Neprilysin (NEP), an integral plasma membrane ectopeptidase, plays an important role in turning off peptide signaling events at the cell surface. In preliminary studies we have observed that the absence of NEP predisposes mice to exaggerated pulmonary vascular remodeling in response to chronic hypoxia. The distal migration of pulmonary arterial smooth muscle cells (PASMCs) is a prominent feature of the stimulated change observed. The migration of vascular cells is controlled and regulated by activation of signal transduction pathways. Using bombesin, one of the substrates of NEP, as our model peptide, we were interested in understanding the regulation of migration in PASMCs derived from NEP knockout (KO) mice. We hypothesized that the increased migration of PASMCs from NEP KO mice was regulated by selected kinases. PASMCs were isolated from the pulmonary arteries of NEP KO and age matched wild-type (C57BL6) mice and migration was assessed using the Boyden chamber assay. PASMCs from wild-type and NEP KO mice were exposed to normoxia (20% oxygen) or hypoxia (3% oxygen) for 24h and migration was measured in the absence or presence of 0.1% serum, bombesin and inhibitors to selected kinases (protein kinase C [PKC] RO-31-8220 10μmol/L), (phosphatidyl inositol 3-kinase [PI3K] wortmannin 50nmol/L) and (Rho kinase Y27362 10μmol/L). The data are represented as fold increase in migration compared to that in PASMCs from wild type mice in the absence of serum. PASMCs from NEP KO mice have enhanced migration at baseline (0% and 0.1% serum) and in response to bombesin (10nmol/L), [Table 1
The baseline increase in migration of NEP KO cells was sensitive to Rho kinase and, to a lesser extent, PI3K inhibitors. The response to bombesin was attenuated by inhibitors to, Rho kinase, PI3K and PKC in KO cells. In contrast, inhibition in wild-type cells to bombesin induced cell migration was inhibited by Rho kinase and PI3K but not by PKC. To account for this differential effect, we have also looked at the membrane translocation of various isoforms of PKC in response to bombesin and found that PKC-delta was differentiallly activated in NEP KO cells and may contribute to the increase in migration in response to bombesin. Based on these studies, we conclude that the activation of Rho kinase, PI3-kinase and PKC-delta may play an important role in the enhanced migration of KO cells and could contribute to increase in remodeling observed in chronically hypoxic NEP KO mice.