Suppression of human lens epithelial cell proliferation by proteasome inhibition, a potential defense against posterior capsular opacification.
OBJECTIVE
Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway.


METHODS
HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-beta2 (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 muM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition.


RESULTS
TGF-beta2 suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-beta2 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-beta2, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. 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. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132.


CONCLUSIONS
Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-beta2, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.
Awasthi
Niranjan
N
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ), New Jersey Medical School, Newark, NJ 07101-1709, USA.
Wagner
B J
BJ
D015153:Blotting, Western; D002386:Cataract / Q000517:prevention & control*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007903:Lens Capsule, Crystalline / Q000187:drug effects*; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering / Q000494:pharmacology; D013778:Tetrazolium Salts; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053781:Transforming Growth Factor beta2
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D018809:Proliferating Cell Nuclear Antigen; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering; C509041:TGFB2 protein, human; D013778:Tetrazolium Salts; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D016222:Fibroblast Growth Factor 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017228:Hepatocyte Growth Factor; D051357:Cyclin-Dependent Kinase 2; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde