Coordinated Regulation of Extracellular Matrix Synthesis by the MicroRNA-29 Family in the Trabecular Meshwork

Abstract

The microRNA-29 (miR-29) family has emerged, in various tissues, as a key modulator of extracellular matrix (ECM) homeostasis. In this study, the authors investigate the role of the miR-29 family in the regulation of ECM synthesis in the trabecular meshwork (TM) under basal and TGF-β2 stimulatory conditions. Human TM cells were incubated with 2.5 ng/mL activated, recombinant human TGF-β2 for 24, 48, and 72 hours. A specific pharmacologic inhibitor was used to block SMAD3 function in the context of TGF-β2 stimulation. Changes in the expression of the miR-29 family were assessed by real-time PCR. The effect of miR-29 molecules and inhibitors on ECM levels was determined by immunoblot analysis. All three members of the miR-29 family were expressed in cultured TM cells. Although the incubation of TM cells with TGF-β2 induced miR-29a and suppressed miR-29b levels, no significant effect was observed on miR-29c expression. Additional studies revealed that SMAD3 modulates miR-29b expression under basal and TGF-β2 conditions. Subsequent gain- and loss-of-function experiments demonstrated that the miR-29 family functions as a critical suppressor of various ECM proteins under basal and TGF-β2 stimulatory conditions. The findings derived from this study identify the miR-29 family as a critical regulator of ECM expression in the TM and suggest that its modulation by TGF-β2 may be important in controlling ECM synthesis. Together, these data provide further insight into the complex regulatory mechanisms mediating TGF-β2 signaling and ECM production in the TM.