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

Abstract

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of ␤ 1 and ␤ 3 integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells. INTRODUCTIONThe nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates important functions in many cell types, including vascular smooth muscle cells (VSMCs) Lohmann and Walter, 2005;Hofmann et al., 2006;Lincoln et al., 2006). In addition to its potent vasorelaxant properties, it has been associated with vascular proliferative diseases, such as atherosclerosis and restenosis (von der Leyen et al., 1995;Janssens et al., 1998;Varenne et al., 1998;von der Thusen et al., 2004). Several studies (an excellent summary is given in Lincoln et al., 2006) suggested that activation of cGKI-dependent pathways has antimitogenic effects in smooth muscle cells (SMCs) in culture (Garg and Hassid, 1989;Boerth et al., 1997;Chiche et al., 1998;Brophy et al., 2002) and might be vasculoprotective in the intact animal (Anderson et al., 2000;Sinnaeve et al., 2002). Despite a large body of evidence that active cGKI acts antiproliferative, Hassid and coworkers reported that NO/ cGMP amplified the proliferative response of fibroblast growth factor-2 in primary VSMCs (Hassid et al., 1994). These results were supported by a report that NO and cGMP analogues activated rather than inhibited the mitogen-activated protein (MAP) kinase pathway in freshly isolated rat aortic VSMCs (Komalavilas et al., 1999). An increased MAP kinase activity has been associated with an increase in cell proliferation. The antiproliferative effect of cGMP/cGKI signaling could not be confirmed by a study on atherosclerosis in an in vivo mouse model (Wolfsgruber et al., 2003). This study suggested a proatherogenic role of VSMC cGKI. Furthermore, the recent analysis of several murine models for restenosis did not show significant effects of the presence or absence of cGKI in VSMCs on restenosis (Lukowski et al., 2008).Previous in vitro and in vivo studies led to the hypothesis that cGKI may affect different functions in primary and cultured VSMCs. A loss or reduction of cGKI, as it may occur during passage of VSMCs, is associated with the development of a syn...

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

“…cell number, relative cell number. (31)(32)(33). Therefore, it was tested whether Rp-PET could inhibit cGMP/cGKI-stimulated VSMC growth and phosphorylation of VASP.…”

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

“…Effects of NO/ cGMP/PKG signaling on differentiation/proliferation of vascular smooth muscle in response to growth factors, vasoactive peptides, physical damage and other stimuli have also been clearly demonstrated. However, these effects are still poorly understood and seem to vary depending on the vessels from which the cells originate, conditions under which studies are conducted (primary cultures versus passaged cells), stage of cell differentiation, and the challenge/stimulus used (Dumitrascu et al, 2006;Lincoln et al, 2006;Bouallegue et al, 2007;Lukowski et al, 2008;Weinmeister et al, 2008;Hofmann et al, 2009). …”

“…Full treatment of these topics is beyond the scope of this review. Moreover, NO/cGMP/ PKGI effects may differ substantially among cells within an intact tissue or in the same cells prepared as primary cultures or multiply passaged cells in culture (Lincoln et al, 2006;Weinmeister et al, 2008). Described sections III.A-III.G is a modest compilation of mechanisms that have been shown to involve input from the NO/cGMP/PKGI pathway, and the number and complexity of these continue to expand.…”

“…PKGI phosphorylation of RhoA at Ser-188 blocks the action of this protein in myriad processes; these include signaling through Rho-associated protein kinases to foster increased contraction in vascular smooth muscle, regulation of cellular adhesion, and relief of inhibition of insulin receptor substrate-1 to enhance insulin signaling through activation of the downstream phosphoinositide 3-kinase-Akt cascade (Ellerbroek et al, 2003;Dhaliwal et al, 2007;Weinmeister et al, 2008;Haas et al, 2009;Nossaman and Kadowitz, 2009). In vascular smooth muscle in the absence of cGMP signaling, RhoA translocates from the cytosol to the plasma membrane, where it increases the activity of the Rhoassociated protein kinase.…”

Noninvasive Continuous Monitoring of Adipocyte Differentiation: From Macro to Micro Scales

“…Error bars are the standard error, statistical significance between time points (using analysis of variance): p < 0.0001. which is different but adopts concepts from other commonly used techniques applied in quantitative biology, e.g. a hemocytometer to determine the number of living cells in a culture (Strober, 1997) and pre-engraved gridded plates or gridded stickers (Weinmeister et al, 2008;Finlayson & Freeman, 2009;Neubrand et al, 2010;Doerner et al, 2015;Kraus et al, 2016;Macias-Romero et al, 2016;Thompson et al, 2016;Toneff et al, 2016) for locating, identifying, counting, and tracking movement of cells, cell clusters, or of other organisms. These devices are not used or provide the option to map or estimate the adipogenic propagation.…”