Abstract
Abstract: :
Purpose: Several age–related disease models suggest that oxidation directly inhibits the proteasome (PSM) as part of their pathophysiology. However, recent work in the lens suggests that the ubiquitin (Ub)–PSM system retains activity even in the oldest lens regions and with cataract–associated oxidation. This lens PSM activity may contribute to cataract insults. Certain syndromes of human lens opacity and blindness are believed to be the result of TGFß induced gene expression. The TGFß cataract has also been demonstrated extensively in animal models. In addition to epithelial–mesenchymal transition (EMT)–related gene expression shown in these models, TGFß induces reactive oxygen species in several other tissues. The present study addresses the mechanism of excess TGFß signaling, a probable early insult leading to cataract, and its reliance on the Ub–PSM system. Methods: HLE B–3 cells were treated with TGFß alone or with the proteasome inhibitors MG–132 and lactacystin. Expression levels of EMT–related and oxidation genes were measured by semi–quantitative RT–PCR. Apoptosis was quantitated with FACS analysis of Annexin–FITC stained cells. Western blots were performed with anti–SnoN antibody. Results: TGFß induced the expression of α–SMA, Fibronectin, and ß–ig mRNA in HLE B–3 cells and increased the level of Annexin positive staining. TGFß also induced a time dependent decrease in the level of the Smad repressor, SnoN. γ–GCS mRNA decreased under the same TGFß treatment. PSM inhibitor co–treatment blocked the induction of α–SMA mRNA, the loss of SnoN protein, the decrease in γ–GCS mRNA, and the increase in Annexin positive staining. Conclusions: The HLE B–3 cell line responds to TGFß treatments by activating EMT genes and reducing expression of γGCS. This response is blocked by inhibiting the PSM. This evidence suggests that TGFß utilizes the Ub–PSM system in the lens, and may contribute to the oxidation seen in age–related cataract. The PSM inhibitor effect suggests that the PSM is a novel target for medical therapy for cataract.
Keywords: proteolysis • growth factors/growth factor receptors • cataract