Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

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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.