MAPK Phosphorylation of Connexin 43 Promotes Binding of Cyclin E and Smooth Muscle Cell Proliferation

Robert M. Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA.
Circulation Research (Impact Factor: 11.02). 05/2012; 111(2):201-11. DOI: 10.1161/CIRCRESAHA.112.272302
Source: PubMed


Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis.
To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.
We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury.
We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.

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    • "tance regulator ( CFTR ) regulates GJC possibly via a complex mechanism involv - ing c - Src , modulating voltage sensitivity and gating . Further , functional interaction of gap junctions , CFTR and glutamate receptors ( GluRs ) were with cyclin E ( CCNE1 ) , for example after MAPK phosphory - lation , promoting smooth muscle cell proliferation ( Johnstone et al . , 2012b ) . Cx43 also competes with cyclin D1 for bind - ing to heat shock protein 70 ( HSP70 ) ( Hatakeyama et al . , 2013 ) . Further , degradation of connexins was linked to bind - ing to tumor susceptibility gene 101 ( TSG101 ) , an ubiquitin – conjugating enzyme associated with the cell cycle , turnover of proteins , and transcriptional re"
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    • "Endothelium-derived hyperpolarizing factor Edwards et al., 1998; Hutcheson et al., 1999; Yamamoto et al., 1999; Beny and Schaad, 2000; Kansui et al., 2004; Mather et al., 2005; Rath et al., 2012; Howitt et al., 2013 Vascular conducted responses Kruger et al., 2002; Dora et al., 2003b; Simon and McWhorter, 2003; Wolfle et al., 2007; Figueroa and Duling, 2008 Feedback on vasoconstriction Dora et al., 1997; Straub et al., 2011 Smooth muscle cell proliferation Chadjichristos et al., 2006; Liao et al., 2007; Johnstone et al., 2010; Zhong et al., 2012 a Smooth muscle cell differentiation Kwak et al., 2003; Chadjichristos et al., 2008; Shen et al., 2010; Gairhe et al., 2011, 2012 b Endothelial cell proliferation Larson et al., 1997; Kwak et al., 2001; Yeh et al., 2006; Nakano et al., 2008; Wang et al., 2008 c Endothelial cell dysfunction Xie and Hu, 1994; Makino et al., 2008; Chadjichristos et al., 2010; Wang et al., 2012; Ebong and Depaola, 2013 Endothelial cell migration Pepper and Meda, 1992; Pepper et al., 1989; Kwak et al., 2001 a Evidence that gap junction independent pathways are involved in this regulation (Johnstone et al., 2012b). b Evidence that although connexin expression and/or gap junction communication levels correlated to disease they do not appear to be directly linked to phenotypic modulation (Matsushita et al., 2007; Behringer et al., 2012). "
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    Pharmacological reviews 02/2014; 66(2):513-69. DOI:10.1124/pr.112.007351 · 17.10 Impact Factor
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    • "In brief, Cx43 interacts with TGF-b and regulates TGF-b/Smad signaling by triggering the release of Smad2/3 and increasing the phosphorylation of Smad2. Johnstone et al. [52] demonstrated that MAPK phosphorylates Cx43 and this causes it to interact with cyclin E in vascular smooth muscle cells (VSMC), leading to effects on proliferation (Fig. 2). Previously, they showed that Cx43 becomes phosphorylated at its MAPK serine after atherogenic stimuli, which is associated with VSMC proliferation [53]. "
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    FEBS letters 01/2014; 588(8). DOI:10.1016/j.febslet.2014.01.001 · 3.17 Impact Factor
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