MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation - PubMed (original) (raw)

. 2012 Jul 6;111(2):201-11.

doi: 10.1161/CIRCRESAHA.112.272302. Epub 2012 May 31.

Brett M Kroncke, Adam C Straub, Angela K Best, Clarence A Dunn, Leslie A Mitchell, Yelena Peskova, Robert K Nakamoto, Michael Koval, Cecilia W Lo, Paul D Lampe, Linda Columbus, Brant E Isakson

Affiliations

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

Scott R Johnstone et al. Circ Res. 2012.

Abstract

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.

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

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 (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.

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Figures

Figure 1

Figure 1. In vivo co-localization of Cx43 and cell cycle proteins

Representative immunofluorescence images show carotid vessels from control or PDGF treated C57Bl/6 mice (A–B). Immunofluorescence of Cx43 (red) and cyclin E (green) with blue indicating nuclei (DAPI), “*” indicates the luminal side of the vessels and the scale bar is 20 µm. Immunofluorescent co-localization of proteins is demonstrated by orange staining (overlay of green and red staining) in each section (A). Immunofluorescence, proximity ligation assay for Cx43 and cyclin E, where red staining indicates co-localization of the proteins within 40nm of each other (B). In B, autofluorescence of the internal elastic lamina (IEL, green) and nuclei (blue), “*” indicates the luminal side of the vessels and the scale bar is 20 µm. Immune-TEM with expanded view (red box) of a control carotid taken from a C57Bl/6 mouse (C). The nucleus (Nuc), smooth muscle cell (SMC), IEL, endothelial cell (EC) and red blood cells (RBC) are identified. In C, the red box highlights the larger 25 nm beads labeling Cx43 (yellow arrowhead) and 15 nm beads for cyclin E (red arrowhead) in VSMC layers, bar represents 2 µm. Representative higher magnification iTEM images in D and E of carotids taken from control or PDGF treated C57Bl/6 or ApoE−/− mice immuno-detected for Cx43 or Cx43-P (as labeled, 25 nm gold beads, yellow arrowhead), cyclin E (polyclonal, 8–10 nm gold beads red arrowhead) or CDK2 (monoclonal, 15 nm beads, green arrowhead). Co-localization demonstrated in representative images by the blue arrows. In the corresponding graphs average distance between beads are shown as Cx43 to cyclin E (red bars) or Cx43 and CDK2 (green bars) or Blue bars where interactions are being measured for control (Con), PDGF (PD) or ApoE−/− (Apo) mice. In E, Cx43-P expression is only shown for PDGF and ApoE−/− mice as it is not detected in controls, therefore no statistical significance was determined. In D and E, “***” indicates P<0.001 as compared to controls (n=20 interactions), the scale bar is 200 nm.

Figure 2

Figure 2. In vitro identification of Cx43 co-localization with cell cycle proteins

Total protein and membrane fraction lysates from Cx43+/+ VSMC grown in low serum and treated with PDGF harvested were at specific timepoints over a 24 hours timecourse. Samples were analyzed by Western blot with antibodies against Cx43, Cx43-P, cyclin E, CDK2, GAPDH or β-tubulin and by co-immunoprecipitation with cyclin E coated beads and detection of Cx43 by Western Blot (A). Specificity of Western blot detection was shown using antibody coated beads treated with lysis buffer only to eliminate IgG contamination (A). Expression of Rb, phosphorylated retinoblastoma (pRb)-780, and pRb-807/811 were detected in total protein and membrane fraction lysates from PDGF timecourse experiments (B). Total protein lysates from Cx43+/+ VSMC grown in low serum, pre-treated with ERK inhibitors (U0126 and Roscovitine) or DMSO then treated with PDGF for 24 hours were analyzed by Western blot with antibodies against Cx43, Cx43-P, cyclin E or CDK2 and by co-immunoprecipitation with cyclin E coated beads and detection of Cx43 by Western Blot (C, upper panel). VSMC proliferation of Cx43+/+ VSMC grown in low serum, pre-treated with ERK inhibitors (U0126 and Roscovitine) or DMSO then treated with PDGF for 24 hours was assayed by flow cytometry (C, lower panel). In A–C, black arrowheads identify the expected molecular weight for each protein, colored arrowheads correspond to molecular weights: green is 25kDa, blue is 37 kDa, red is 50kDa, yellow is 100 kDa. In C, “***” indicates P<0.001, n=4.

