Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ

Vijay Krishna Raghunathan, Joshua T. Morgan, Britta Dreier, Christopher M. Reilly, Sara M Thomasy, Joshua Wood, Irene Ly, Binh C. Tuyen, Marissa Hughbanks, Christopher J Murphy, Paul Russell

Research output: Contribution to journalArticle

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Abstract

PURPOSE. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma. METHODS. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/ TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing. RESULTS. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP- 1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA. CONCLUSIONS. The expression of YAP/TAZ and ECM-relatedgenes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.

Original languageEnglish (US)
Pages (from-to)378-386
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume54
Issue number1
DOIs
StatePublished - Jan 2013

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Extracellular Matrix
Trabecular Meshwork
Genes
Proteins
Glaucoma
Angiopoietins
14-3-3 Proteins
Hydrogels
Aqueous Humor
Dexamethasone
Small Interfering RNA
Up-Regulation
Down-Regulation
Steroids

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ. / Raghunathan, Vijay Krishna; Morgan, Joshua T.; Dreier, Britta; Reilly, Christopher M.; Thomasy, Sara M; Wood, Joshua; Ly, Irene; Tuyen, Binh C.; Hughbanks, Marissa; Murphy, Christopher J; Russell, Paul.

In: Investigative Ophthalmology and Visual Science, Vol. 54, No. 1, 01.2013, p. 378-386.

Research output: Contribution to journalArticle

Raghunathan, Vijay Krishna ; Morgan, Joshua T. ; Dreier, Britta ; Reilly, Christopher M. ; Thomasy, Sara M ; Wood, Joshua ; Ly, Irene ; Tuyen, Binh C. ; Hughbanks, Marissa ; Murphy, Christopher J ; Russell, Paul. / Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ. In: Investigative Ophthalmology and Visual Science. 2013 ; Vol. 54, No. 1. pp. 378-386.
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abstract = "PURPOSE. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma. METHODS. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/ TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing. RESULTS. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP- 1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA. CONCLUSIONS. The expression of YAP/TAZ and ECM-relatedgenes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.",
author = "Raghunathan, {Vijay Krishna} and Morgan, {Joshua T.} and Britta Dreier and Reilly, {Christopher M.} and Thomasy, {Sara M} and Joshua Wood and Irene Ly and Tuyen, {Binh C.} and Marissa Hughbanks and Murphy, {Christopher J} and Paul Russell",
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AU - Raghunathan, Vijay Krishna

AU - Morgan, Joshua T.

AU - Dreier, Britta

AU - Reilly, Christopher M.

AU - Thomasy, Sara M

AU - Wood, Joshua

AU - Ly, Irene

AU - Tuyen, Binh C.

AU - Hughbanks, Marissa

AU - Murphy, Christopher J

AU - Russell, Paul

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N2 - PURPOSE. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma. METHODS. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/ TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing. RESULTS. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP- 1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA. CONCLUSIONS. The expression of YAP/TAZ and ECM-relatedgenes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.

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