Abstract
Purpose :
Glucocorticoid-induced actin cytoskeletal reorganization in the trabecular meshwork (TM) cells is thought to participate in increased resistance to aqueous humor outflow through the trabecular pathway. However, our understanding of the molecular mechanisms regulating actin cytoskeletal organization in TM cells remains incomplete. Here, we report identification of several molecules known to directly regulate actin cytoskeletal crosslinking whose levels are upregulated in dexamethasone (Dex) treated human TM cells.
Methods :
To identify proteins, whose levels were dysregulated in response to Dex treatment of human TM cells, we analyzed the cytoskeletome and nuclear protein fractions derived from Dex-treated (0.5 µM for 7 days) and control TM cells using non-label quantitative proteomics analyses. The findings from these proteomics analyses were independently validated by immunoblot and immunofluorescence analyses in different (donor) strains of human TM cells.
Results :
Human TM cells treated with Dex consistently revealed a preferential and significant increase in the levels of proteins regulating actin cytoskeletal organization, cell adhesion and fibrogenic activity in both the cytoskeletome and nuclear protein fractions. Proteins including ArgBP2 (SORBS2), CAP/Ponsin (SORBS1), LIMCH1 (LIM and calponin homology domains 1), palladin, septin 11, calponin-3, FAT1, SIPA1L1, Phospholipid phosphatase 3, DIAPH2 and CTGF which are directly involved in actin cytoskeletal organization, cell adhesion, contraction, Rho GTPase activation and fibrogenic activity exhibited significantly increased levels in Dex treated cells. Several of these proteins exhibit colocalization with actin stress fibers and focal adhesions in TM cells.
Conclusions :
The unbiased and global proteomics approach we utilized in this study to determine dexamethasone-induced changes in proteins of the cytoskeletal enriched and nuclear protein fractions of human TM cells identified several proteins directly involved in actin cytoskeletal crosslinking and cell adhesive interactions. Collectively, these findings provide not only novel insights into the molecular basis for glucocorticoid-induced actin cytoskeletal organization in TM cells but also identify novel potential therapeutic targets for lowering ocular hypertension in glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.