Figure 3

Figure 3. Cx43 interactions with cyclin E are dependent on MAPK phosphorylation in vitro

The expression of vascular connexins Cx43, Cx37 and Cx45 in VSMC isolated from newborn mouse aortas was identified by Western blotting (A). Site directed mutagenesis was performed for the MAPK sites in both full length Cx43 (a.a.1–382) and Cx43CT (a.a. 236–382) for phospho-mimetic (Aspartate, Cx43MK4D, Cx43CTMK4D) and null phosphorylation (Cx43MK4A, Cx43CTMK4A) constructs (B). Primary VSMC from Cx43−/− mice were transfected with Cx43 plasmids and expression confirmed by Western blotting of Cx43 (Cx43) (C). Levels of expression were quantified against loading for GAPDH (n=3, C). Lysates from Cx43−/− VSMC transfected with each of the Cx43 plasmids and treated with PDGF were incubated with cyclin E coated beads then protein detection performed by Western blot analysis of Cx43 (n=3, D). Cx43CT-GST and cyclin E1-GST proteins were purified by GST bead purification followed by cleavage (Cx43CT proteins only) or elution (glutathione, cyclin E only). Samples were further purified to >90% purity by size exclusion chromatography (SEC) with Cx43CT detected at approximately 17 kDa and cyclin E at 75 kDa due to the addition of the 25kDa GST tag (E). Purified proteins for Cx43CT, Cx43CTMK4A, Cx43CTMK4D and cyclin E were assessed for in vitro binding via ANSEC either as solo proteins or in combination (Cx43CT + cyclin E). Eluted fractions were analyzed for the expression of Cx43 (Cx43) or cyclin E (polyclonal) by Western blotting (n=2, F). Following ANSEC analysis, samples from elution fraction 15 from the Cx43CTMK4D + cyclin E samples were cross-linked using BS3 followed by Western blot analysis (G). In C–E, black arrowheads identify the expected molecular weight for each protein, colored arrowheads correspond to molecular weights: green is 25kDa, blue is 37 kDa, red is 50kDa, Yellow is 100 kDa.

Figure 4

Figure 4. Cx43 MAPK phosphorylation is critical in VSMC proliferation in vitro

VSMC isolated from Cx43+/+, Cx43+/− and Cx43−/− mice were grown in low serum media for 72 hours then treated with EDU (control) or EDU + PDGF (PDGF) and VSMC proliferation measured as a percentage of total cells via flow cytometry (A). Cx43−/− VSMC were transfected with controls (lipofectamine and empty vector) or with the Cx43 plasmids (Cx43, Cx43MK4A, Cx43MK4D). Cells were then treated with EDU + PDGF and VSMC proliferation measured as a percentage of total cells via flow cytometry (B). In graphs, “*” indicates P<0.05, “**” indicates P<0.01 and “***” indicates P<0.001 (A, n=6 and B, n=3).

Figure 5

Figure 5. MAPK phosphorylation of Cx43 is critical in VSMC proliferation in vivo

Representative immunofluorescence images from control and PDGF treated carotids from C57Bl/6 and Cx43-MK4A mice (A). Phosphorylated Cx43 protein expression (Cx43-P) following PDGF treatments was analyzed by immunofluorescence (A). In each image red represents Cx43-P, green represents autofluorescence from the internal elastic lamina, blue indicates nuclei (DAPI) and “*” represents the luminal side of the vessels, scale bar is 20 µm. In B, (top panel), electron micrographs show the ultrastucture of VSMC layers in the carotids from C57Bl/6 and Cx43-MK4A mice under control or PDGF treated conditions, scale bar is 2 µm. SM-actin (SM-22α) protein expression following PDGF treatments was analyzed by immunofluorescence (B, lower panel). In each image magenta represents SM22-α, blue indicates nuclei (DAPI), “*” represents the luminal side of the vessels and scale bar is 20 µm. In C, representative i-TEM images of carotid VSMC from control or PDGF treated Cx43-MK4A mice show Cx43 (Cx43, 25 nm gold beads, yellow arrows), cyclin E (polyclonal, 8–10 nm gold beads red arrows) or CDK2 (monoclonal, 15 nm beads, green arrows). In the corresponding graphs average distance between beads are shown as Cx43 to cyclin E (red bars) or Cx43 to CDK2 (green bars). No-co-localization was demonstrated for control (Con) or PDGF vessels. In vivo cell proliferation measured EDU incorporation (Red) in the nuclei (Blue, DAPI) of C57Bl/6 and Cx43-MK4A mice under control or PDGF treated conditions. Proliferation of VSMC was measured as the number of nuclei incorporating EDU as compared to total nuclei counted (n=4, D). In D, “***” indicates P<0.001 as compared to controls.

Figure 6

Figure 6. MAPK phosphorylation is required for neointima formation following carotid injury

Cross sections from C57Bl/6 and Cx43-MK4A mice under control or injury conditions were stained by H&E for analysis of neointima formation (A). Expression of smooth muscle actin (SM22-α, magenta) was detected in media and neointima layers by immunofluorescence (B). Measures for the areas corresponding to neointima and media layers were used to calculate the ratio in control and injured C57Bl/6 and Cx43-MK4A mice (C n=7). Immunofluorescent detection of Cx43 (D) and Cx43-P (E) in control and injured C57Bl/6 and Cx43-MK4A mice. Scale bars in panels A, B, D and E are 50 µm. In C, “*” indicates P<0.05, “**” indicates P<0.01.

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