Abstract
Purpose: :
Matrix metalloproteinases (MMP) are a family of matrix degrading enzymes involved in various biological processes. Previous work from our lab determined the involvement of MMP-2 and 9 in TGFβ induced epithelial to mesenchymal transition (EMT) in lens epithelial cells (LEC). Our recent studies have suggested the Myocardin Related Transcription Factor-A (MRTF-A) works downstream of TGFβ in causing EMT. In the following experiments we have furthered our investigation of MRTF-A and explored the potential relationship between MMP-9 and MRTF-A in the EMT of LECs.
Methods: :
Rat lens explant cultures were used as model system for all experiments. The explants were treated with TGFβ, recombinant human MMP-9 (rhMMP9) and MMP-2/9 inhibitor for 48 hours and immunostained for αSMA and MRTF-A. Immunofluorescent micrographs were analyzed with ImageJ. Cytoplasmic and nuclear intensities of cells were measured to determine the localization of MRTF-A in the cell. Factorial ANOVA test was performed to determine significance. Expression of αSMA was used as an indicator for EMT.
Results: :
In untreated explant cultures MRTF-A remains predominantly cytoplasmic. Treatment of explants with TGFβ (6ng/ml) reveals primarily nuclear MRTF-A with a significant decrease in cytoplasmic MRTF-A. Quantification with ImageJ reveals a marked increase of 77% in nuclear MRTF-A with a significant decrease of 80% in cytoplasmic MRTF-A. The significant increase in nuclear MRTF-A with TGFβ treatment corresponds with an increase in αSMA. When co-treated with an MMP-2/9 (25µM) inhibitor and TGFβ, explants show an increase in αSMA staining that is greater than control but considerably less than TGFβ treatments. This data is also in agreement with a significant decrease in nuclear MRTF-A (11%) and a significant increase in cytoplasmic MRTF-A (59.5%). When treated with rhMMP-9 (1.25µg/ml) alone there is an increase in αSMA production comparable to TGFβ treatment. Quantification confirms a corresponding significant increase in nuclear MRTF-A (26%) with a decrease in cytoplasmic MRTF-A (25.5%).
Conclusions: :
Our previous work (ARVO 2011, 3931) had demonstrated expression and involvement of MRTF-A in TGFβ induced EMT in LECs. Our current work has extended this and quantified the difference in intracellular localization of MRTF-A using ImageJ software in order to further establish the relationship. We have further ascertained that addition of MMP-2/9 specific inhibitors can successfully suppress nuclear migration of MRTF-A with a corresponding decrease in αSMA expression by the cells. Furthermore, nuclear migration of MRTF-A and EMT can be induced in LECs by treatment with rhMMP-9 alone. Together, our data show that MMP-9 works upstream of MRTF-A in TGFβ induced EMT of LECs.
Keywords: EMT (epithelial mesenchymal transition) • transcription factors