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Hannah C. Webber, Jaclyn Bermudez, Anirudh Sethi, Abbot F Clark, Weiming Mao; Cross-inhibition between TGFβ and Wnt pathways in the Trabecular Meshwork through Smad4-β-catenin interaction. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4686.
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© ARVO (1962-2015); The Authors (2016-present)
TGFβ/SMAD signaling pathway activator TGFβ2 and the Wnt signaling antagonist sFRP1 are both increased in the trabecular meshwork (TM) of primary open angle glaucoma (POAG) patients. They both increase intraocular pressure (IOP) in perfusion cultured bovine and human eyes as well as in mouse models. In non-TM cells and other diseases, TGFβ and Wnt signaling crosstalk controls cell signaling, gene expression, and physiology. Here we studied the cross-inhibition between the TGFβ and canonical Wnt pathways in the TM.
Lentiviral based luciferase assays were performed in primary non-glaucomatous TM (NTM) cell strains. Cells were treated with 100ng/ml Wnt3a, 5ng/ml TGFβ2, or both for 24 hours after transduction with TGFβ or Wnt pathway lentiviral reporter viruses. Plasmid-based luciferase assays were performed in HTM5 cells using the TGFβ or Wnt pathway reporter plasmid together with siRNA against Smad4 or β-catenin. Western blots were used to confirm siRNA knockdown, to study nuclear translocation of β-catenin, p-Smad2/3, Smad4, and to probe conditioned media for extracellular matrix proteins in primary NTM cells treated with Wnt3a, TGFβ2, or both for 24 hours. Co-Immunoprecipitation (Co-IP) was used to study Smad4-β-Catenin association in the nuclear fraction of HTM5 cells using the same treatment regime.
We found that co-treatment with Wnt3a and TGFβ2 significantly decreased TGFβ2-induced TGFβ pathway activity and Wnt3a-induced Wnt pathway activity in primary HTM cells (p<0.05). This cross-inhibition was abolished with knockdown of Smad4 and β-catenin. Western blots showed that Wnt3a induced nuclear translocation of β-catenin and Smad4, but not p-Smad2/3. However, TGFβ2 induced nuclear translocation of β-catenin, Smad4, and p-Smad2/3. Co-IP showed that Smad4 complexes with β-catenin in the nucleus. Co-treatment with TGFβ2 and Wnt3a also resulted in a dampened expression of a subset of extracellular matrix proteins including fibronectin and SPARC in the TM.
Canonical Wnt signaling and TGFβ/SMAD signaling pathways cross-inhibit each other in the TM, leading to regulation of extracellular matrix proteins. This inhibition may be through a Smad4/β-catenin interaction.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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