Glucocorticoids Preferentially Influence Expression of Nucleoskeletal Actin Network and Cell Adhesive Proteins in Human Trabecular Meshwork Cells

Abstract

Clinical use of glucocorticoids is associated with increased intraocular pressure (IOP), a major risk factor for glaucoma. Glucocorticoids have been reported to induce changes in actin cytoskeletal organization, cell adhesion, extracellular matrix, fibrogenic activity, and mechanical properties of trabecular meshwork (TM) tissue, which plays a crucial role in aqueous humor dynamics and IOP homeostasis. However, we have a limited understanding of the molecular underpinnings regulating these myriad processes in TM cells. To understand how proteins, including cytoskeletal and cell adhesion proteins that are recognized to shuttle between the cytosolic and nuclear regions, influence gene expression and other cellular activities, we used proteomic analysis to characterize the nuclear protein fraction of dexamethasone (Dex) treated human TM cells. Treatment of human TM cells with Dex for 1, 5, or 7 days led to consistent increases (by ≥ two-fold) in the levels of various actin cytoskeletal regulatory, cell adhesive, and vesicle trafficking proteins. Increases (≥two-fold) were also observed in levels of Wnt signaling regulator (glypican-4), actin-binding chromatin modulator (BRG1) and nuclear actin filament depolymerizing protein (MICAL2; microtubule-associated monooxygenase, calponin and LIM domain containing), together with a decrease in tissue plasminogen activator. These changes were independently further confirmed by immunoblotting analysis. Interestingly, deficiency of BRG1 expression blunted the Dex-induced increases in the levels of some of these proteins in TM cells. In summary, these findings indicate that the widely recognized changes in actin cytoskeletal and cell adhesive attributes of TM cells by glucocorticoids involve actin regulated BRG1 chromatin remodeling, nuclear MICAL2, and glypican-4 regulated Wnt signaling upstream of the serum response factor/myocardin controlled transcriptional activity.

Glypican‐4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling

“…Palladin, whose levels were significantly elevated in the cytoskeletal fraction and total cell lysates of Dex treated TM cells compared to control cells (Table 1, Supporting Information: Figures and ), is a known actin interacting and crosslinking protein (Goicoechea et al, 2008; Gurung et al, 2016). Interestingly, unlike other cytoskeletal and cell adhesive proteins described above, in our previous study on the effects of Dex on the nuclear proteome profile in Dex treated TM cells, we did not find changes in the levels of palladin (Bachman et al, 2022). Additionally, some of the Dex induced proteins including LIMCH1, CNN3, ArgBP2, and palladin have been found to be tyrosine phosphorylated in normal TM cells (Maddala & Rao, 2020).…”

“…Cells were subsequently washed with cytoskeletal buffer (10-mM 2-[N-morpholino] ethane sulfonic acid [MES], containing 150-mM NaCl, 5-mM EDTA, 5-mM MgCl2, and 5-mM glucose [pH 6.1]), and permeabilized for 10 min with 0.5% Triton X-100 in PBS, and blocked with serum-containing buffer (10% FBS in PBS with 0.02% sodium azide) before immunostaining with primary antibodies (Supporting Information: Table S1). Immunostaining was performed overnight in serum-containing buffer with 0.2% saponin, followed by the appropriate secondary antibodies conjugated with Alexa fluorophores 488 or 568 for 2 h at room temperature (Bachman et al, 2022). For actin staining, permeabilized HTM cells were treated with Phalloidin-Tetramethylrhodamine B isothiocyanate (Millipore Sigma) (Bachman et al, 2022).…”

“…Immunostaining was performed overnight in serum-containing buffer with 0.2% saponin, followed by the appropriate secondary antibodies conjugated with Alexa fluorophores 488 or 568 for 2 h at room temperature (Bachman et al, 2022). For actin staining, permeabilized HTM cells were treated with Phalloidin-Tetramethylrhodamine B isothiocyanate (Millipore Sigma) (Bachman et al, 2022). Cell nuclei were counterstained with Hoechst (Hoechst 33258, Molecular Probes).…”

“…Human TM cells (minimum of two strains and their duplicates) grown on gelatin (2%) coated glass coverslips (control and treated) were rinsed with cold PBS and fixed with 4% paraformaldehyde as we described previously (Bachman et al, 2022).…”

Identification and verification of Hub Biomarkers and Immune-Related Pathways Participating in the Trabecular Meshwork after using Corticosteroid

“…In this work, we identi ed 30 DEGs that might be involved in IOP increasing after using corticosteroids. The enriched GO biological process terms assigned to these genes were mainly enriched in positive regulation of cytokine production 16,17 , regulation of extracellular matrix organization 18 , actin lament bundle assembly, actin lament bundle organization 19 , and T-helper 2 cell cytokine production 18 . KEGG pathway analysis showed signi cant enrichment of Phenylalanine metabolism 20 .…”