June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Characterization of PRG4 expression, localization, and function in trabecular meshwork cells
Author Affiliations & Notes
  • Colleen M McDowell
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Timur Mavlyutov
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Alyssa Morin
    University of Connecticut Health Center, Connecticut, United States
  • Emily Anne Hicks
    McMaster University, Ontario, Canada
  • Aftab Taiyab
    McMaster University, Ontario, Canada
  • Alarifi Abdulaziz
    University of Calgary, Alberta, Canada
  • Gregory Jay
    Brown University, Rhode Island, United States
  • Heather Sheardown
    McMaster University, Ontario, Canada
  • Tannin Schmidt
    University of Connecticut Health Center, Connecticut, United States
  • Footnotes
    Commercial Relationships   Colleen McDowell, None; Timur Mavlyutov, None; Alyssa Morin, None; Emily Anne Hicks, None; Aftab Taiyab, None; Alarifi Abdulaziz, None; Gregory Jay, Lubris BioPharma (F), Lubris BioPharma (I), Lubris BioPharma (P); Heather Sheardown, None; Tannin Schmidt, Lubris BioPharma (F), Lubris BioPharma (I), Lubris BioPharma (C), Lubris BioPharma (P)
  • Footnotes
    Support  NIH NEI1R01EY026529-01
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3458. doi:
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      Colleen M McDowell, Timur Mavlyutov, Alyssa Morin, Emily Anne Hicks, Aftab Taiyab, Alarifi Abdulaziz, Gregory Jay, Heather Sheardown, Tannin Schmidt; Characterization of PRG4 expression, localization, and function in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3458.

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

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Abstract

Purpose : Proteoglycan 4 (PRG4), or lubricin, is a mucin-like glycoprotein that is present on the ocular surface and in tears. PRG4 possesses anti-inflammatory properties and binds to and antagonizes toll-like receptor 4 (TLR4), reducing inflammation and inflammatory cytokine expression. Exogenous PRG4 inhibits TGFβ1 fibrosis in synovial and cardiac fibroblasts, while endogenous expression of PRG4 can be increased by TGFβ1. Interestingly, PRG4 mRNA expression is upregulated by TGFβ2 in trabecular meshwork (TM) cells. We recently identified that TGFβ2 and TLR4 signaling crosstalk is important in the regulation of glaucomatous TM damage. However, knowledge of PRG4’s protein expression and function in TM cells is lacking. The objectives of this work were to a) characterize PRG4 secretion by TM cells and the effect of TGFB2 and/or TLR4 inhibition, and b) visualize PRG4 localization in the TM.

Methods : Primary and transformed human TM cells were treated with a selective TLR4 inhibitor (TAK-242, 15µM), TGFβ2 (5ng/ml), and/or rhPRG4 (150 µg/ml, Lubris BioPharma LLC). Conditioned medium was collected and PRG4 quantified by indirect ELISA and visualized by western blotting. Fibronectin (FN) was visualized by immunocytochemistry and quantified using ImageJ analysis. PRG4 was immunolocalized in TM of C57BL/6J mice and PRG4 deficient mice. Whole globes were fixed, embedded, and sectioned, followed by immunolabeling with an anti-PRG4 Ab. Exclusion of the primary Ab was used as a negative control.

Results : TM cells synthesize and secrete PRG4 protein in culture, and this expression is upregulated by TGFB2, and blocked by the presence of a TLR4-inhibitor (n=2 primary cell strains, each repeated in triplicate, p<0.05 for each strain). Treatment with rhPRG4 inhibited TGFβ2-induced FN expression (n=3 independent experiments GTM3 TM cells, p<0.05). PRG4 expression was detected throughout the ciliary body and trabecular meshwork of C57BL/6J murine globes. No staining was observed in the absence of primary Ab, or in the PRG4 deficient mice.

Conclusions : PRG4 is expressed by TM cells in vitro, and this expression can be biochemically regulated. rhPRG4 inhibited TGFB2 stimulated FN, indicating PRG4 can regulate the fibrotic response in TM cells. Future studies will further elucidate PRG4’s function, be it biomechanical and/or biological, in the TM and the role it plays in glaucoma development and potential treatments.

This is a 2020 ARVO Annual Meeting abstract.

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