Downregulation Of Cx43 Expression Research Articles

Downregulation of Connexin 43 Expression by High Glucose Induces Senescence in Glomerular Mesangial Cells

Connexin 43 (Cx43) plays an important role in cell differentiation and growth control, but whether it can be regulated by high glucose and whether it can mediate in glomerular mesangial cells (GMC) the phenotype alterations that are induced by high glucose still remain to be explored. In this study, RNA interference and gene transfer techniques were used to knock down and overexpress Cx43 gene in rat GMC to determine the contribution of Cx43 to GMC senescence that was induced by high glucose. The results show that high glucose (30 mM) not only downregulated Cx43 mRNA and protein expression (P<0.05) but also increased the percentage of senescence-associated beta-galactosidase (SA-beta-gal) stained cells and expression of p21cip1 and p27kip1 (P<0.05), indicating that high glucose promoted rat GMC senescence. Knocking down Cx43 gene expression significantly increased the percentage of SA-beta-gal stained cells and p27kip1 and p21cip1 expression in GMC (P<0.05), whereas overexpression of Cx43 significantly decreased the percentage of SA-beta-gal stained cells (P<0.05). These results demonstrate for the first time that downregulation of Cx43 expression by high glucose promotes the senescence of GMC, which may be involved in the pathogenesis of diabetic nephropathy.

High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes.

To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy. In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures. Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9). High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.

Brain macrophages inhibit gap junctional communication and downregulate connexin 43 expression in cultured astrocytes.

Astrocytes are typically interconnected by gap junction channels that allow, in vitro as well as in vivo, a high degree of intercellular communication between these glial cells. Using cocultures of astrocytes and neurons, we have demonstrated that gap junctional communication (GJC) and connexin 43 (Cx43) expression, the major junctional protein in astrocytes, are controlled by neuronal activity. Moreover, neuronal death downregulates these two parameters. Because in several brain pathologies neuronal loss is associated with an increase in brain macrophage (BM) density, we have now investigated whether coculture with BM affects astrocyte gap junctions. We report here that addition of BM for 24 h decreases the expression of GJC and Cx43 in astrocytes in a density-dependent manner. In contrast, Cx43 is not detected in BM and no heterotypic coupling is observed between the two cell types. A soluble factor does not seem to be involved in these inhibitions because they are not observed either in the presence of BM conditioned media or in the absence of direct contact between the two cell types by using inserts. These observations could have pathophysiological relevance as neuronal death, microglial proliferation and astrocytic reactions occur in brain injuries and pathologies. Because astrocyte interactions with BM and dying neurons both result in the downregulation of Cx43 expression and in the inhibition of GJC, a critical consequence on astrocytic phenotype in those situations could be the inhibition of gap junctions.