Effects of high glucose-induced Cx43 downregulation on occludin and ZO-1 expression and tight junction barrier function in retinal endothelial cells

Abstract

Purpose: To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability.

Methods: Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid.

Results: Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability.

Conclusions: Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.

Keywords: connexins; gap junctions; tight junctions.

Figure 1

Western blot analysis of Cx43, ZO-1, and occludin protein levels and gap junction intercellular communication activity in RRECs grown in N, HG, N transfected with Cx43 siRNA, and N transfected with scrambled siRNA. (A) Cells grown in HG medium showed decreased Cx43, ZO-1, and occludin expression compared with those grown in N medium. Cells grown in N medium and transfected with Cx43 siRNA also showed reduced Cx43, ZO-1, and occludin expression compared to cells grown in N medium. (B) A bar graph shows cumulative data indicating significant reduction in ZO-1 and occludin expression in cells grown in HG medium or cells transfected with Cx43 siRNA, bar graph, black: Cx43, white: ZO-1, gray: occludin. (C) Scrape load dye transfer assay after scrape loading indicated reduced transfer of Lucifer yellow into contiguous cells in RRECs grown in HG or transfected with Cx43 siRNA compared with cells grown in N medium or cells transfected with scrambled siRNA. Fluorescent dye was detected in two to three layers in either side of the scrape line in cells transfected with Cx43 siRNA compared with four to five layers in cells grown in N medium or cells transfected with scrambled siRNA, indicating reduced GJIC activity in cells transfected with Cx43 siRNA. Magnification ×100. (D) A bar graph shows significant reduction in the number of dye-coupled cell layers in cells grown in HG condition or cells transfected with Cx43 siRNA. (E) A bar graph shows cumulative data from co-immunoprecipitation assays indicating significant reduction in ZO-1 and occludin expression in cells grown in HG medium or cells transfected with Cx43: Cx43 siRNA; scram: scrambled siRNA. Data are presented as mean ± SD, *P < 0.05; n = 3 for co-immunoprecipitation assays; all other experiments n = 4.

Figure 2

Effect of high glucose and Cx43 siRNA on Cx43, ZO-1, and occludin immunoreactivity in RREC. (A) Representative images of Cx43 immunostaining show decrease in Cx43 plaques in cells grown in high-glucose medium and cells transfected with Cx43 siRNA compared with cells grown in N medium or cells transfected with scrambled siRNA and grown in N medium. (B) Cells grown in high-glucose medium or cells transfected with Cx43 siRNA show reduced ZO-1 immunostaining and increase number of breaks (arrows) in ZO-1 at tight junctions as compared with cells grown in N medium or cells transfected with scrambled siRNA and grown in N medium. (C) Cells grown in high-glucose medium and cells transfected with Cx43 siRNA show reduced occludin immunostaining as compared with cells grown in N medium or cells transfected with scrambled siRNA and grown in N medium. Images were captured at 800 ms exposure. Scale bar: 25 μm.

Figure 3

Cx43 downregulation alone increases cell monolayer permeability in RRECs. Cells grown in HG medium showed increased monolayer permeability compared with cells grown in N medium. Importantly, cells grown in N medium and transfected with Cx43 siRNA showed increased monolayer permeability compared with cells grown in N medium only. No difference in monolayer permeability was observed in cells grown in N medium compared with those transfected with scram siRNA. Data are presented as mean ± SD, *P < 0.05, n = 4.

Figure 4

Transfection with Cx43 plasmid upregulated Cx43 level and reduced HG-induced increase in cell monolayer permeability. (A) Cells transfected with the pEGFPN1 (Cx43p) showed increased expression of Cx43 in normal condition as well as in high-glucose condition. This increase in Cx43 expression through plasmid transfection resulted in increased ZO-1 expression and occludin expression in both normal- and high-glucose condition. Transfection with dominant negative Cx43 (DN) was sufficient to decrease Cx43 expression, resulting in a concomitant decrease in both ZO-1 and occludin expression. (B) A bar graph shows cumulative data indicating that cells transfected with Cx43 cDNA showed a significant increase in Cx43, and those transfected with DN Cx43 exhibited a decrease in Cx43 expression. Both ZO-1 and occludin expression increased and decreased relative to Cx43 expression. Data are presented as mean ± SD, *P < 0.05; n = 4. (C) Cells grown in HG medium exhibited increased monolayer permeability compared with cells grown in N medium. Importantly, transfection with Cx43 plasmid protected cells against HG-induced excess cell monolayer permeability. Additionally, transfection with DN Cx43 cDNA increased cell monolayer permeability in N medium and exacerbated monolayer permeability in cells grown in HG medium, n = 6. (D) Representative images showing GJIC is increased following Cx43p transfection and reduced following Cx43 DN transfection. (E) A bar graph shows cumulative data from four experiments. A significant increase in the number of dye-coupled cell layers was observed in cells transfected with Cx43p and conversely, a reduction in the number of dye-coupled cell layers was observed following Cx43 DN transfection. Data are presented as mean ± SD, *P < 0.05. Cx43, Cx43 plasmid.