Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

发育中的肺损伤常引起周围肺血管平滑肌增生 细胞（SMC）和肺血管早熟肌化，从而限制肺血流和 引起肺动脉高压。旨在增加肺部 cGMP 水平的药物，例如吸入 一氧化氮已被观察到可减少异常肺动脉 SMC 增殖并改善 动物模型中新生肺部发育受损。然而，这些疗法的保护作用是 不完整。 该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： cGMP 调节肺动脉平滑肌细胞 (PASMC) 的分化和增殖。循环GMP 主要通过刺激 cGMP 依赖性蛋白激酶 I (PKGI) 来调节 PASMC 表型。新兴 有证据表明 cGMP 刺激 PKGI 核定位和核蛋白磷酸化 CREB 和 ATF-1 等转录因子调节 SMC 表型。最近我们观察到 cGMP 刺激的 PKGI 蛋白水解产生 COOH 末端组成型活性激酶片段 PKGI¿， 它易位至 SMC 细胞核，对于基因表达的反式激活至关重要。研究的 PKGI 蛋白水解裂解位点和底物诱饵的作用表明前蛋白转化酶是 负责 SMC 中的 PKGI 核定位。然而，调节 PKGI 蛋白水解和 PKGI 的核定位以及 PKGI 在调节 SMC 表型中的作用尚不清楚。我们的中央 假设 PKGI 蛋白水解和 PKGI 核定位是核 PKGI 的关键决定因素 信号传导和 SMC 分化和增殖。我们将在下面的大鼠 PASMC 中测试这一假设 目标。具体目标 1 描述了前蛋白转化酶在调节 PKGI 蛋白水解中的具体作用 和核 PKGI 信号传导。我们将使用 RNAi 来识别哪些前蛋白转化酶调节 PKGI 蛋白水解。我们还将使用前蛋白转化酶抑制剂来测试这些蛋白酶如何调节 PASMC 分化和增殖。具体目标 2 检查调节核定位的机制 PKGI¿。高尔基体在 PKGI 蛋白水解中的作用以及 PKGI 中主动核转运的作用 将评估核 PKGI 信号传导。具体目标3测试PKGI蛋白水解的功能意义 和核 PKGI 区室在调节 PASMC 分化和增殖中的作用，并测试是否 受伤的发育中的肺部缺乏 PKGI 蛋白水解作用。 本拨款申请中提出的研究将提供有关该机制的新信息 调节 cGMP 驱动的 SMC 表型调节。他们可能会找出可能的途径 血管损伤异常并为预防肺血管损伤的治疗提供新靶点 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.