Temporal changes in MMP mRNA expression in the lens epithelium during anterior subcapsular cataract formation.
Transforming growth factor beta (TGFbeta) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFbeta1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFbeta for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (alpha-SMA) immunoreactivity that was evident following 4 days of treatment with TGFbeta. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFbeta treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.
Nathu
Zahra
Z
Department of Pathology and Molecular Medicine, McMaster University, Health Sciences Centre, Hamilton, ON, Canada.
Dwivedi
Dhruva J
DJ
Reddan
John R
JR
Sheardown
Heather
H
Margetts
Peter J
PJ
West-Mays
Judith A
JA
D000199:Actins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000032:analysis / Q000378:metabolism; D002386:Cataract / Q000201:enzymology*; D002460:Cell Line; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007903:Lens Capsule, Crystalline / Q000201:enzymology*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000235:genetics / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000235:genetics*; D042282:Microdissection; D023421:Models, Animal; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000199:Actins; D015820:Cadherins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9