June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Activation of Glypican-4 regulated Wnt5/PCP pathway in dexamethasone treated trabecular meshwork cells
Author Affiliations & Notes
  • Vasantha Rao
    Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States
    Pharmacology, Duke University School of Medicine, Durham, North Carolina, United States
  • Camelia Eldawy
    Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States
  • William Backman
    Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States
  • Nikolai P Skiba
    Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States
  • Rupalatha Maddala
    Ophthalmology, Duke University School of Medicine, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Vasantha Rao None; Camelia Eldawy None; William Backman None; Nikolai Skiba None; Rupalatha Maddala None
  • Footnotes
    Support  NIH Grants: R01EY018590 and R01-EY028823
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3304 – A0404. doi:
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      Vasantha Rao, Camelia Eldawy, William Backman, Nikolai P Skiba, Rupalatha Maddala; Activation of Glypican-4 regulated Wnt5/PCP pathway in dexamethasone treated trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3304 – A0404.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Glucocorticoids induce ocular hypertension by increasing resistance to aqueous humor outflow through the trabecular pathway however, the molecular basis for this etiology remains poorly understood. Here we report the activation of glypican-4-regulated Wnt5/PCP pathway in dexamethasone (Dex) treated trabecular meshwork cells, which offers a novel insight into glucocorticoid induced cytoskeletal changes in the trabecular meshwork.

Methods : Dex treated (0.5 µM for 7 days) and control cells from three different human TM cell strains were used to prepare nuclear protein and cytoskeletal-enriched fractions. These fractions were then subjected to label free quantitative proteomics analysis. The effects of Dex and TGF-β2 on glypican-4 (GPC4) protein levels were determined by immunoblot and immunofluorescence analysis, while levels of Wnt5A and phospho-JNK were evaluated by immunoblot analysis.

Results : Using a 2-fold change relative to corresponding controls and a significance of P<0.05 as criteria, we identified differentially expressed proteins in both the nuclear protein and cytoskeletome fractions of Dex treated cells. The levels of GPC4, a cell surface heparan sulfate proteoglycan, and regulator of Wnt signaling were significantly elevated in both cytoskeletome and nuclear protein fractions of Dex treated TM cells compared to control cells. Both immunoblot and immunofluorescence analyses revealed distribution of GPC4 to the cytosol and nuclear fractions of TM cells. Additionally, significant increases in the levels of GPC4 were noted in the total cell lysates of human TM cells treated with Dex and TGF-β2 relative to control cells. Intriguingly, the cytoskeletome fraction of human TM cells revealed the presence of only Wnt5A, Wnt5B, Wnt9A and WLS, and relatively high levels of DAAM2, Frizzled-7, PTK7, and Scribble, which are key regulators of the Wnt/RhoA/PCP pathway. Moreover, Dex treatment also resulted in increased levels of Wnt5A and JNK phosphorylation (indicators of activation of the Wnt/PCP pathway) in TM cells.

Conclusions : This study identifies the Glypican-4/Wnt5/RhoA induced PCP pathway as one of the predominant upstream signaling pathways regulating actin cytoskeletal reorganization and cell adhesive changes in glucocorticoid treated TM cells, unravelling a new molecular target for management of IOP in glaucoma.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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