Existence of the canonical wnt signaling pathway in the human trabecular meshwork

Weiming Mao, J. Cameron Millar, Wan Heng Wang, Sean M. Silverman, Yang Liu, Robert J. Wordinger, Jeffrey S. Rubin, Iok Hou Pang, Abbot F. Clark

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

PURPOSE. We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS. Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS. WNT3a induced b-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS. There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

Original languageEnglish (US)
Pages (from-to)7043-7051
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume53
Issue number11
DOIs
StatePublished - Oct 1 2012
Externally publishedYes

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Trabecular Meshwork
Wnt Signaling Pathway
Luciferases
Catenins
Actin Cytoskeleton
Intravitreal Injections
Adenoviridae
Genes
Microscopy
Proteins
Western Blotting

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Mao, W., Cameron Millar, J., Wang, W. H., Silverman, S. M., Liu, Y., Wordinger, R. J., ... Clark, A. F. (2012). Existence of the canonical wnt signaling pathway in the human trabecular meshwork. Investigative Ophthalmology and Visual Science, 53(11), 7043-7051. https://doi.org/10.1167/iovs.12-9664

Existence of the canonical wnt signaling pathway in the human trabecular meshwork. / Mao, Weiming; Cameron Millar, J.; Wang, Wan Heng; Silverman, Sean M.; Liu, Yang; Wordinger, Robert J.; Rubin, Jeffrey S.; Pang, Iok Hou; Clark, Abbot F.

In: Investigative Ophthalmology and Visual Science, Vol. 53, No. 11, 01.10.2012, p. 7043-7051.

Research output: Contribution to journalArticle

Mao, W, Cameron Millar, J, Wang, WH, Silverman, SM, Liu, Y, Wordinger, RJ, Rubin, JS, Pang, IH & Clark, AF 2012, 'Existence of the canonical wnt signaling pathway in the human trabecular meshwork', Investigative Ophthalmology and Visual Science, vol. 53, no. 11, pp. 7043-7051. https://doi.org/10.1167/iovs.12-9664
Mao, Weiming ; Cameron Millar, J. ; Wang, Wan Heng ; Silverman, Sean M. ; Liu, Yang ; Wordinger, Robert J. ; Rubin, Jeffrey S. ; Pang, Iok Hou ; Clark, Abbot F. / Existence of the canonical wnt signaling pathway in the human trabecular meshwork. In: Investigative Ophthalmology and Visual Science. 2012 ; Vol. 53, No. 11. pp. 7043-7051.
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abstract = "PURPOSE. We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS. Western immunoblotting and/or immunofluorescent microscopy were used to study {\ss}-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS. WNT3a induced b-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS. There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.",
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AU - Liu, Yang

AU - Wordinger, Robert J.

AU - Rubin, Jeffrey S.

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AU - Clark, Abbot F.

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N2 - PURPOSE. We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS. Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS. WNT3a induced b-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS. There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

AB - PURPOSE. We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS. Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS. WNT3a induced b-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS. There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

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