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Urmimala Raychaudhuri, April Nettesheim, Angela Dixon, Paloma Borrajo Liton; Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3464.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigate the role of CTSB on ECM remodeling and outflow physiology.
Experiments were conducted in primary cultures of human TM cells. Caveolae fractions were obtained by Opti-prep gradient method using the Caveolae/Rafts isolation kit (Sigma). CTSB expression was specifically silenced in hTM cells via siRNA (siCTSB). CTSB activity was pharmacologically inhibited using Ca074 and Ca074Me (10µM, 24 h). Protein expression levels of CTSB, Cav1, annexin A2, SMA, and components of the proteolytic cascade (uPA, uPAR, PAI-1), were evaluated in fractionated fractions, whole cell lysates, and/or conditioned media by Western-blot and/or ELISAS. mRNA expression was quantified by qPCR. Degradation of ECM components was monitored by flow cytometry using DQ-ECM substrates.
WB analysis demonstrated the presence of the mature form of CTSB (pro-CTSB) and its cell surface receptor Annexin2, together with the proteolytic cascade components uPA and uPAR in purified caveolae fractions from human TM cells. Cav1 expression was used as a positive fractioning control. Co-localization of CTSB with Cav1 was additionally confirmed by immunofluorescence. Interestingly, silencing CTSB expression or inhibition of CTSB activity by Ca074Me resulted in increased expression of PAI-1 at both protein (2.67±1.02 fold, p<0.01, n=3) and mRNA levels (3.2±0.97 fold, p<0.001, n=3), as well as those of the fibrotic marker SMA (siCTSB: 3.15±1.43 fold, p<0.01, n=3; Ca074Me: 5.63±1.7 fold, p<0.0001, n=4) compared to respective controls. Inhibition of CTSB activity also resulted in decreased degradation of gelatin (71±18%, p<0.05, n=4) and collagen I (47±11%, p<0.05, n=4).
Our studies strongly demonstrate (1) the presence of CTSB and components of the proteolytic cascade within caveolae in TM cells, and (2) a role of CTSB in modulating the proteolytic cascade and fibrosis. CTSB activity might constitute a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.
This is a 2020 ARVO Annual Meeting abstract.
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