Regulation Of Matrix Metalloproteinase And TIMP-1 Expression By TGF-β₁ In Human RPE Cells

Objective To investigate the expression of MMP-2, -9 and TIMP-1 in cultured human retinal pigment epithelial (RPE) cells, to discuss the regulation of MMP-2, MMP-9 and TIMP-1 by TGF-β₁, and to assess altered MMP activity as a determinant in the migration of RPE cells during TGF-β₁-mediated stimulation, so as to search for new easily-performed genetic therapy way for proliferative vitreoretinopathy (PVR).Methods 1) The third-sixth passage RPE cells cultures were treated with TGF-β₁ at different concentration (0.01, 0.1, 1, 10 ng/ml), expression studies were performed with semi-quantitative RT-PCR analyses. 2) The cultured RPE cells were treated with TGF-β₁ at different concentration (0.01, 0.1, 1, 10 ng/ml), and the conditioned media were collected after 36h, or for different time (24, 36, 48h), and the conditioned media were collected. The activities of MMP-2 and MMP-9 were determined by quantitative gelatin zymography. 3) Permeable chamber assays were performed in the presence or absence of the broad-spectrum MMP inhibitor, Doxycycline. RPE cells were displayed increased transmigration through matrigel-coated filters after exposition for 36 hours to 10ng/ml TGF-β₁. Results 1) MMP-2, -9 and TIMP-1 mRNA were expressed in cultured RPE cells. The expression of MMP-2 mRNA was stimulated by TGF-β₁ at 0.01, 0.1, 1, 10 ng/ml in a dose-dependent manner. The expression of TIMP-1 mRNA was down-regulated by TGF-β₁ at 0.01, 0.1. Along with increase of TGF-β₁ (1, 10ng/ml), the expression of TIMP-1 mRNA was gently up-regulated. Compared with control group, there was no significant difference. 2) Between the two gelatinases, MMP-2 and MMP-9, the 68kDa MMP-2 in the conditioned medium was detected by zymography. The MMP-2 secretion was markedly up-regulated by TGF-β₁. The MMP-2 activity was stimulated by TGF-β₁ at 0.01,0.1, 1, 10 ng/ml in a dose-dependent manner, and the secretion of MMP-2 was time-dependently increased by TGF-β₁. In contrast, the 92kDa MMP-9 was not detected. 3) Without Doxycycline, migration of RPE cells stimulated by TGF-β₁ can be significantly facilitated. The number of migrated RPE cells increased about 27%. Transmigration induced by TGF-β₁ was suppressed by Doxycycline, and the migration of RPE cells were decreased through microporous membranes in a dose-deppendent manner. The number of migrated RPE cells reduced 50-70% with increased Doxycycline concentration. Conclusion 1) Cultured human retinal pigment epithelial cells can express MMP-2, -9 and TEMP-1. 2) Expression of MMP-2 is modulated by TGF-β₁, leading to increasing release of MMPs from RPE cells. 3) TEMP-1 expression are not subject to pronounced alterations induced by TGF-β₁, leading to a directional shift in the balance between MMP and TEMP. 4) Changes in MMP expression and activity apparently play a significant role in facilitating migration of RPE cells. 5) TGF-β₁-mediated intracellular signaling pathyways, in particular by selective chemical MMP inhibitors, is a novel strategy to treat the development of PVR.