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B-Crystallin
1 From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; the 2 Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany; and the 3 Department of Biochemistry, University of Njimegen, Njimegen, The Netherlands.
PURPOSE. The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein 
B-crystallin in the RPE in response to oxidative stress and to explore whether B-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells.
METHODS. Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of B-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42°C for 20 minutes) or oxidant-mediated injury (50–300 µM H2O2 for 1 hour). Induction of B-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of B-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing B-crystallin cDNA or a control vector without B-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining.
RESULTS. B-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in B-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of B-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with B-crystallin were more resistant to H2O2-induced cellular injury.
CONCLUSIONS. B-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of B-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.
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