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.

Keywords: chromatin; glaucoma; glucocorticoids; intraocular pressure; nucleoskeleton; proteomics; trabecular meshwork; transcription.

FIGURE 1

Confirmation of purity of nuclear protein fractions isolated from human trabecular meshwork (TM) cells. (A,B) depict immunoblotting results for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and nuclear pore protein (NPP) in cytosolic and nuclear protein fractions, respectively, isolated from human TM cells treated with dexamethasone (Dex) for a period of 5- or 7-days. The nuclear protein fractions show strong staining for NPP but no immunopositive bands for GAPDH, confirming the purity of the nuclear extracts. C1 and C2 and D1 and D2 represent control replicates 1 and 2, and Dex treated replicates 1 and 2, respectively. The nuclear protein fractions from 5- and 7-day Dex treated TM cells were isolated using the commercial kit and in house procedures, respectively, as described in the Methods section.

FIGURE 2

Gene Ontology enrichment analysis of proteins elevated in the nuclear protein fraction of Dex treated (for 7 days) human TM cells. Panels (A–C) depict Gene Ontology (GO) enrichment analyses for biological process, molecular function, and cellular components, respectively (using ShinyGO v0.75 online), of proteins exhibiting a ≥2-fold increase in three or more samples of TM cells treated for 7 days with Dex. Analysis was performed using a P-value cutoff (FDR) of 0.05, and results are shown for the top 30 pathways identified via GO analysis.

FIGURE 3

Immunoblotting analysis based confirmation of elevated levels for selected proteins in the nuclear protein fraction derived from the 7-day Dex-treated human TM cells. (A,B) show the representative immunoblots for the indicated proteins and quantitative changes in the levels of these proteins (histograms), respectively, in the nuclear protein fraction isolated from the TM cells treated with Dex for 7 days. ∗p < 0.05. n = sample number. Immunoblotted proteins were normalized to the nuclear pore protein (NPP).

FIGURE 4

Immunofluorescence based distribution pattern of proteins whose levels were increased in the nuclear protein fraction of human TM cells treated with Dex. Several of the indicated proteins were detected in both the cytosol and nucleus of human TM cells under untreated condition. Arrows indicate the cell nucleus. Scale bars indicate magnification.

FIGURE 5

Immunoblotting analysis based confirmation of elevated levels of selected proteins in the nuclear protein fraction derived from the 5-day Dex-treated human TM cells. (A,B) show the representative immunoblots for the indicated proteins and quantitative changes in levels of these proteins (histograms), respectively, in the nuclear protein fraction isolated from TM cells treated with Dex for 5 days. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. n = sample number. Immunoblotted proteins were normalized to the nuclear pore protein (NPP).

FIGURE 6

GO enrichment analysis of proteins exhibiting upregulated levels in nuclear protein fractions of human TM cells treated with Dex for 5- and 7-day periods. Several proteins exhibited similar trends (≥2-fold increase) in changes in levels between the 5- and 7-day Dex treated samples, and enrichment analysis for Biological process identified preferential upregulation of proteins involved in actin stress fiber formation, bundling and organization, cell adhesion, and vesicle transport. This analysis was performed using a P-value cutoff (FDR) of 0.05, and results are shown for the top 30 pathways.

FIGURE 7

Increased levels of transcriptional (SRF, MRTF-A) and chromatin regulators (BRG1, MTA1, and MeCP2) in Dex treated human TM cells. (A,B) Total cell lysates derived from Dex treated (7 days) TM cells showed a significant increase in the levels of SRF and MRTF-A relative to controls. (C) Nuclear distribution of BRG1, MTA1, and MeCP2 in human TM cells. (D,E) Increased levels of BRG1, MTA1, and MeCP2 proteins in the nuclear protein fraction of Dex treated (5 days) TM cells. *p < 0.05; **p < 0.01. Scale bars indicate image magnification.

FIGURE 8

BRG1 deficiency decreases the levels of various proteins in TM cells under both basal and Dex stimulated conditions. (A,B) BRG1 siRNA and a corresponding scrambled siRNA control were used to determine the role of the chromatin remodeling protein BRG1 in the expression of the indicated proteins in TM cells. Cells were initially treated with siRNA for 24 h prior to the addition of Dex or vehicle and continuation of incubation for another 48 h. Cell lysates were prepared for immunoblotting based quantification of the proteins of interest. As shown in the figure, treatment with BRG1 siRNA significantly reduced the levels of BRG1 and other indicated proteins in TM cells under both normal and Dex treated conditions. *p < 0.05; ****p < 0.0001. Sc si: Scrambled siRNA; Brg1 si: BRG1 siRNA. Lanes 1 and 2 indicate replicate samples.

FIGURE 9

Schematic illustration of the mechanisms involved in regulating the expression of actin cytoskeletal network, contractile, cell adhesive, and vesicle trafficking proteins in Dex treated TM cells. BRG1 (regulator of chromatin remodeling), MICAL2 (regulator of nuclear actin depolymerization), and Glypican-4 (regulator of Wnt/PCP signaling) dependent pathways appear to collectively influence gene expression via stimulating SRF/MRTF-A transcriptional activity (a known master regulator of actin cytoskeletal and contractile protein expression) under these conditions, thereby playing a potential role in modulation of aqueous humor outflow and intraocular pressure. GR: Glucocorticoid receptor.