alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. 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 cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold 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 greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.
