Abstract
Purpose :
Actomyosin contraction and extracellular matrix-cell adhesive interactions are recognized to influence aqueous humor outflow through the trabecular meshwork and thereby regulate intraocular pressure. This study aims to understand how (i) actomyosin contraction and ECM-cell adhesive interactions regulate the expression of cytoskeletal and cell adhesive proteins in the TM, and (ii) identify the actin dependent transcriptional mechanisms regulating expression of cytoskeletal and cell adhesive proteins in TM cells.
Methods :
Both pharmacological and molecular approaches were used to explore whether serum response factor (SRF) and its co-transcriptional regulator (MRTF-A), master regulators of expression of cytoskeletal, contractile and cell adhesive proteins, play a crucial role in TM physiology. Pharmacological inhibition (MRTF-A inhibitor CCG-22740; 10 µM for 48 hrs) and siRNA-based methodology was used to target the activity and expression of MRTF-A and MICAL2 (a known regulator of MRTF-A nuclear translocation) and evaluate effects on the levels of cytoskeletal proteins using biochemical and proteomics analyses.
Results :
Since dexamethasone and TGF-β2, which are known ocular hypertensive agents, induced increases in the levels of various cytoskeletal and cell adhesive proteins in TM cells, we asked whether actin dependent MRTF-A and nuclear actin depolymerizing protein (MICAL2) mediate these effects. Human TM cells treated with CCG-22740 in the presence of dexamethasone (0.5 µM) exhibited significant decreases in the levels of CO1A1, laminin, agrin, Zyxin, transgelins, Tensin1, CTGF, FND3B, CNN3, Septin9, FND3A, GPC4, TSP1, CDH6, syndican-2, LIMCH1, CNN3, CTGF and α-SMA compared to controls treated with Dex alone. Some of these same proteins were significantly decreased in TM cells treated with CCG-22740 in the presence of TGF-β2. Additionally, MRTF-A siRNA also led to significant decreases in the levels of some of the same proteins mentioned above. Moreover, treatment of TM cells with MICAL2 siRNA resulted in decreased levels of LIMCH1, CTGF and α-SMA in TM cells.
Conclusions :
Collectively, these results reveal a critical role for a SRF/MRTF-A-mediated transcriptional mechanism in regulating the levels of various cytoskeletal, contractile, cell adhesion and ECM proteins in TM cells treated with Dex and TGF-β2.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.