Abstract
alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA 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 alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.
Highlights
␣A-Crystallin (␣A) is a member of the small heat shock protein family and has the ability to prevent denatured proteins from aggregating in vitro
The extralenticular expression of ␣-crystallins and their ϳ40% sequence identity with Drosophila HSP27 [1, 2] suggest that during evolution ␣-crystallin may have been recruited as a refractive protein in the lens through a gene-sharing mechanism involving duplication of an ancestral small heat shock protein (sHSP) gene [3]
Like other sHSPs, ␣A and ␣B polypeptides can act as molecular chaperones in vitro, preventing protein aggregation induced by heat and other stresses [4]
Summary
31252–31261, 1998 Printed in U.S.A. The Molecular Chaperone ␣A-Crystallin Enhances Lens Epithelial Cell Growth and Resistance to UVA Stress*. ␣A remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of ␣A was not associated with its relocalization to the nucleus These results indicate that ␣A has important cellular functions in the lens over and above its well characterized role in refraction. SHSPs are believed to be important for normal cell growth and differentiation (for example, HSP27 protects cells during stress by preserving actin microfilaments and preventing apoptotic cell death [13,14,15]), little information is available regarding the cellular functions of this family in general and ␣-crystallins in particular. We show that a photoprotective phenotype can be conferred on lens epithelial cells in culture by the expression of ␣A-crystallin
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