Role of the ubiquitin-proteasome pathway in downregulation of the gap-junction protein Connexin43 by TNF-{alpha} in human corneal fibroblasts.

Purpose. Fibroblasts in the corneal stroma communicate with each other through gap junctions and form a three-dimensional-network structure. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) downregulates the gap-junction protein connexin43 (Cx43) and thereby inhibits gap-junctional intercellular communication (GJIC) in corneal fibroblasts. The authors examined the role of the ubiquitin-proteasome system in the TNF-alpha-induced degradation of Cx43 in these cells. Methods. Human corneal fibroblasts were cultured with TNF-alpha in the absence or presence of the proteasome inhibitor MG132. The expression of Cx43 was detected by immunofluorescence and immunoblot analyses. GJIC was monitored by observing the intercellular diffusion of the fluorescent dye Lucifer yellow. The ubiquitination of Cx43 was evaluated by immunoprecipitation and immunoblot analysis. Results. TNF-alpha induced a decrease both in the amount of Cx43 as detected by immunoblot analysis and in the extent of specific staining for this protein as revealed by immunofluorescence analysis in corneal fibroblasts. These effects of TNF-alpha were inhibited by MG132. MG132 also attenuated the TNF-alpha-induced inhibition of GJIC in these cells. In addition, TNF-alpha induced the ubiquitination of Cx43 in corneal fibroblasts. Conclusions. The ubiquitin-proteasome pathway contributes to the degradation of Cx43 and the inhibition of GJIC induced by TNF-alpha in corneal fibroblasts. The ubiquitin-proteasome system may thus play an important role in the disruption of corneal homeostasis associated with corneal inflammation.