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

J Biol Chem. 2001 Nov 16;276(46):43435-45. doi: 10.1074/jbc.M102195200. Epub 2001 Sep 6.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Blotting, Northern
  • Blotting, Western
  • Camptothecin / pharmacology
  • Cell Line
  • Cell Nucleus / metabolism
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Crystallins / biosynthesis*
  • Crystallins / genetics*
  • DNA / metabolism
  • Dose-Response Relationship, Drug
  • Down-Regulation*
  • Epithelial Cells / metabolism*
  • Gene Expression Regulation
  • Genes, Reporter
  • Genes, bcl-2*
  • Humans
  • Lens, Crystalline / metabolism*
  • Mice
  • Oxidative Stress
  • Precipitin Tests
  • Protein Binding
  • Proto-Oncogene Mas
  • RNA / metabolism
  • Rabbits
  • Staurosporine / pharmacology
  • Time Factors
  • Transfection
  • Water / metabolism*

Substances

  • Crystallins
  • MAS1 protein, human
  • Proto-Oncogene Mas
  • Water
  • RNA
  • DNA
  • Chloramphenicol O-Acetyltransferase
  • Staurosporine
  • Camptothecin