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P<P, published online ahead of print November 11, 2009
(Investigative Ophthalmology and Visual Science. )
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.09-3573
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Role of the Ubiquitin-Proteasome Pathway in Down-Regulation of the Gap-Junction Protein Connexin43 by TNF-alpha in Human Corneal Fibroblasts

Kazuhiro Kimura1 and Teruo Nishida2

1Ocular Pathophysiology, Yamaguchi University School of Medicine, Ube, Japan 2Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube City, Japan

Correspondence: Kazuhiro Kimura, Email: k.kimura{at}yamaguchi-u.ac.jp

Abstract

Purpose: Fibroblasts in the corneal stroma communicate with each other via gap junctions and form a three-dimensional network structure. The proinflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) down-regulates the gap-junction protein connexin43 (Cx43) and thereby inhibits gap-junctional intercellular communication (GJIC) in corneal fibroblasts. We have now 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 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.

Key Words: corneal fibroblasts • cytokine • connexins






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