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Padmanabhan P Pattabiraman, Sai Supriya Vuda, Jaeyoung Heo, Haarika Reddy, Carol B Toris, Douglas J Rhee; Clusterin-Cathepsin K Signaling in Trabecular Meshwork Outflow Pathway Regulates Intraocular Pressure. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5157.
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© ARVO (1962-2015); The Authors (2016-present)
Understand the role of the secretory protein, clusterin, and its target cysteine protease, cathepsin k, in the regulation of intraocular pressure (IOP).
Time-dependent regulation of clusterin by ocular hypertension inducing transforming growth factorβ2 (TGFβ2) (8ng/ml for 1, 2, 24, and 48h) was performed in human trabecular meshwork (HTM) cells using qPCR and immunoblotting. Effect of - A) clusterin loss in HTM cells by siRNA (siCLU) and in Clu-/- mice were assessed for changes in actin, α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) like collagen-1A and fibronectin. B) Adenovirus-induced expression of clusterin (AdCLU) and cathepsin k (AdCTSK) on actin, ECM, CTSK expression and activity, and TGFβ2 levels in HTM cells were determined by immunofluorescence, qPCR, immunoblotting, and ELISA assays. C) AdCLU expression in outflow pathway tissue on IOP and ECM changes were assessed in human anterior segments and in wild-type mice (C57BL6-SVJ129). Students t-test was used for statistical analyses and results were significant if p<0.05 with a sample size of N=3-8 in each experiment.
In HTM cells, TGFβ2 decreased clusterin mRNA and protein significantly at 24h and 48h. Clusterin loss in HTM cells by siClu and in Clu-/- mice increased actin stress fibers, α-SMA and ECM levels and distribution in TM. AdCLU expression in HTM cells increased clusterin and its secretion resulting in rounded cells with intact nuclei, decreased stress fibers and ECM indicating the loss in cell tension. Perfusion of AdCLU in cadaveric human anterior segments or AdCLU injection into mice anterior chamber lowered IOP significantly [29.2±5% (mean±SEM)]. IOP lowering effect in mice was found 3 days post-injection and lasted up to 10 days correlating to elevated clusterin in mice AH. The IOP decrease corroborated with lower ECM staining in TM tissue both ex vivo and ex vivo. AdCLU significantly increased CTSK mRNA, protein and activity. Increasing CTSK expression using AdCTSK resulted in decreased actin stress fibers, ECM assembly, fibril formation and expression and significant decrease in TGFβ2 mRNA and protein levels.
We have identified clusterin and its effector CTSK as critical IOP lowering targets by maintaining optimal cell-matrix interactions in the AH outflow pathway. Dysregulation of clusterin-CTSK functions in the TM pathway can contribute to ocular hypertension and POAG.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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