Inhibition of Cx43 attenuates ERK1/2 activation, enhances the expression of Cav­1 and suppresses cell proliferation.

ABSTRACT

In addition to being an important component of the gap junction, connexin 43 (Cx43) has been shown to regulate other cellular functions, including cell proliferation. This regulatory role of Cx43 may be important in therapeutic situations, including wound healing or ischemic injuries. Caveolin­1 (Cav­1) has been shown to regulate angiogenesis. The aim of the present study was to analyze whether Cx43 counter­regulates Cav­1 in controlling the proliferation and migration of endothelial cells. The inhibition of Cx43 with niflumic acid, flufenamic acid and 18­α­glycyrrhetinic acid in cultured human umbilical vein endothelial cells resulted in decreased phosphorylation of extracellular signal­regulated kinase (ERK)1/2 and increased expression of Cav­1, as shown by western blot analysis. Furthermore, the inhibition of Cx43 resulted in a 50±7% decrease in cell proliferation, determined using a crystal violet assay, a 48±5% decrease in migration, determined using a migration assay, and a 49±6% decrease in endothelial tube formation, determined using a Matrigel assay, compared with the control. Similar results were obtained following specific inhibition of Cx43 by mimetic peptides (Gap26 and Gap27). Inhibition of the mitogen­activated protein kinase kinase/ERK pathway with PD­98059 resulted in an increased expression of Cav­1 and a reduction in the expression of Cx43. Furthermore, cell proliferation, migration and tube formation in endothelial cells were impaired. By contrast, downregulation of the protein expression of Cav­1 by small interference RNA resulted in increased expression of Cx43 and phosphorylation of ERK1/2. Accordingly, the number of cells in the Cav­1 treated­group increased by 35±5% compared with the controls. The data of the present study showed that Cav­1 suppressed cell proliferation by inhibiting the activity of Cx43, which is upstream of ERK1/2. The downregulation of Cav­1 protein resulted in loss of the inhibitory activity of Cav­1 on cell proliferation and led to increased cell proliferation. This counter­regulatory effect of Cx43 may be of importance in therapeutic angiogenesis.