TGFβ regulates collagen deposition by modulating PTEN expression and activity in trabecular meshwork cells

Abstract

Purpose : So far no therapeutic target has proved to be clinically useful in preventing the excess deposition of extracellular matrix (ECM) in the trabecular meshwork (TM) of glaucoma patients. Phosphatase and tensin homolog (PTEN) is now considered a major regulator of ECM deposition. Since there is a significant increase of active transforming growth factor-β2 (TGFβ2) in the aqueous humor of primary open-angle glaucoma patients, and TGFβ is known to modulate the expression of PTEN, we investigated whether TGFβ2 regulates ECM deposition in the TM cells by modulating the expression and activity of PTEN.

Methods : Human donor TM cells in culture were treated with 5ng/ml of TGFβ2 for 12, 24, or 48 hrs. Protein and mRNA were extracted and expression of PTEN was studied by immunoblot and real-time PCR, respectively. Phosphorylation of PTEN was examined to analyze its activity. PTEN activity was inhibited using small molecule inhibitor VO-OHpic to study its effect on collagen deposition in TM cells. PTEN was also overexpressed in TM cells to study its effect on collagen deposition induced by TGFβ2.

Results : Treatment with TGFβ2 significantly increased PTEN (2.15 fold, p<0.05) and collagen mRNA (4.1 fold, p<0.0001) in human TM cells at 24 hrs (N=3). TGF-beta also significantly increased PTEN protein expression (2.05 fold, p<0.001) and, as expected, increased expression of collagen protein. However, phosphorylation of PTEN also increased significantly (1.76 fold, p<0.05), indicating that its activity is inhibited. This further correlated with increased phosphorylation of AKT. Inhibition of PTEN activity using VO-OHpic increased collagen deposition by TM cells (11.9 fold). Overexpression of PTEN decreased collagen deposition induced by TGFβ2 in TM cells.

Conclusions : Our previous study has shown that deletion of the PTEN gene induced excess ECM deposition by dermal fibroblasts. Decreased activity of PTEN by phosphorylation is thus a novel mechanism by which TGFβ2 induces collagen deposition in TM cells. Regulation of PTEN activity could serve as a therapeutic target with high potential to prevent excess deposition of ECM in the TM of glaucoma patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.