Mutated BIGH3 induces apoptosis in Human Corneal and HeLa cells through an integrin recognition site

Abstract

Abstract: : Purpose:To determine the effects of overexpression of mutated BIGH3 in HeLa and human corneal epithelial (HCE) cells Methods:Six mutations known to be responsible for autosomal dominant corneal dystrophies linked to chromosome 5 were generated in an expression vector containing the BIGH3 gene and transfected in HeLa and HCE cells. The expression and secretion of the various BIGH3-EGFP fusion proteins were controlled by Western blot analysis. Apoptotic cells were identified by Hoechst/propidium iodide staining and LDH activity was measured in the medium of transfected cells. In HCE, apoptosis was also visualized by the characteristic DNA laddering pattern. Serial truncated BIGH3 proteins and site specific mutations were generated in order to determine the exact region that mediated apoptosis Results:The overexpressed BIGH3 fusion protein was secreted independently of its mutation status and was clearly observed in the culture medium by Western blot analysis. Overexpression of mutated BIGH3 induced apoptosis in both cell lines. Although all the disease-causing mutations tested in this experiment did induce apoptosis, the strongest effect was observed with the R124C and R555W mutations. In order to determine the region of the BIGH3 protein implicated in mediating apoptosis, a protein lacking the C-terminal region was generated. Overexpression of the truncated protein was not able to induce apoptosis suggesting that a region located in this domain was necessary for apoptosis to occur. Specific mutation of amino acids Asp-Ile (DI) at amino acid position 617-618 was sufficient to prevent induction of apoptosis Conclusion:. Overexpression of mutated BIGH3 induces apoptosis in HeLa and HCE cells through an integrin-related pathway that uses the DI domain of the 4th internal Fas domain described by Kim et al. (J. Biol. Chem. 2000). This work suggests that apoptosis is an element in the pathophysiology of 5q31-linked corneal dystrophies

Keywords: 323 apoptosis/cell death • 341 cell death/apoptosis • 370 cornea: basic science