Ubiquitin–Proteasome Pathway Function Is Required for Lens Cell Proliferation and Differentiation

Abstract

The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the differentiation of lens epithelial cells into lens fibers. bFGF-induced cell proliferation was monitored in rat lens epithelial explants by bromodeoxyuridine (BrdU) incorporation. Indicators of lens differentiation included expression of crystallins, lens major intrinsic protein 26 (MIP26), CP49, and filensin and morphologic changes such as cell multilayering and elongation or loss of nuclei. Clasto-lactacystin-beta-lactone, the proteasome-specific inhibitor, was used to study the role of the proteasome in controlling the proliferation and differentiation processes. Explants treated with bFGF initially underwent enhanced proliferation, as indicated by BrdU incorporation and multilayering of the epithelial cells. By 4 days of bFGF treatment, most cells withdrew from the cell cycle, as indicated by diminished BrdU incorporation. After 7 days of treatment with bFGF, lens epithelial explants displayed characteristics of lens fibers, including higher ratios of crystallins to other cytoplasmic proteins and expression of large quantities of MIP26, CP49, and filensin. Adding the proteasome inhibitor to the medium simultaneously with bFGF (day 0) or at day 4 prohibited or delayed bFGF-induced cell proliferation and differentiation. This was indicated by reduced BrdU incorporation and decreased expression of beta- and gamma-crystallins, MIP26, CP49, and filensin. Proteasome inhibition also significantly decreased the number of layers and the sizes of differentiating fibers. These data show that proteasome activity is required not only for lens cell proliferation but also required for the transition from the epithelial phenotype to the fiber phenotype.