Connexin 43 overexpression induces lung cancer angiogenesis in vitro following phosphorylation at Ser279 in its C-terminus

Abstract

Blocking angiogenesis can inhibit tumor growth and metastasis. However, the mechanism underlying regulation of lung cancer angiogenesis remains unclear. The gap junction protein connexin 43 (Cx43) is implicated in angiogenesis. The aim of the present study was to determine the role of Cx43 in angiogenesis in vitro and its signaling pathways. Human pulmonary microvascular endothelial cells were transfected with Cx43-targeting siRNA or Cx43-overexpressing recombinant plasmid vector. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine Cx43, zonula occludens-1 (ZO-1), E-cadherin, β-catenin, von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression levels, respectively. Tyr265, Ser279, Ser368, and Ser373 phosphorylation levels in the C-terminus of Cx43 and intracellular and membranal Cx43 contents were determined using western blotting. Additionally, immunofluorescence, tube formation, Cell Counting Kit-8, and Transwell migration assays were performed. The results revealed that compared with that in the control samples, Cx43, ZO-1, E-cadherin, β-catenin, vWF, and PAI-1 mRNA and protein expression were significantly increased in the Cx43 overexpression group and significantly decreased in the Cx43-knockdown group. Moreover, the phosphorylation level of Ser279 as well as cell proliferation and migration rates were markedly increased in the Cx43 overexpression group, and tube formation revealed that the potential of angiogenesis was also increased. Conversely, in the Cx43-knockdown group, the phosphorylation level of Ser279 and cell proliferation and migration rates were reduced, and the potential of angiogenesis was greatly impaired. Under Cx43 overexpression, membranal Cx43 content was significantly increased, whereas under Cx43 knockdown, it was significantly reduced. Therefore, Cx43 overexpression could induce pulmonary angiogenesis in vitro by promoting cell proliferation and migration and activating ZO-1, E-cadherin, β-catenin, vWF, and PAI-1. This may be achieved by promoting phosphorylation and activation of the intracellular signal site Ser279 at the C-terminus of Cx43.

Keywords: C terminus; angiogenesis; connexin 43; human pulmonary microvascular endothelial cells; phosphorylation activation.

Figure 1.

Relative expression levels of various mRNAs in different groups determined using RT-qPCR (the resulting data are presented as the mean ± standard deviation; *P<0.01 vs. NC; n=3 in each group). RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, normal control group; Cx43, connexin 43; ZO-1, zonula occludens-1; vWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1; siRNA, small-interfering RNA.

Figure 2.

Relative expression levels (normalized with NC) of various proteins in different groups as determined through western blotting (the resulting data are presented as the mean ± standard deviation; *P<0.01 vs. NC; n=5 in each group). (A) Densitometric analysis and (B) representative western blots. Groups: a, NC; b, Cx43-siRNA; and c, Cx43. NC, normal control group; Cx43, connexin 43; ZO-1, zonula occludens-1; vWF, von Willebrand factor; PAI-1, plasminogen activator inhibitor-1; siRNA, small-interfering RNA.

Figure 3.

Relative expression levels (normalized with NC) of various proteins in different groups as determined using western blotting (the resulting data are presented as the mean ± standard deviation; *P<0.01 vs. NC; ns, P>0.05 vs. NC; n=5 in each group). (A) Densitometric analysis and (B) representative western blots. Groups: a, NC; b, Cx43-siRNA; c, Cx43. NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA; ns, not significant.

Figure 4.

Relative intracellular and membranal expression levels of Cx43 in different groups as determined using western blotting (the resulting data are presented as the mean ± standard deviation; *P<0.05 vs. NC; **P<0.01 vs. NC; n=5 in each group). (A) Densitometric analysis and (B) representative western blots. Groups: a, NC; b, Cx43-siRNA; c, Cx43. NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 5.

Average fluorescence intensity of various groups following immunofluorescence assay (the resulting data are presented as the mean ± standard deviation; *P<0.01 vs. NC; n=5 in each group). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 6.

Cell morphology among various groups following immunofluorescence assay (magnification, ×400; scale bar, 20 µm). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 7.

Number of branches in different groups following tube formation assay (the resulting data are presented as the mean ± SD; *P<0.01 vs. NC; n=30 in each group). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 8.

Cell morphology observed via tube formation assay (magnification, ×50; scale bar, 250 µm). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 9.

Comparison of the average dye transfer distance among different groups using scrape loading/dye transfer assay (the resulting data are presented as the mean ± SD; *P<0.01 vs. NC; n=8 in each group). Units are in µm. NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 10.

Fluorescence observed in various groups using scrape loading dye transfer assay (magnification, ×100; scale bar, 100 µm). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 11.

Comparison of the OD value among different groups via CCK-8 assay (the resulting data are presented as the mean ± SD; *P<0.01 vs. NC; n=3 in each group). OD, optical density; CCK-8, Cell Counting Kit-8; NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Figure 12.

Comparison of the number of migrating cells in the different groups with Transwell migration assay (the resulting data are presented as the mean ± SD; *P<0.01 vs. NC; n=5 in each group). (A) Representative images of migrating cells (magnification, ×100; scale bar, 25 μm) and (B) analysis of number of migrating cells. NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.