Abstract
Purpose :
To understand how dexamethasone influences the expression of actin cytoskeletal, contractile, and fibrogenic proteins in trabecular meshwork cells, in this study we focused on identifying the upstream regulators of gene expression in dexamethasone treated cells.
Methods :
Human TM cells treated with dexamethasone (Dex, 0.5 µM) for 24 hrs, 5 and 7 days along with their respective controls were extracted for the nuclear protein fraction and analyzed for the differential profile of proteins by non-label quantitative LC-MS-MS proteomics. Distribution and changes in Brg1 levels in TM cells treated with Dex were evaluated by immunofluorescence and immunoblot analyses. Effects of Brg1 inhibition and deficiency on the levels of client proteins in TM cells were determined using a chemical inhibitor (PFI-3) and siRNA of Brg1, respectively.
Results :
The protein levels of Brg1/SMARCA4, the catalytic subunit of the SWI/SNF family of chromatin remodeling complex, were significantly increased in the Dex treated TM cell nuclear protein fraction compared to control cells based on mass spec and immunoblot analyses. Brg1, an actin binding nuclear protein was distributed predominantly in the nucleus. Brg1 and several of other components of the SWI/SNF complex were readily detectable in the cytoskeletome fraction of the human TM cells indicating an association between the SWI/SNF chromatin-remodeling complex and nucleoskeletal actin and actin related proteins. Inhibition of Brg1 activity and suppression of Brg1 expression significantly decreased the protein levels of cell adhesion proteins (ArgBP2, CAP/ponsin), contractile protein (CNN3), fibrogenic protein (CTGF) and MICAL2 (regulator of SRF/MRTF-A transcriptional activity) and glypican-4 (regulator of Wnt signaling) in human TM cells.
Conclusions :
This study identifies Brg1, a catalytic subunit of the SWI/SNF family chromatin-remodeling complex as one of the upstream regulators of the expression of actin cytoskeletal, contractile and cell adhesion proteins in dexamethasone treated human TM cells. Thus, Brg1 and the SWI/SNF chromatin-remodeling complex may be promising targets to lower intraocular pressure in steroid-induced glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.