Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells

Abstract

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo.The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal-regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC. Vascular smooth muscle cell (VSMC)1 proliferation and migration are associated with several vascular diseases such as atherosclerosis and restenosis following vascular injury (1-5). VSMC from several species when cultured in vitro undergo a change in phenotype from a contractile state to a synthetic state similar to the changes that occur with VSMC in vivo in response to vascular injury (6 -8). Several lines of evidence suggest that nitric oxide (NO) inhibits VSMC proliferation (9 -13), suggesting that signal transduction pathways regulated by NO may be important in VSMC phenotypic modulation. NO stimulates the production of cyclic GMP (14), which, in turn, regulates several functions of VSMC such as smooth muscle relaxation (15). The major receptor protein for cGMP in VSMC is cGMP-dependent protein kinase (PKG), a serine/ threonine kinase that catalyzes the phosphorylation of important proteins that regulate intracellular Ca 2ϩ levels and relaxation of vascular smooth muscle (16). Our laboratory has recently demonstrated that PKG also plays a major role in the regulation of the phenotype and morphology of VSMC (17, 18).The expression of PKG is highly variable in VSMC. When adult rat aortic SMC are subcultured in vitro, PKG expression is reduced to nearly un...

Progressive and Inhibitory Cell Cycle Proteins Act Simultaneously to Regulate Neurotrophin-mediated Proliferation and Maturation of Neuronal Precursors

“…Although the MAPK pathway is a recognized signal transducer for neurotrophins, only recently have both ANP type -A and -C (CNP) and cGMP been shown to utilize this pathway. Both inhibition 73,74 and activation [75][76][77][78] of the MAPK pathway are seen in response to these molecules. The mechanisms underlying these responses are not fully understood.…”

“…PKG directly stimulates MAPK pathway activity, and also up-regulates levels of MAP Kinase Phosphatase-1 (MPK-1), a known feedback inhibitor of the MAPK pathway. [75][76][77]83 The proliferative response of vascular smooth muscle cells to insulin depends on sustained activation of the MAPK pathway that occurs through a reduction in MPK-1 mRNA. 83 Insulin also inhibits secretion of CNP, a potent anti-proliferative agent in this cell type.…”

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

“…Modulation of PKG activity by these methods has revealed significant roles for the NO -activated PKG-dependent signal transduction pathway in cell growth regulation (Cordelier et al, 1997;Chiche et al, 1998;Kim et al, 1999;Komalavilas et al, 1999;Gu et al, 2000). In many cases, this appears to be due to the intersection of PKG signaling with the MAPK and stress-activated kinase-dependent signal transduction pathways (Lander et al, 1996;Suhasini et al, 1998;Go et al, 1999;Komalavilas et al, 1999;Browning et al, 2000) and expression of the cyclindependent kinase inhibitor P21 Waf1/Cip1 (e.g. Gu et al, 2000).…”

“…The in vitro study of PKG is hampered by its rapid downregulation in most cell types when grown in culture (Chiche et al, 1998;Komalavilas et al, 1999). Thus, the role of PKG in cellular growth regulation has relied on the use of primary cultures, transfection to overexpress PKG or the use of pharmacological activators and inhibitors of PKG or sGC.…”

Diarrhea or colorectal cancer: Can bacterial toxins serve as a treatment for colon cancer?

“…These results are in direct contrast with those reported previously using GC-C agonists (i.e., uroguanylin, STa) and͞or specific phosphodiesterase inhibitors in colon cancer cells (13,(40)(41)(42)(43). Also, in other cell types, such as vascular smooth muscle cells (VSMC) and hepatocytes, nitric oxide and certain natriuretic peptides have been shown to activate PKG by concurrent increases in [cGMP] int , and induce apoptosis (44,45). The mechanisms by which these apoptotic events occur have not yet been resolved, though activation of specific members of the mitogen-activated protein kinase (MAPK) cascades have been suggested (44).…”