Downregulation of MMP-2 and -9 by proteasome inhibition: a possible mechanism to decrease LEC migration and prevent posterior capsular opacification.
OBJECTIVE
The proliferation, epithelial-mesenchymal transition (EMT), and migration of residual lens epithelial cells (LECs) after cataract surgery leads to the development of posterior capsular opacification (PCO). The authors have shown that proteasome inhibition suppresses LEC proliferation and EMT. The present study investigates the prevention of LEC migration by proteasome inhibition through the suppression of matrix metalloproteinase (MMP) expression and activity.


METHODS
HLE B-3 and primary human LEC migration assays were performed using polycarbonate membrane inserts and 20% fetal bovine serum (FBS) as chemoattractant. Cultured cells were treated with 1 ng TGF-beta(2), with or without MG132 (proteasome inhibitor) or GM 6001 (MMP inhibitor). Capsular bags with intraocular lenses (IOLs) were prepared from human donor eyes and cultured in serum-free DMEM. The capsular bags were then treated with 1 or 10 ng/mL TGF-beta(2), with or without MG132 (2.5 or 10 muM, respectively). The medium was sampled and replaced every 2 days and analyzed for MMP-2 and -9 activities by SDS-PAGE zymography. Protein and RNA expression were analyzed by Western blotting and RT-PCR, respectively.


RESULTS
Proteasome inhibition blocks LEC migration in HLE B-3 and primary human LECs. To further evaluate the mechanism of decrease in LEC migration by proteasome inhibition, the authors measured MMP-2 mRNA and protein expression and MMP-2 and -9 activities. In HLE B-3 cells, TGF-beta(2) increased MMP-2 mRNA and protein levels; these increases were inhibited by MG132 cotreatment. Medium from HLE B-3 cultures showed MMP-2 and -9 activities, which were induced by TGF-beta(2) treatment and inhibited by MG132 co-treatment. TGF-beta(2) treatment also increased MMP-2 and -9 activities in IOL capsular bag cultures; these were progressively decreased by proteasome inhibition.


CONCLUSIONS
Proteasome inhibition decreases LEC migration. This inhibition is correlated with decreased MMP-2 and -9 activities, observed both with and without TGF-beta(2) treatment. These findings support proteasome inhibition as a therapeutic strategy to prevent PCO.
Awasthi
Niranjan
N
Departments of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ, USA. niranjan.awasthi@utsouthwestern.edu
Wang-Su
Shuh Tuan
ST
Wagner
B J
BJ
D015153:Blotting, Western; D002386:Cataract / Q000517:prevention & control*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology; D006801:Humans; D007903:Lens Capsule, Crystalline / Q000187:drug effects*; D007908:Lens, Crystalline / Q000166:cytology*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015853:Cysteine Proteinase Inhibitors; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9