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.

Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

“…Further exploration of these data by pathway enrichment analysis revealed mechanisms associated with the Wnt signaling pathway and EMT to be discordantly activated in these cancers. Existing evidence derived solely from experimental studies has demonstrated physical binding of GPC4 to Wnt ligands and its regulatory role in Wnt signaling [22,[28][29][30]. Differentially expressed GPC4 can undergo shedding from cell surfaces by the action of either phospholipases or proteases [47].…”

“…The mechanism of action of GPC4 in cancer is also relatively unknown. Studies show that GPC4 influences various mitogenic signaling pathways including TGF-β [26], TLR4/NF-kB [27], Wnt [22,[28][29][30], Mmp14 [31], and FGF2 [32], all of which have the potential to affect cancer progression. GPC4 is also a part of signaling network in CNS, regulating forebrain development by the positive modulation of FGF signaling [33] and promoting fiber sprouting of neurons via mTOR [34].…”

Glucocorticoid-Induced Ocular Hypertension and Glaucoma

“… 85 Also, Rao’s group recently reported that glypican-4 mediates DEX and Wnt5a-induced changes in TM cell cultures. 86 …”