Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene.

Ying-Wei Mao,John Reddan,David Wan-Cheng Li,Hua Xiang,Stanley Korsmeyer,Juan Wang

doi:10.1074/jbc.m102195200

Abstract

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.

Highlights

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors
Expression of the ␣B-crystallin Gene Is Distinctly Down-regulated in pSFFV-Bcl-2-N/N1003A Cells—To further explore why BCL-2 cannot prevent pSFFV-Bcl-2-N/N1003A cells from H2O2-induced apoptosis, we investigated whether expression of the lens crystallin genes was affected by BCL-2
Associated with the protection of cell death by exogenous mouse ␣B-crystallin expression, activation of caspase-3 found in pSFFV-Bcl-2-N/N1003A cells was largely repressed (Fig. 8E). These results suggest that the down-regulation of ␣B-crystallin in N/N1003A cells by BCL-2 is responsible for the attenuated ability of the pSFFV-Bcl-2-N/N1003A cells to protect against H2O2-induced apoptosis

Summary

Introduction

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Our results demonstrate that BCL-2 can down-regulate expression of the ␣B-crystallin gene in rabbit lens epithelial cells through modulating transactivity of LEDGF and possibly other transcription factors.

Results

Conclusion