Oxidized Phospholipid Species Promote in Vivo Differential Cx43 Phosphorylation and Vascular Smooth Muscle Cell Proliferation

Regulation of both the expression and function of connexins in the vascular wall is important during atherosclerosis. Progression of the disease state is marked by vascular smooth muscle cell (VSMC) proliferation, which coincides with the reduced expression levels of connexin 43 (Cx43). However, nothing is currently known about the factors that regulate post-translational modifications of Cx43 in atherogenesis, which could be of particular importance, due to the association between site-specific Cx43 phosphorylation and cellular proliferation. We compared the effects of direct carotid applications of two oxidized phospholipid derivatives, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), on Cx43 expression and phosphorylation, and on cell proliferation. Since both POVPC and PGPC have been shown to act through different intracellular pathways, we hypothesized that each oxidized phospholipid species could induce differential Cx43 phosphorylation events in the cytoplasmically located carboxyl-terminal region of the protein, which could potentially enhance cell proliferation. Application of POVPC caused a reduction in VSMC Cx43 levels, enhanced its phosphorylation at serine (pS) 279/282, and increased VSMC proliferation both in vivo and in vitro. Treatment with PGPC enhanced VSMC pS368 levels with no associated change in proliferation. These oxidized phospholipid-induced Cx43 post-translational changes in VSMCs were consistent with those identified in ApoE−/− mice. Taken together, these results demonstrate that post-translational phosphorylation of Cx43 could be a key factor in the pathogenesis of atherosclerosis. Regulation of both the expression and function of connexins in the vascular wall is important during atherosclerosis. Progression of the disease state is marked by vascular smooth muscle cell (VSMC) proliferation, which coincides with the reduced expression levels of connexin 43 (Cx43). However, nothing is currently known about the factors that regulate post-translational modifications of Cx43 in atherogenesis, which could be of particular importance, due to the association between site-specific Cx43 phosphorylation and cellular proliferation. We compared the effects of direct carotid applications of two oxidized phospholipid derivatives, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), on Cx43 expression and phosphorylation, and on cell proliferation. Since both POVPC and PGPC have been shown to act through different intracellular pathways, we hypothesized that each oxidized phospholipid species could induce differential Cx43 phosphorylation events in the cytoplasmically located carboxyl-terminal region of the protein, which could potentially enhance cell proliferation. Application of POVPC caused a reduction in VSMC Cx43 levels, enhanced its phosphorylation at serine (pS) 279/282, and increased VSMC proliferation both in vivo and in vitro. Treatment with PGPC enhanced VSMC pS368 levels with no associated change in proliferation. These oxidized phospholipid-induced Cx43 post-translational changes in VSMCs were consistent with those identified in ApoE−/− mice. Taken together, these results demonstrate that post-translational phosphorylation of Cx43 could be a key factor in the pathogenesis of atherosclerosis. One of the markers of atherogenesis is a change in connexin 43 (Cx43) protein expression in the vascular smooth muscle cells (VSMCs) of the large vessels.1Kwak BR Mulhaupt F Veillard N Gros DB Mach F Altered pattern of vascular connexin expression in atherosclerotic plaques.Arterioscler Thromb Vasc Biol. 2002; 22: 225-230Crossref PubMed Scopus (204) Google Scholar, 2Kwak BR Veillard N Pelli G Mulhaupt F James RW Chanson M Mach F Reduced connexin43 expression inhibits atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice.Circulation. 2003; 107: 1033-1039Crossref PubMed Scopus (149) Google Scholar, 3Solan JL Lampe PD Key connexin 43 phosphorylation events regulate the gap junction life cycle.J Membr Biol. 2007; 217: 35-41Crossref PubMed Scopus (129) Google Scholar Reductions in VSMC Cx43 protein expression and functionality has been associated with enhanced VSMC proliferation and differentiation, which is a central component of atherogenic progression.1Kwak BR Mulhaupt F Veillard N Gros DB Mach F Altered pattern of vascular connexin expression in atherosclerotic plaques.Arterioscler Thromb Vasc Biol. 2002; 22: 225-230Crossref PubMed Scopus (204) Google Scholar, 4Kwak BR Jongsma HJ Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions.Mol Cell Biochem. 1996; 157: 93-99Crossref PubMed Scopus (134) Google Scholar Connexin 43 expression and functionality is controlled in a large part through post-translational phosphorylation of sites within its cytoplasmically located carboxyl terminal region. Differential phosphorylation events of the Cx43 carboxyl terminus can influence Cx protein expression,5Doble BW Dang X Ping P Fandrich RR Nickel BE Jin Y Cattini PA Kardami E Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes.J Cell Sci. 2004; 117: 507-514Crossref PubMed Scopus (103) Google Scholar targeting,6Martin PE Blundell G Ahmad S Errington RJ Evans WH Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels.J Cell Sci. 2001; 114: 3845-3855Crossref PubMed Google Scholar function,3Solan JL Lampe PD Key connexin 43 phosphorylation events regulate the gap junction life cycle.J Membr Biol. 2007; 217: 35-41Crossref PubMed Scopus (129) Google Scholar, 7Pahujaa M Anikin M Goldberg GS Phosphorylation of connexin43 induced by Src: regulation of gap junctional communication between transform