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TGFβ2 regulates human trabecular meshwork cell contractility via ERK and ROCK pathways with distinct signaling crosstalk dependent on the culture substrate

Haiyan Li, Jessica L. Henty-Ridilla, Preethi S. Ganapathy, View ORCID ProfileSamuel Herberg
doi: https://doi.org/10.1101/2021.07.01.450718
Haiyan Li
aDepartment of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY 13210, USA
bDepartment of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
eBioInspired Institute, Syracuse University, Syracuse, NY 13244, USA
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Jessica L. Henty-Ridilla
cDepartment of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
dDepartment of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
eBioInspired Institute, Syracuse University, Syracuse, NY 13244, USA
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Preethi S. Ganapathy
aDepartment of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY 13210, USA
dDepartment of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
eBioInspired Institute, Syracuse University, Syracuse, NY 13244, USA
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Samuel Herberg
aDepartment of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY 13210, USA
bDepartment of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
cDepartment of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
eBioInspired Institute, Syracuse University, Syracuse, NY 13244, USA
fDepartment of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, USA
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  • ORCID record for Samuel Herberg
  • For correspondence: herbergs@upstate.edu
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Abstract

Transforming growth factor beta 2 (TGFβ2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma. Receptor binding of TGFβ2 activates non-canonical extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both broadly affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate pathologic HTM cell contractility associated with glaucomatous dysfunction is unclear. Here, we investigated the molecular mechanism underlying TGFβ2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways. We compared soft biomimetic hydrogels composed of collagen type I, elastin-like polypeptide, and hyaluronic acid with conventional stiff glass coverslips. Results show that HTM cell morphology and filamentous (F)-actin organization was affected by the underlying culture substrate: TGFβ2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, α-smooth muscle actin, fibronectin, and phospho-myosin light chain in cells grown on hydrogels compared to glass. Importantly, we showed that ERK inhibition further increased TGFβ2-induced phospho-myosin light chain levels in HTM cells on hydrogels, but not on glass, which translated into hypercontractility of three-dimensional (3D) HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGFβ2-induced hydrogels. This suggests that ERK signaling negatively regulates ROCK-mediated HTM cell contractility, and that impairment of this crosstalk balance contributes to the pathologic contraction associated with the glaucomatous stressor TGFβ2. These findings emphasize the critical importance of using 3D tissue-mimetic extracellular matrix substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 01, 2021.
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TGFβ2 regulates human trabecular meshwork cell contractility via ERK and ROCK pathways with distinct signaling crosstalk dependent on the culture substrate
Haiyan Li, Jessica L. Henty-Ridilla, Preethi S. Ganapathy, Samuel Herberg
bioRxiv 2021.07.01.450718; doi: https://doi.org/10.1101/2021.07.01.450718
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TGFβ2 regulates human trabecular meshwork cell contractility via ERK and ROCK pathways with distinct signaling crosstalk dependent on the culture substrate
Haiyan Li, Jessica L. Henty-Ridilla, Preethi S. Ganapathy, Samuel Herberg
bioRxiv 2021.07.01.450718; doi: https://doi.org/10.1101/2021.07.01.450718

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