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

Abstract

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.

Keywords: Wnt pathway; cytoskeleton; eye; glucocorticoid; glypican; intraocular pressure.

FIGURE 1

Gene Ontology analysis of Dex upregulated proteins in the cytoskeletal fraction of HTM cells. Panels (a, c, and d) depict enrichment of Dex upregulated proteins in HTM cells for Biological process, Cellular components and Molecular function, respectively as deduced from GO analysis using the ShinyGO 0.76 online tool. (b) Networks output of Dex induced cytoskeletome proteins for Biological process in TM cells. HTM, human TM; TM, trabecular meshwork.

FIGURE 2

Distribution profile and colocalization of Dex-induced proteins with the actin cytoskeleton, and the effects of dasatinib (Abl and Src kinase inhibitor) treatment in HTM cells. (a) HTM cells grown under 2% serum and treated with Dex (0.5 μM for 7 days) were co-stained for F-actin (with Rhodamine-phalloidin/red) and the indicated individual proteins (immunostaining) then imaged with confocal microscopy. (b) HTM cells grown under 2% serum treated with dasatinib (1 μM for 24 h) revealed altered distribution profiles of CNN3, LIMCH1, ArgBP2 and palladin and colocalization of these proteins with F-actin. Bars in the bottom right panels represent image magnification. HTM, human TM.

FIGURE 3

Dexamethasone induces the levels of GPC4, Wnt5a, p-JNK, and p-MYPT1 in HTM cells. (a) Analysis of cytoskeletome fractions derived from HTM cells treated with Dex (0.5 μM for 7 days) reveals significant increases (calculated as fold-change in expression) in the levels of GPC4, Wnt5a, and phospho-JNK, relative to controls. α-actinin 1 was probed as loading control. (b) HTM cells treated with Dex as described above were found to exhibit increased immunostaining for GPC4 (arrows) compared to control cells. The bar in the right panel represents image magnification. (c) HTM cells treated with Dex (0.5 μM for 7 days) and ethanol (control) were evaluated for changes in the levels of expression of GPC4, Wnt5a, and Wnt5b by q-RT-PCR analysis. Dex treated TM cells showed significant increase in the levels of GPC4 and Wnt5a expression compared to control cells. N = 3. To determine the influence of Dex and Wnt5a effects on GPC4 levels and Wnt/PCP signaling in TM cells, HTM cells were treated with either Wnt5a alone (100 ng/ml for 24 h) or Dex (0.5 μM for 48 h)/Wnt5a (24 h) and the total cell lysates derived from the treated samples and their respective controls were analyzed for the levels of GPC4, p-JNK, and p-MYPT1 by immunoblot analysis. (d) HTM cells treated in the combination of both Wnt5a and Dex showed significant increase in the levels of GPC4, p-JNK, and p-MYPT1 compared to Wnt5a alone treated and control cells. *p < 0.05, **p < 0.01, ***p < 0.001. N = 4. HTM, human TM; LC, loading control; PCP, planar cell polarity; TM, trabecular meshwork.

FIGURE 4

Glypican-4 deficiency reduces actin stress fibers and focal adhesions in TM cells. To determine the role of GPC4 in TM cell morphology and actin cytoskeletal organization, HTM cells treated with siRNA of GPC4 (60 pmoles) for 72 h in comparison with scrambled (Sc) siRNA control exhibited altered cell shape (a), decreased actin stress fibers ([b] stained with rhodamine-phalloidin) and focal adhesions ([b] Immunostained with vinculin; the bar in the right panel represents image magnification), and significant decrease in the levels GPC4, Wnt5a, phospho-paxillin, phospho-MYPT1 and p-focal adhesion kinase (c, d). GAPDH was probed for loading control. ScsiRNA: Scrambled siRNA. N = 6. HTM, human TM; TM, trabecular meshwork.

FIGURE 5

Glypican-4 deficiency impairs Dex and Wnt5a induced actin cytoskeletal reorganization and cell adhesive interactions in TM cells. To determine whether GPC4 deficiency impacts Dex and Wnt5a induced actin cytoskeletal changes and cell adhesive interactions, in separate sets of plates, HTM cells were treated initially with siRNA of GPC4 or scrambled (Sc) siRNA control. After 24 h of siRNA treatment, to one set of the plates, Dex (0.5 μM) was added and incubated for 48 h. In a second set of siRNA treated cells, Wnt5a (100 μg/ml) was added after 48 h and incubated for additional 24 h. After the treatments, the cell lysates were analyzed for the levels of GPC4, p-JNK, p-MYPT1 and Wnt5a. (a, b) The levels of GPC4, p-JNK, p-MYPT1, and Wnt5a were significantly decreased in both siRNA of GPC4 alone and its combination with Dex treatment compared to the sc-siRNA treated (alone) and in combination with Dex treated cells. Similarly, (c, d) the levels of GPC4, p-JNK and p-MYPT were significantly decreased in the siRNA of GPC4 treated alone and its combination with Wnt5a treated cells compared to the Sc- siRNA alone and its combination with Wnt5a treated cells. N = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. LC is a loading control. Additionally, under above-described treatment conditions, (e) actin stress fibers (rhodamine-phalloidin staining) and focal adhesions (vinculin immunostaining) were reduced markedly in GPC4 siRNA alone and its combination with Dex or Wnt5a treated HTM cells compared to the Sc-siRNA alone and its combination with Dex or Wnt5a treated cells. Bars in the right panels represent image magnification. GPsi, GPC4 siRNA; Scsi, Scrambled siRNA.

FIGURE 6

A schematic illustration of possible involvement and interactions between chromatin remodeling, transcription factors, and GPC4-dependent Wnt/PCP signaling in regulating expression and reorganization of actin cytoskeletal proteins and cell adhesive proteins in Dex treated TM cells, to collectively influence barrier activity of the TM, AH outflow and IOP. GC, glucocorticoid; GR, glucocorticoid receptor; IOP, intraocular pressure; TM, trabecular meshwork.