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


OBJECTIVE
To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation.


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 (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.


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.
Robert M. Berne Cardiovascular Research Center, Charlottesville, VA 22908, USA.
Scott R
SR
Johnstone
Brett M
BM
Kroncke
Adam C
AC
Straub
Angela K
AK
Best
Clarence A
CA
Dunn
Leslie A
LA
Mitchell
Yelena
Y
Peskova
Robert K
RK
Nakamoto
Michael
M
Koval
Cecilia W
CW
Lo
Paul D
PD
Lampe
Linda
L
Columbus
Brant E
BE
Isakson
D000818:Animals; D000831:Animals, Newborn; D050197:Atherosclerosis / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation; D018031:Connexin 43 / Q000378:metabolism* / Q000502:physiology; D019927:Cyclin E / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology
D018031:Connexin 43; D019927:Cyclin E; C543076:GJA1 protein, mouse; D020928:Mitogen-Activated Protein Kinases