Abstract
Abstract: :
Purpose: Previous work using lens epithelial cells derived from αA and αB–crystallin knockout mice suggests that these chaperones may contribute to the organization and stability of the microtubule cytoskeleton during mitosis. In wild type cells, αA and αB–crystallins accumulate at specific stages of the cell cycle. This study was designed to determine the mechansim of the increase in their expression. Methods: Primary lens epithelial cells were cultured from 1–2 months old mice. Cells were synchronized by serum starvation in the G0 phase, and restimulated by the addition of serum to re–enter the cell cycle, and progress to G1, S, G2 and M phases. Expression of αA and αB–crystallins was quantified by real time PCR methods using gene–specific primers, and by immunoblot analyses. Synthesis of αA–crystallin was analyzed by metabolic labeling with [35–S] methionine supplemented medium, immunoprecipitation with an α–crystallin antibody and SDS–PAGE. Cells were immunolabeled with αA antibody and analyzed by flow cytometry at different stages of the cell cycle. Cell cycle stages in synchronized cells were confirmed by flow cytometry and morphological analysis. Results: Serum–starved cells had as much as ten–fold higher αA and αB–crystallin expression than cells in G1 phase, as demonstrated by immunoblot analysis. The expression of αA and αB–crystallin again increased three–fold as the cells entered the S phase, preceding mitosis. The enhanced accumulation resulted in part from increased synthesis of αA–crystallin, as shown by [35–S] methionine–labeling of immunoprecipitated αA–crystallin on SDS–PAGE. The increase in αA–crystallin expression was confirmed by flow cytometric analysis. Higher αA–crystallin expressing cells increased from 18% in G1 to 42% in S phase, whereas lower αA–crystallin expressing cells decreased from 43% in G1 to 2% in S phase. Increase in αB–crystallin expression in progressing from G1 to S phase was paralleled by an increase in mRNA expression as shown by quantitative real–time RT–PCR analysis. These results demonstrate for the first time that in lens epithelial cells, αA and αB–crystallin expression and synthesis are enhanced preceding mitosis during the cell cycle. Conclusions: Since the absence of αA and αB– crystallin in lens epithelial cells has been associated with increased cell death or genomic instability, our results indicating that the αA– and αB–crystallin expression increases in S phase of the cell cycle are significant. This increase may be important for optimal lens epithelial growth, and implies important roles for these proteins.
Keywords: chaperones • crystallins • proliferation