Regulation of cGMP-dependent Protein Kinase Expression by Soluble Guanylyl Cyclase in Vascular Smooth Muscle Cells

Abstract

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in nonpassaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the ␣ 1 and ␤ 1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NOresponsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.The nitric oxide (NO)-cGMP signaling pathway plays a critical role in vascular function (1, 2). Cyclic GMP is a second messenger that is generated by cytoplasmic soluble guanylyl cyclases (sGCs), 1 which are activated by NO, or particulate guanylyl cyclases, which are activated by peptide hormones such as atrial natriuretic peptide. sGC is a heterodimer that consists of two subunits, termed ␣ and ␤, which are linked by disulfide bonds, and has a prosthetic heme group covalently bound to the heterodimer (1, 2). NO activates cGMP synthesis by binding to the heme, leading to the conversion of GTP to cGMP. Although two isoforms of each subunit have been cloned from rat and bovine lung, the a 1 b 1 heterodimer has been found in most tissues and cell types, including vascular smooth muscle cells (VSMC) (3-6). NO and cGMP are involved in many physiological processes such as VSMC relaxation, apoptosis, proliferation, migration, and extracellular matrix production (7-10). An important cGMP receptor protein expressed in VSMC is cGMP-dependent protein kinase (PKG), which is ultimately responsible for mediating the effects of cGMP in vascular smooth muscle relaxation (11). There are two distinct genes encoding PKG in mammalian cells, the type I and type II genes (12, 13). Type I, which is the gene product expressed in VSMC, exists as two isoforms, PKG I␣...

Cyclic Guanosine Monophosphate-dependent Protein Kinase I Promotes Adhesion of Primary Vascular Smooth Muscle Cells

“…Many in vitro studies demonstrated an antiproliferative effect of cGMP and cGKI signaling in subcultured VSMCs (Garg and Hassid, 1989;Hassid et al, 1994;Li and Sun, 2005), VSMC-derived cell lines (Capey et al, 2007) or VSMCs that have been transfected with cGKI (Boerth et al, 1997;Browner et al, 2004;Dey et al, 2005). In agreement with these studies, we find that activation of the cGMP/cGKI pathway slightly inhibited proliferation of passaged murine VSMCs.…”

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…This is accompanied by a decline in PKGI activity. A similar decline in PKGI protein as well as PKGI mRNA has been reported by Browner et al (2004) in cultured bovine aortic smooth muscle cells exposed to inflammatory cytokines, NO-donors, or cGMP analogs.…”

“…In all instances, phosphotransferase activity of the respective kinase is increased, and in a few instances, the biological action results at least in part from cross-activation of the "other" kinase. There are many well documented examples of this "cross-activation" in biological processes (Jiang et al, 1992;Kurjak et al, 1999;White et al, 2000;Sellak et al, 2002;Barman et al, 2003;Browner et al, 2004;Burnette and White, 2006;Wörner et al, 2007). However, cross-activation does not seem to occur in some cells; its incidence may reflect effects of selective subcellular microdomains and compartmentation of proteins involved in these pathways (Massberg et al, 1999;Weber et al, 2007).…”

“…In addition to the normal mass action effect of cGMP elevation, this process is mediated by several mechanisms in the cGMP-signaling pathway: 1) increased breakdown of cGMP as a result of cGMP activation of catalytic activities by binding to allosteric sites of certain PDEs (PDEs 2 and 5) (Figs. 6 and 7) (Bender and Beavo, 2006a;Conti and Beavo, 2007); 2) increased breakdown of cGMP resulting from phosphorylation of the regulatory domain (PDE3 and PDE5) (Ahmad et al, 2000;Koesling et al, 2005;Francis et al, 2009); 3) increased binding of cGMP to allosteric sites in PDEs resulting in sequestration of cGMP (PDEs 2 and 5) (Gopal et al, 2001;Kotera et al, 2004a); 4) desensitization of NO-GC Mayer et al, 2009), 5) down-regulation of PKG protein level (Lincoln et al, 2001;Browner et al, 2004;Dey et al, 2009), and 5) up-regulation of PDEs such as PDE1 and PDE5 (T. Kono, S. Francis, and J. Corbin, unpublished observations; Kim et al, 2001).…”

Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity

“…Nonetheless, the finding that prolonged treatment of cultured VSMC with nitrovasodilators decreases cGKI expression (43) suggests a possible role for NO hyporesponsiveness downstream of NOactivated cGMP production, specifically decreased cGMP sensitivity. Prolonged NO and cGMP treatment-mediated downregulation in cGMP sensitivity and cGKI expression in VSMC is further supported by the finding that sGC and cGKI reciprocally regulate one another's level of expression (3). Prolonged NO and cGMP treatment decreases cGMP sensitivity and cGKI expression in lamb pulmonary vein, verifying a significant role for this downstream system in modulating NO responsiveness in unambiguously contractile VSMC (17).…”