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. 2012 Jun 21:3:220.
doi: 10.3389/fphys.2012.00220. eCollection 2012.

Control of vascular smooth muscle cell growth by connexin 43

Chintamani N Joshi  1 , Danielle N Martin, Patti Shaver, Chaitanya Madamanchi, Barbara J Muller-Borer, David A Tulis
Affiliations

Control of vascular smooth muscle cell growth by connexin 43

Chintamani N Joshi et al. Front Physiol. .

Abstract

Connexin 43 (Cx43), the principal gap junction protein in vascular smooth muscle cells (VSMCs), regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC) and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY), or the Cx inducer diallyl disulfide (DADS) significantly reduced proliferation after 72 h compared with vehicle controls. Bromodeoxyuridine uptake revealed reduction (p < 0.05) in DNA synthesis after 6 h and flow cytometry showed reduced (40%) S-phase cell numbers after 16 h in DADS-treated cells compared with vehicle controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at MAPK-sensitive Serine (Ser)255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared with controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays important roles in the regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSM.

Keywords: Cx43; cAMP; cGMP; protein kinases; vascular smooth muscle cells.

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Figures

Figure 1
Figure 1
Effects of cyclic nucleotide or Cx43 stimulation on VSMC proliferation. (A) In rat primary VSMCs in the presence of 10% serum for 72 h and using the MTT assay, 8Br-cAMP (100 μM), the sGC stimulator BAY (100 nM) or the garlic-derived Cx inducer DADS (50 μM) significantly reduced rat primary VSMC numbers compared with vehicle controls. Data are mean ± SEM with an n = 6. (B) Using hemocytometry after 72 h, both 8Br-cAMP and BAY significantly reduced cell numbers by ∼40%, and the presence of DADS reduced cell numbers by ∼20% (p = 0.06). Data are mean ± SEM of two independent experiments each with n = 3–4. (C) MTT assay performed on VSMCs after 12 h shows no observable changes in any treatment group. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with normalized vehicle controls.
Figure 2
Figure 2
Effects of cyclic nucleotide or Cx43 stimulation on VSMC DNA synthesis. In rat primary VSMCs in the presence of 10% serum for 6 h, DADS (50 μM) significantly reduced the rate of DNA synthesis by 20% compared with vehicle controls estimated by BrdU incorporation. Data are mean ± SE from three independent experiments each with an n = 6. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05compared with normalized vehicle controls.
Figure 3
Figure 3
Effects of 8Br-cAMP and DADS on 10% FBS-stimulated VSMC cell cycle progression. Using flow cytometry after 16 h incubation, a 40% reduction in the percentage of cells in the S-phase was observed in the presence of DADS (50 μM) with no significant changes in the G0/G1 or G2/M phases compared with vehicle controls. Data are mean ± SEM with an n = 3. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05compared with vehicle controls.
Figure 4
Figure 4
Total Cx43 expression in VSMCs. (A) In-cell Western blotting for total Cx43 expression in rat primary VSMC homogenates. Incubation of rat primary VSMCs for 270 min in the presence of 8Br-cAMP (100 μM), 8Br-cGMP (100 μM), BAY (100 nM), or DADS (50 μM) significantly stimulated total Cx43 expression normalized to α-tubulin. Data are from two independent experiments each performed in triplicate. (B) After 24-h incubation the stimulation of Cx43 was significantly increased only after DADS treatment with no observable changes for the 8Br-cAMP, 8Br-cGMP, or BAY treatment groups compared with vehicle controls. For these experiments n = 6 for each treatment. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with normalized vehicle controls. (C) A representative Western blot showing total Cx43 in green and α-tubulin in red using IR-labeled antibodies. These data were obtained from a single Western blot with a column (unrelated to the current study) removed for clarity. (D) Photomicrograph of confluent VSMCs following immunocytochemical staining for total Cx43. Punctate appearance of Cx43 indicating the presence of gap junctions is predominantly at cell-to-cell contacts (indicated by arrows).
Figure 5
Figure 5
Results from in-cell Western analyses showing effects of kinase inhibition on total Cx43 expression in rat primary VSMCs following incubation in the presence of (A) 8Br-cAMP (100 μM), (B) 8Br-cGMP (100 μM), (C) BAY (100 nM), and (D) DADS (50 μM) for 270 min. (A) Inhibition of PKA (by PKI), PKG (by DT2), or PKC (by CalC) each independently subdued 8Br-cAMP-induced increase in Cx43 expression. (B,C) Only PKG and PKC inhibition reversed the effects of 8Br-cGMP and BAY, respectively. (D) Stimulation of Cx43 expression in the presence of DADS was not dependent on PKA, PKG, or PKC. (E) Treatment with any individual kinase inhibitor did not affect total basal Cx43 expression compared with vehicle controls. Data are mean ± SE from n = 4–7 for each treatment. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with normalized vehicle controls; #p < 0.05 comparing inhibitors to agonists alone normalized to vehicle controls.
Figure 6
Figure 6
Gap junction intercellular communication (GJIC) as an indicator of functionality estimated by fluorescence recovery after photobleaching (FRAP). (A) Incubation of rat primary VSMCs for 15 min in the presence of 8Br-cAMP (100 μM) or DADS (50 μM) significantly increased GJIC. Data are mean ± SE from an n = 5–9 for each treatment. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with normalized vehicle controls. (B–E) Representative photomicrographs (630×) of confluent VSMCs undergoing FRAP with the targeted cell outlined. (B) Before photobleaching; (C) immediately after photobleaching; (D) 2.5 min after photobleaching; and (E) 4.5 min after photobleaching at the conclusion of fluorescence recovery.
Figure 7
Figure 7
Effect of kinase inhibition on (A) 8Br-cAMP- and (B) DADS-stimulated GJIC estimated by FRAP. Rat primary VSMCs were incubated for 15 min in the presence of the following PKA, PKG, and PKC inhibitors: PKI (10 μM), DT-2 (10 μM), and Calphostin-C (100 nM), respectively, followed by 8Br-cAMP (100 μM) or DADS (50 μM) alone for 15 min. (A) Stimulation of GJIC by 8Br-cAMP was effectively inhibited by PKC inhibition alone but not by inhibition of PKA or PKG. (B) DADS-mediated GJIC stimulation was partially (non-significantly) reversed by PKA or PKG inhibition. Data are mean ± SE from an n = 4 for the inhibitors treatment and an n = 6–9 for control, 8Br-cAMP and DADS. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with normalized vehicle controls, #p < 0.05 compared with cAMP-treated cells.
Figure 8
Figure 8
In-cell Western analyses of site-specific Cx43 phosphorylation in rat primary VSMCs in the presence of (A) 8Br-cAMP and (B) DADS. (A) In the presence of 8Br-cAMP, phosphorylation of the MAPK-sensitive Ser279 was significantly increased compared to vehicle controls. (B) The presence of DADS significantly increased phosphorylation at the MAPK sensitive Ser255 and Ser279 sites, p34cdc2 kinase-sensitive Ser262, and the PKC-sensitive Ser368. Data are mean ± SE from three independent experiments with an n = 4 for Ser279 phosphorylation, and n = 3–4 for Ser255, 262, and 368 phosphorylation. Student–Newman–Keuls method for multiple comparisons following one-way ANOVA was used. *p < 0.05 compared with respective vehicle controls.
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