Inhibition Of Rho/Rho Kinase Induces Expression of the Small Heat Shock Protein AlphaB-crystallin in Porcine Lens Epithelial Cells

Abstract

Abstract: : Purpose: The small heat shock protein alphaB-crystallin has been shown to interact with actin cytoskeleton and intermediate filaments. Little is known, however, regarding the cellular mechanisms regulating such interactions. Here we explored the role of the Rho/Rho kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. Methods:Primary lens epithelial cells isolated from porcine lenses were treated with either Rho/Rho kinase inhibitors (C3 exoenzyme, Y-27632 or lovastatin), latrunculin, the PKC inhibitor (GF109203X) or transfected with adenovirus encoding a dominant negative mutant of Rho kinase ( DNRK). Actin stress fibers were stained with rhodamine-phalloidin, whereas focal adhesions and alphaB-crystallin were immunolocalized using vinculin monoclonal antibody or alphaB-crystallin polyclonal antibody, respectively, in conjunction with FITC or TRITC conjugated secondary antibodies. AlphaB-crystallin levels were determined by Western blot analysis. Results: In porcine lens epithelial cells, alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit redistribution in response to actin depolymerization induced by Rho/Rho kinase inhibitors or by DNRK. In contrast, Rho/Rho kinase inhibitors and DNRK caused a dramatic decrease in actin stress fibers and focal adhesions. Interestingly, alphaB-crystallin levels were increased in lovastatin, Y-27632 or DNRK treated lens cells and this response was found to be dose dependent in the case of Y-27632 (10 to 100 µM). Nocodazole (microtubule depolymerizing agent) treatment resulted in increased alphaB-crystallin levels while latrunculin and PKC inhibitor (GF1098203X) were without effect. Cyclohexamide or genistein pretreatment blocked the Rho kinase inhibitor (Y-27632) induced increase in alphaB-crystallin protein levels. Increased alphaB-crystallin was associated with activation of P38 MAP kinase upon Y-27632 treatment. Conclusion: These results indicate that Rho /Rho kinase inactivation activates alphaB-crystallin synthesis and this response is sensitive to tyrosine phosphorylation of MAP kinases such as P38. Further, alphaB-crystallin induction appears to be better correlated with Rho/Rho kinase inactivation than to the actin depolymerization per se.

Keywords: 378 crystallins • 580 signal transduction • 383 cytoskeleton