Wnt induction of SMAD/TGFβ signaling in the Trabecular Meshwork

Purpose: Primary Open Angle Glaucoma is a progressive, irreversibly blinding disease, the leading risk factor of which is increased intraocular pressure (IOP) thought to be due to an inherent pathological change in the trabecular meshwork (TM) tissue. Canonical Wnt signaling genes are expressed in the TM, the primary site for regulation of aqueous humor outflow and therefore IOP. Canonical Wnt signaling activation has been found to regulate IOP, but the mechanism behind this phenomenon remains unknown. Extracellular matrix deposition in the TM caused by increased activation of the TGF-β pathway by the TGF-β2 ligand has been associated with increased IOP and with primary open angle glaucoma. In other cell types and diseases, evidence exists for crosstalk between the TGF-β and Wnt signaling pathways. Our study aims to pinpoint the affect of Wnt signaling on the glaucoma-associated TGF-β pathway in the TM. Methods: Lentivirus-based luciferase assays were conducted in normal trabecular meshwork (NTM) and glaucomatous trabecular meshwork (GTM) cells by using TGF-β/SMAD or TCF/LEF (Wnt) signaling reporter vectors. Trabecular meshwork cells were all kind gifts from Novartis. Cell were treated with or without 100ng/ml Wnt3a or 5ng/ml TGF-β2. In some experiments, siRNAs were also used to knock down smads in NTM cells. Western immunoblotting was performed on nuclear and cytosolic fractions of NTM and GTM cells with corresponding primary as well as secondary antibodies. Results: In NTM cells, Wnt3a treatment increased TGFβ/SMAD pathway reporter activity (n=5 p less than 0.05) but TGF-β2 did not affect and even slightly decreased TCF/LEF (Wnt) signaling activity, although this decrease was not statistically significant (n=5, P greater than 0.05). SiRNA knockdown of SMAD pathway mediator smad3 decreased Wnt3a-induced SMAD/TGFβ signaling activity (n=6 p less than 0.05) in NTM cells. However, nuclear fractions of NTM and GTM cells showed translocation of smad4 (co-smad) into the nucleus upon Wnt3a treatment but not smad2 or smad3. Nuclear fractions also showed translocation of β-catenin by TGFβ2 treatment. Conclusions: The Wnt pathway ligand Wnt3a is able to activate SMAD/TGFβ transcriptional activity in TM cells, but not vice versa. This activation seems to involve translocation of only smad4. We hypothesize that a protein complex consisting of β-catenin and smad4 can form in the TM. By selectively recruiting other smad proteins into the complex during SMAD activation, the SMAD/TGFβ pathway can be differentially regulated. Defining how Wnt and SMAD signaling pathways crosstalk in the TM is imperative in defining the role of Wnt signaling in IOP regulation, and could lead to discovery of a therapeutic target for regulation of TGF-β pathway and therefore regulation of POAG.