Differential Regulation of Components of the Ubiquitin-Proteasome Pathway during Lens Cell Differentiation

PURPOSE: To investigate the role for the ubiquitin-proteasome pathway in controlling lens cell proliferation and differentiation and the regulation of the ubiquitin conjugation machinery during the differentiation process. METHODS: bFGF-induced lens cell proliferation and differentiation was monitored in rat lens epithelial explants by bromodeoxyuridine (BrdU) incorporation and expression of crystallins and other differentiation markers. Levels of typical substrates for the ubiquitin-proteasome pathway, p21(WAF) and p27(Kip), were monitored during the differentiation process, as were levels and activities of the enzymes involved in ubiquitin conjugation. RESULTS: Explants treated with bFGF initially underwent enhanced proliferation as indicated by BrdU incorporation. Then they withdrew from the cell cycle as indicated by diminished BrdU incorporation and accumulation of p21(WAF) and p27(Kip). bFGF-induced cell proliferation was prohibited or delayed by proteasome inhibitors. Lens epithelial explants treated with bFGF for 7 days displayed characteristics of lens fibers, including expression of large quantities of crystallins. Whereas levels of E1 remained constant during the differentiation process, the levels of ubiquitin-conjugating enzyme (Ubc)-1 increased approximately twofold, and the thiol ester form of Ubc1 increased approximately threefold on 7 days of bFGF treatment. Levels of Ubc2 increased moderately on bFGF treatment, and most of the Ubc2 was found in the thiol ester form. Although levels of total Ubc3 and -7 remained unchanged, the proportions of Ubc3 and -7 in the thiol ester form were significantly higher in the bFGF-treated explants. Levels of Ubc4/5 and -9 also increased significantly on treatment with bFGF, and more than 90% of Ubc9 was found in the thiol ester form in the bFGF-treated explants. In contrast, levels of Cul1, the backbone of the SCF type of E3s, decreased 50% to 70% in bFGF-treated explants. CONCLUSIONS: The data show that proteolysis through the ubiquitin-proteasome pathway is required for bFGF-induced lens cell proliferation and differentiation. Various components of the ubiquitin-proteasome pathway are differentially regulated during lens cell differentiation. The downregulation of Cul1 appears to contribute to the accumulation of p21(WAF) and p27(Kip), which play an important role in establishing a differentiated phenotype.