Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells

Abstract

Abstract-Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-I␣ or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKGdeficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation. (Circ Res. 1998;82:139-146.)Key Words: nitric oxide Ⅲ phenotype Ⅲ matrix protein Ⅲ vascular disease Ⅲ atherosclerosis Ⅲ restenosis

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

“…Transfection of RASMC-The transfection of RASMC with the bovine PKG I␣ cDNA was performed as described previously (17,18). Cells at passage 3 were transfected with 5 g of recombinant pcDNA1-neo/PKG I␣ vector or empty (control) pcDNA1-neo vector using 10 l of Transfectam reagent with precipitation of the DNA-liposome complex for 15 min at room temperature.…”

“…The transfected cells from passage 6 -12 were used for the experiments. For the experiments reported here, at least three different clones of cells expressing physiological levels of PKG and three different clones of cells transfected with empty vector and not expressing PKG were examined, as reported previously (17,18).…”

“…Coincident with the loss of expression of PKG, VSMC assume the more synthetic phenotype. Transfection of the PKG-deficient VSMC with cDNAs encoding either PKG I␣ or the catalytic domain of type I PKG reverted the morphology from synthetic to the original contractile morphology (17,18).…”

“…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).…”

Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner

“…Endothelial cell expression of TSP1 was induced by hypoxia or inhibition of NO synthesis by using N-nitro-L-arginine methyl ester (24). Subsequent studies in mesangial and smooth muscle cells verified that cGMP and cGMP-dependent protein kinase negatively regulate TSP1 gene expression (25,26). It is less clear whether TSP1 can itself regulate NO signaling in endothelial cells.…”