Mechanisms of PKG-induced PASMC Differentiation

描述（由申请人提供）：肺损伤导致肺外周动脉肺动脉平滑肌细胞（PASMC）过度增殖和肺动脉高压，在婴儿和儿童中发病率很高。实验室研究表明，吸入一氧化氮（NO）可减轻发育中的肺血管疾病。尽管NO的保护机制尚不完全清楚，但NO调节PASMC的部分机制是通过刺激可溶性鸟苷酸环化酶合成cGMP，从而刺激cGMP依赖性蛋白激酶（PKG）。PKG通过降低细胞增殖、增加收缩蛋白表达和刺激细胞凋亡诱导PASMC分化。该拨款提案的广泛、长期目标是确定和表征PKG调节PASMC分化的分子机制。最近，我们在酵母双杂交实验中发现了两个新的RING finger蛋白TRIM39R和AXOT蛋白（AXOT），它们与PKG相互作用。由于PKG和RING finger蛋白都能调节细胞分化，它们可能具有相似的信号通路，RING finger蛋白可能介导PKG的许多细胞调节活动。该建议的中心假设是PKG通过与RING finger蛋白和调节蛋白相互作用来调节PASMC的分化。特异性目的1研究TRIM39R如何调节PKG对PASMC增殖、分化和凋亡的调节。特异性目的2定义了PKG和AXOT之间的分子相互作用，并定义了AXOT在PASMC中调节PKG重要的非血管舒张作用中的作用。Specific aim 3利用一种新的实验方法，根据PKG在PASMC中磷酸化的差异，鉴定出新的RING finger和转录调节蛋白。本研究将为PKG如何调节PASMC的增殖、分化和凋亡提供重要的新机制信息。他们很可能提供信息，将导致新的治疗靶点的发展，可用于预防肺血管疾病在受伤的肺。

DESCRIPTION (provided by applicant): Lung injury causes excessive pulmonary artery smooth muscle cell (PASMC) proliferation in the peripheral arteries of the lung and pulmonary hypertension and significant morbidity in infants and children. Laboratory studies indicate that inhaled nitric oxide (NO) attenuates pulmonary vascular disease in the developing lung. Although the protective mechanisms of NO are incompletely understood, NO regulates PASMC in part by stimulating soluble guanylate cyclase to synthesize cGMP, which stimulates cGMP-dependent protein kinase (PKG). PKG has been observed to induce PASMC differentiation by decreasing cell proliferation, increasing the expression of contractile proteins, and stimulating apoptosis. The BROAD, LONG-TERM OBJECTIVES of this grant proposal are to identify and characterize molecular mechanisms whereby PKG regulates PASMC differentiation. Recently, we identified two novel RING finger proteins, TRIM39R and axotrophin (AXOT), that interact with PKG in a yeast two-hybrid assay. Since PKG and RING finger proteins both regulate cell differentiation, they may share similar signaling pathways and RING finger proteins may mediate many of the cell regulatory activities of PKG. The central hypothesis of the proposal is that PKG regulates PASMC differentiation by interacting with RING finger proteins and regulatory proteins. Specific aim 1 examines how TRIM39R modulates PKG's regulation of PASMC proliferation, differentiation, and apoptosis. Specific aim 2 defines the molecular interaction between PKG and AXOT and defines the role of AXOT in modulating PKG's important non-vasodilatory actions in PASMC. Specific aim 3 utilizes a novel experimental approach to identify new RING finger and transcription regulating proteins phosphorylated by PKG in PASMC, based on their differential phosphorylation in cells. The studies proposed in this grant application will provide important new mechanistic information about how PKG modulates PASMC proliferation, differentiation, and apoptosis. They are likely to provide information that will lead to the development of novel therapeutic targets that can be used to prevent pulmonary vascular disease in the injured lung.