Purchase this article with an account.
O. Alcazar, G. Striker, S. W. Cousins, M. E. Marin-Castano; Hydroquinone-Induced Extracellular Matrix Turnover Deregulation Is Overcome by MMP-14 and TIMP2 Overexpression in Human Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5057.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In recent years, there has been cumulative evidence that oxidant cigarette smoke-related compounds may be involved in the development of dry AMD. In particular, hydroquinone (HQ) has been shown to diminish the activity of the matrix metalloproteinase 2 (MMP-2), which is a key enzyme for extracellular matrix turnover of the Bruch’s membrane. It is believed that active MMP-2 results from the interaction of the inactive pro-MMP-2 with MMP-14 and TIMP2 in a ternary complex. We postulate that overexpression of MMP-14 and TIMP2 in RPE cells may prevent, at least in part, the decrease in MMP-2 activity and increase in collagen IV accumulation after incubation with HQ. In this study we aimed at determining the impact of overexpressing MMP-14 and TIMP2 onto ARPE-19 cells stimulated with HQ at different times.
MMP-14 and TIMP2 cDNAs were amplified by RT-PCR, using RNA from human kidney and specific primers, and subcloned into an expression vector. Transient transfections were performed on ARPE-19 cells using Lipofectamine2000. Forty-eight hours after transfection, cells were incubated with HQ for either 6 or 18 hours, and further collected for protein determination and/or RNA isolation. Immunoblotting, zymography and semi-quantitative RT-PCR analyses were then performed.
MMP-2 activity modestly decreased after exposure of mock-transfected ARPE-19 cells to HQ for 6 hours, and there was not significant change induced by overexpression of either MMP-14 or TIMP2, alone or in combination. However, a longer incubation time (18 hours) with HQ induced a more dramatic impairment in MMP-2 activity. Importantly, MMP-14 and TIMP2 overexpression, alone and in combination, contributed to recover the MMP-2 activity to mock-transfected control levels. Of note, total MMP-2 protein expression remained unchanged at both incubation times (6 and 18 hours) with HQ. In addition, mock-transfected ARPE-19 cells showed decreased levels of collagen IV transcripts when exposed to HQ for 18 hours. Interestingly, cells transfected with MMP-14, TIMP2, and combination of both did not show any significant dysregulation in the transcriptional levels of collagen IV.
Our data suggest that both MMP-14 and TIMP2 contribute to maintain adequate levels of MMP-2 activity in ARPE-19 cells when challenged with the cigarette smoke-related oxidant HQ.
This PDF is available to Subscribers Only