MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation

<p>Rationale: 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.</p> <p>Objective: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.</p> <p>Methods and Results: 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 (Cx43CT) and produced null phosphorylation Ser>Ala (Cx43MK4A/Cx43CTMK4A) and phospho-mimetic Ser>Asp (Cx43MK4D/Cx43CTMK4D) 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.</p> <p>Conclusions: 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.</p&gt

MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation

Mitogen activated protein kinase (MAPK) phosphorylation of connexin 43 (Cx43) is associated with proliferation of vascular smooth muscle cells (VSMC) in atherosclerosis. We aimed to investigate whether MAPK phosphorylation of Cx43 directly regulates VSMC proliferation. Using in vivo models of VSMC proliferation e.g. carotid treatments with platelet-derived growth factor–BB (PDGF), we identified that MAPK phosphorylated Cx43 interacts with the cell cycle control proteins cyclin E and CDK2. To confirm this in vitro we isolated primary Cx43–/– VSMC and transfected these with constructs for Cx43 containing null phosphorylation (alanine) or phospho-mimetic (aspartate) mutations of the MAPK serines. Co-immunoprecipitation and proliferation studies in transfected cells combined with analytical size exclusion studies of purified Cx43 C-terminus and cyclin E proteins demonstrated that MAPK phosphorylation of Cx43 is critical for its binding with cyclin E and for VSMC proliferation in vitro. Finally, using a novel knock-in mouse containing Cx43 MAPK alanine mutations (Cx43-MK4A), we showed that the null-phosphorylation mutation disrupts interactions between Cx43 and cyclin E and VSMC proliferation in vivo. We conclude that MAPK phosphorylated Cx43 is a novel interacting partner of cyclin E and is required to promote VSMC proliferation