Differential protective activity of alpha A- and alpha B-crystallin in lens epithelial cells

alphaA- and alphaB-crystallins are molecular chaperones expressed at low le vels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- an d alphaB-crystallins in lens epithelial cells have not been studied in deta il. In this study, the relative ability of alphaA- and alphaB-crystallin, i n protecting lens epithelial cells from apoptotic cell death was determined , The introduction of alphaA-crystallin in the transformed human lens epith elial (HLE) B-3 lens epithelial cell line (which expresses low endogenous l evels of alphaB-crystallin) led to a nearly complete protection of cell dea th induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor cu. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA -/-alphaB-/- double knockout mouse lens epithelia by infecting primary cell s with Ad12-SV40 hybrid virus. The transformed cell line alpha AYBKO1 deriv ed from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Ce lls expressing different amounts of either alphaA-crystallin or alphaB-crys tallin were isolated. The ability of alphaA- or aB-crystallin to confer pro tection from apoptotic cell death was determined by annexin labeling and fl ow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to threefold higher than that of alphaB-crystallin, Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its gre ater protective effect against apoptosis suggests that it may play a signif icant role in protecting lens epithelial cells from stress.