Over-Expression of SARG Triggers Apoptosis in Human Corneal Epithelial Cells

Abstract

Purpose:: SARG (specifically androgen regulated gene) exhibits expression in the basal cells of the stratified corneal epithelium. Little is known about the role of SARG in vivo. We previously reported that SARG may modulate signal transduction via its C-terminal interaction with 14-3-3zeta in HCE (human corneal epithelial cells). Here we present evidence indicating that excess of SARG may perturb this normal interaction and trigger an apoptotic response in HCE cells.

Methods:: Among a series of mSARG deletion clones, the region responsible for interaction with 14-3-3 ζ was mapped via a bacterial 2-hybrid system to the C-terminal domain of mSARG. Clones containing and lacking this region were employed in transient overexpression experiments in HCE and HeLa cell lines using the GeneJammer^TM transfection reagent. Apoptosis was assessed via morphological changes, immunostaining with caspase-3 antibody and TUNEL assays.

Results:: Transient overexpression of full-length and C-terminal mSARG in HCE and HeLa cells caused significant cell loss in 5 days. In contract, minimum or little cell loss in control experiments withtransfection of plasmid carrying N-terminal SARG-EGFP fusion. Cell blebbing and chromosomal condensation were more prevalent among those lines transfected with the mSARG C-terminal region in 2 days after transfection. These results indicated that apoptosis triggered by mSARG may be mediated via SARG C-terminal region. To confirm that these observations were the result of apoptosis and not simple cytoxicity (either from over-expression or from the transfection reagent), TUNEL assays were carried out on these cultures as well as caspase-3 immunostaining. These experiments demonstrated that significant apoptosis occured in those cell cultures which over-expressed the mSARG C-terminal region.

Conclusions:: These data provide supporting evidence for a model of cell death in which the equilibrium between mSARG and 14-3-3 ζ acts to keep HCE cells viable or trigger cell death via an apoptotic mechanism.

Keywords: cornea: epithelium • signal transduction • apoptosis/cell death