Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

The present study was designed to investigate the role of endogenous sulfur dioxide (SO(2)) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO(2) inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO(2) synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO(2) suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO(2) did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO(2) dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO(2) simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO(2) could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO(2)-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO(2) exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.