Abstract
Purpose :
Elevated intraocular pressure (IOP) is the primary risk factor of primary open-angle glaucoma (POAG). TGFβ2 and Wnt signaling pathways are associated with POAG. Excessive TGFβ2 induces pathological changes in the trabecular meshwork (TM). Also, TGFβ2 elevates IOP in mouse and human eyes. In contrast, inhibition of Wnt signaling contributes to POAG. We reported that an elevation of sFRP1 and Dkk1, the Wnt inhibitors, in the POAG TM and/or aqueous humor. Overexpression of either sFRP1 or Dkk1 elevates IOP in mouse and/or human eyes. There is also a cross-inhibition between TGFβ2 and Wnt signaling pathways. Here, we determine the mechanism of this cross-inhibition and if the activation of the canonical Wnt signaling inhibits TGFβ2 signaling and its related pathological changes in the TM.
Methods :
HTM cells were treated with or without TGFβ2, Wnt3a, and/or GSK3β inhibitors. Cytosolic and nuclear fractions were co-immunoprecipitated (Co-IP). Some of the nuclear fractions were used for electrophoretic mobility shift assay (EMSA). Förster resonance energy transfer and fluorescent live imaging (FRET-FLIM) was used to study protein-protein interaction using pHTM cells transduced with Ad5-SMAD4-GFP with or without Ad5-β-catenin-mCherry. Human donor eyes were used for perfusion cultures.
Results :
We treated pHTM cells with TGFβ2 with or without Wnt3a/GSK3β inhibitors and found that in single or in combination, they promoted SMAD4 and β-catenin nuclear translocation. Co-immunoprecipitation assays showed that Smad4 was associated with β-catenin in pHTM cells. FRET-FLIM assays showed a shortening of the fluorescent lifetime of Smad4-GFP in the presence of β-catenin-mCherry in pHTM cells, indicating a direct binding between the two proteins. EMSA assays showed that the Smad4-β-catenin complex did not interfere with DNA binding. Western immunoblotting and immunofluorescence studies showed that the activation of Wnt signaling using Wnt3a or GSK3β inhibitors inhibited TGFβ2-induced TGFβ signaling as well as ECM proteins in pHTM cells. Topical application of CHIR inhibited TGFβ2-induced ocular hypertension in perfusion culture human eyes.
Conclusions :
The activation of either signaling pathways promotes the nuclear translocation of both β-catenin and Smad4 and they form a complex in the nucleus via direct binding. Also, the activation of Wnt signaling inhibits TGFβ2-induced pathological changes in the TM.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.