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D. J. Dwivedi, J. Robertson, Z. Nathu, J. A. West-Mays; Role of MMP-9 and MMP-2 in TGFß-Induced Anterior Subcapsular Cataracts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4221.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown that adenoviral gene delivery of active porcine TGFß1 to the anterior chamber of the rodent eye produces anterior subcapsular cataracts (ASC) within 4 days. The lack of formation of ASC in MMP-9 knock out (KO) mice suggests that MMP-9 expression is required for mediating the alterations associated with TGFß-induced subcapsular cataract formation. Studies were thus aimed at further determining the mechanism by which MMP-9 may mediate the formation of TGFß-induced ASC.
Wild-type (WT) and MMP-9 KO mice on an FVB background, aged 6-8 weeks were injected with recombination deficient adenovirus containing cDNA coding for active porcine TGFß1 (AdTGFß1) or control vector AdGFP containing green fluorescent protein in the anterior chamber of the eye. Animals were sacrificed at day 4 post-injection. Lenses were dissected and real-time quantitative PCR (RT-QPCR) was carried out to quantify changes in expression for candidate genes. Cells of the human lens epithelial cell line, FHL 124, were cultured in M199 media supplemented with 10% FBS and treated with active human recombinant MMP-9 (100ng/ml) for 3 and 6 hours. Untreated cells served as controls. Cell lysates were examined by western blot analysis.
The RT-QPCR results revealed that 4 days post-injection, lenses from the WT mouse eyes receiving AdTGFß1 exhibited elevated levels of MMP-2 and MMP-9 mRNA, whereas the E-cadherin mRNA expression was substantially suppressed as compared to the lenses from eyes injected with the control vector. In comparison, lenses from MMP-9 KO eyes injected with AdTGFß1 at 4 days post injection lacked expression of MMP-9 mRNA, as expected, and exhibited a small increase in MMP-2 mRNA levels and slight decrease in E-cadherin mRNA levels as compared to controls. Further experiments were carried out, the FHL 124 cells were treated with active recombinant human MMP-9 for 3 and 6 hours. Western blots developed with an MMP-2 specific antibody revealed that unlike control cells, which did not exhibit detectable levels of MMP-2 protein, the cells treated with active MMP-9 revealed bands at 72 kd corresponding to MMP-2 at both 3 and 6 hours.
In MMP-9 KO mice, AdTGFß1 caused a slight increase in MMP-2, but did not induce morphological changes characteristic of ASC. This data in combination with the cell culture findings, suggests that MMP-9 may be required to stimulate the secretion of MMP-2 to a level which is required for initiating the formation of ASC. Further studies will determine how MMP-2 is regulated by MMP-9 in ASC.
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