THE MOLECULAR CHAPERONE ALPHA-A-CRYSTALLIN ENHANCES LENS EPITHELIAL-CELL GROWTH AND RESISTANCE TO UVA STRESS

alpha A-Crystallin (alpha A) is a member of the small heat shock prote in (sHSP) family and has the ability to prevent denatured proteins fro m aggregating in vitro. Lens epithelial cells express relatively low l evels of alpha A, but in differentiated fiber cells, alpha A is the mo st abundant soluble protein. The lenses of alpha A-knock-out mice deve lop opacities at an early age, implying a critical role for alpha A in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alpha A in lens epithelial cells, we used t he following two systems: alpha A knock-out (alpha A(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress cuk The growth rate of alpha A(-/-) mouse lens epithelial cells was reduce d by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell. sorter analysis of propidium iodide-st ained cells, indicated a relative deficiency of alpha A(-/-) cells in the G(2)/M phases. Exposure of mouse lens epithelial cells to physiolo gical levels of WA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of ap optotic cells in the alpha A(-/-) cultures was increased 40-fold over wild type. In cells lacking alpha A, WA exposure modified F-actin, but actin was protected in cells expressing alpha A Stably transfected ce ll lines overexpressing human alpha A were generated by transfecting e xtended life span human lens epithelial cells with the mammalian expre ssion vector construct pCI-neo alpha A Cells overexpressing alpha A we re resistant to WA stress, as determined by clonogenic survival. alpha A remained cytoplasmic after exposure to either WA or thermal stress indicating that, unlike other sHSPs, the protective effect of alpha A was not associated with its relocalization to the nucleus. These resul ts indicate that alpha A has important cellular functions in the lens over and above its well characterized role in refraction.