Sustained Activation of Rho GTPases Promotes a Synthetic Pulmonary Artery Smooth Muscle Cell Phenotype in Neprilysin Null Mice

Objective - Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells. Approach and Results - PASMCs isolated from wild-type and NEP -/- mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP -/- PASMCs compared with wild-type. Rac and Rho (ras homology family member) levels and activity were higher in NEP -/- PASMCs, and ShRNA to Rac and Rho restored SM-protein, and attenuated the enhanced migration and proliferation of NEP -/- PASMCs. SM-gene repressors, p-Elk-1, and Klf4 (Kruppel lung factor 4), were higher in NEP -/- PASMCs and decreased by shRNA to Rac and Rho. Costimulation of wild-type PASMCs with PDGF (platelet-derived growth factor) and the NEP substrate, ET-1 (endothelin-1), increased Rac and Rho activity, and decreased SM-protein levels mimicking the NEP knock-out phenotype. Activation of Rac and Rho and downstream effectors was observed in lung tissue from NEP -/- mice and humans with chronic obstructive pulmonary disease. Conclusions - Sustained Rho activation in NEP -/- PASMCs is associated with a decrease in SM-protein levels and increased migration and proliferation. Inactivation of RhoGDI (Rho guanine dissociation inhibitor) and RhoGAP (Rho GTPase activating protein) by phosphorylation may contribute to prolonged activation of Rho in NEP -/- PASMCs. Rho GTPases may thus have a role in integration of signals between vasopeptides and growth factor receptors and could influence pathways that suppress SM-proteins to promote a synthetic phenotype.