶I(PKGI)来调节PASMC的表型。新兴 有证据表明，cGMP刺激的PKGI核定位和核蛋白磷酸化 转录因子，如CREB和ATF-1，调节SMC表型。最近我们观察到， CGMP刺激的PKGI蛋白降解产生COOH末端的结构性活性激酶片段PKGI， 它转位到SMC的核，对基因表达的反式激活至关重要。对中国传统文化的研究 PKGI蛋白水解酶的裂解位点和底物诱骗的作用表明原蛋白转换酶是 负责PKGI？在SMC的核定位工作。然而，调控PKGI蛋白水解酶和 PKGI的核定位和PKGI在调节SMC表型中的作用尚不清楚。我们的中央 假设PKGI蛋白降解和PKGI核定位是核PKGI的关键决定因素 信号与SMC的分化和增殖。我们将在大鼠的PASMC中检验这一假说 目标。特定目的1表征原蛋白转换酶在调节PKGI蛋白分解中的特定作用 和核PKGI信号。我们将使用RNA干扰来确定哪个原蛋白转换酶(S)调节PKGI 蛋白质分解。我们还将使用原蛋白转换酶抑制剂来测试这些酶是如何调节PASMC的 分化和增殖。特定目标2检查了调节核定位的机制 PKGI‘.高尔基体在PKGI蛋白降解和PKGI主动核转运中的作用 将对核PKGI信号进行评估。特异性目标3检测PKGI蛋白水解酶的功能意义 和核PKGI区隔在调节PASMC分化和增殖中的作用 在受损的发育肺中，PKGI蛋白降解不足。 在这项拨款申请中提出的研究将提供有关机制的新信息。 调控cGMP对SMC表型的调控。他们可能会确定可能是 血管损伤异常，为肺血管预防治疗提供新靶点 疾病。

Injury of the developing lung often causes hyperplasia of peripheral pulmonary vascular smooth muscle cells (SMC) and precocious muscularization of pulmonary vessels, thereby restricting lung blood flow and causing pulmonary artery hypertension. Agents directed at increasing lung cGMP levels, such as inhaled nitric oxide gas, have been observed to decrease abnormal pulmonary artery SMC proliferation and improve injured newborn lung development in animal models. However, the protective effect of these therapies is incomplete. The long-term objective of this grant proposal is to further characterize the molecular mechanisms through which cGMP regulates pulmonary artery SMC (PASMC) differentiation and proliferation. Cyclic GMP modulates PASMC phenotype primarily by stimulating cGMP-dependent protein kinase I (PKGI). Emerging evidence suggests that cGMP-stimulated PKGI nuclear localization and phosphorylation of nuclear proteins and transcription factors, such as CREB and ATF-1, regulate SMC phenotype. Recently we observed that cGMP-stimulated PKGI proteolysis generates a COOH-terminal constitutively active kinase fragment, PKGI¿, that translocates to the nucleus of SMC and is critical for the transactivation of gene expression. Studies of the PKGI proteolysis cleavage site and the effect of substrate decoys suggest that proprotein convertases are responsible for PKGI¿ nuclear localization in SMC. However, the mechanisms regulating PKGI proteolysis and PKGI¿ nuclear localization and the role of PKGI¿ in modulating SMC phenotype are unknown. Our central hypothesis is that PKGI proteolysis and PKGI¿ nuclear localization are key determinants of nuclear PKGI signaling and SMC differentiation and proliferation. We will test this hypothesis in rat PASMC in the following aims. Specific aim 1 characterizes the specific role of proprotein convertases in regulating PKGI proteolysis and nuclear PKGI signaling. We will use RNAi to identify which proprotein convertase(s) regulate PKGI proteolysis. We will also use proprotein convertase inhibitors to test how these proteases regulate PASMC differentiation and proliferation. Specific aim 2 examines the mechanisms that regulate the nuclear localization of PKGI¿. The role of the Golgi apparatus in PKGI proteolysis and of active nuclear transport in PKGI¿ in nuclear PKGI signaling will be evaluated. Specific aim 3 tests the functional significance of PKGI proteolysis and nuclear PKGI¿ compartmentation in regulating PASMC differentiation and proliferation and tests whether PKGI proteolysis is deficient in the injured developing lung. The studies proposed in this grant application will provide novel information about the mechanisms regulating cGMP-driven modulation of SMC phenotype. They are likely to identify pathways that may be abnormal in vascular injury and to provide new targets for therapies directed at preventing pulmonary vascular disease.