Human alphaA– and alphaB–Crystallins Modulate Expression and Phosphorylation Status of the Tumor Suppressor, p53

Abstract

Abstract: : Purpose: Alpha–Crystallins, members of the small heat shock protein family, are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that human alphaA– and alphaB–crystallins prevent staurosporine–induced apoptosis through interactions with Bax and Bcl–XS to sequester their translocation into mitochondria (Mao et al., 2003, Cell Death & Differentiation, 11:512–526). Moreover, the two crystallins prevent UVA–induced apoptosis through differential regulation of RAF/MEK/ERK and AKT signaling pathways (Liu et al., 2004. Exp. Eye Res. 79:393–403). Here, we demonstrate that human alphaA– and alphaB–crystallins can also modulate expression and phosphorylation status of p53, a master regulator of apoptosis, to partially contribute to prevention of UVA–induced apoptosis. Methods: Human alphaA–, alphaB–crystallin and the related vector–transfected stable expression clones were established with human lens epithelial cells under G418 screening. Expression of human alphaA– and alphaB–crystallins was determined with Western blot and fluorescence microscopy. The transfected cells were irradiated with UVA with doses previously shown to be implicated in cataractogenesis. Apoptosis in each type of cell was determined by Hoechst staining, and regulation on p53 expression and phosphorylation status was analyzed by Western blot analysis. Results: UVA irradiation slightly down–regulates p53 level but substantially enhances p53 phosphorylation at Ser–15. In stable cell lines expressing both alphaA–and alphaB–crystallins, the level of p53 was distinctly downregulated. Moreover, the two alpha–crystallins also attenuate UVA–induced p53 phosphorylation at Ser–15. Conclusions: Our results demonstrate that the two alpha–crystallins can modulate expression and phosphorylation status of p53 to exert their preventive role against UVA–induced apoptosis. Supported by the Hormel Foundation, University of Minnesota Graduate School and Lotus Scholar Professorship Funds from Hunan Province Government and Hunan Normal University.

Keywords: crystallins • cell death/apoptosis • gene/expression