Abstract
Purpose: :
Posterior capsular opacification (PCO), the most frequent postoperative complication of cataract surgery, is caused by the proliferation, migration and transdifferentiation of the remaining lens epithelial cells (LECs). Cytokines such as TGF–ß, FGF–2 and HGF play an important role in development of PCO. Recently, several reports have recommended that PCO be prevented by inhibiting the proliferation of LECs. This study investigates suppression of LEC proliferation by proteasome inhibition, and its signaling pathway.
Methods: :
Human lens epithelial (HLE B–3) cells and human lens epithelium explants from 17–20 week fetal lenses were cultured in MEM supplemented with 20% FBS. Cells were treated with TGF–ß (1 or 10 ng/ml), FGF–2 (20 or 50 ng/ml), HGF (10 ng/ml) and 10 µM MG132. LEC proliferation was determined using both the WST–1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by western blotting. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition.
Results: :
TGF–ß suppressed proliferation of HLE B–3 cells, while FGF–2 and HGF enhanced proliferation. Proliferation suppression by TGF–ß was blocked by adding FGF–2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF–ß, FGF–2 or HGF. These findings were confirmed by observing PCNA expression, which was decreased by TGF–ß and induced by FGF–2 and HGF. FGF–2 addition cancelled the suppressive effect of TGF–ß. In addition, MG132 decreased PCNA expression, either alone or in the presence of TGF–ß and FGF–2. Similar results were obtained using human lens epithelium explant cultures. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4 or CDK6 protein. Transfection of cells with p21 and p27 siRNA knocked down their protein expression and MG132 treatment could not increase the levels of p21 and p27 over basal levels in control. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA.
Conclusions: :
Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF–ß, FGF–2 and HGF. This process is mediated by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking PCO development.
Keywords: posterior capsular opacification (PCO) • proliferation • enzymes/enzyme inhibitors