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P. B. Liton, C. Luna, G. Li, P. Gonzalez, D. L. Epstein; Characterization of the Expression of Chitinase 3-Like 1 Protein in the Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2061.
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We have previously identified Chi3L1 as one of the more abundant transcripts in trabecular meshwork (TM) cells. Our objective was to confirm and characterize the production of Chi3L1 protein by TM cells, and identify the potential mechanisms involved in its regulation.
The presence of Chi3L1 was evaluated by SDS-PAGE and WB analysis under reducing and non-reducing conditions in the culture media from human and porcine TM primary cultures, in the effluent from perfused porcine anterior segments, and in aqueous humor (AH) samples from porcine and human (control and glaucomatous) eyes, using a specific antibody against Chi3L1. Levels of Chi3L1 mRNA were quantified by qPCR analysis. Regulation of Chi3L1 production was analyzed in primary cultures of porcine TM cells using inhibitors for p38 MAPK (SB202190), JNK (SP600125), ERK (PD98059), NF-kB (lactacystin), and PI3K (wortmannin). The recombinant cytokines TGF-ß1 (2.5-10 ng/ml), TGF-ß2 (2.5-10 ng/ml, IL-6 (50-200 ng/ml), IL-8 (50-200 ng/ml), TNF-alpha (10-50ng/ml), and VEGF (10-50 ng/ml) were used for studying the potential effects on Chi3L1 expression in primary cultures of porcine TM cells
High levels of Chi3L1 protein were found in porcine AH, human AH from both control and glaucoma donors, effluent collected from porcine perfused anterior segments, and culture media from human and porcine TM primary cultures. Constitutive expression of Chi3L1 was significantly abolished in the presence of the p38 MAPK, JNK, ERK, and NF-kB inhibitors, and significantly increased with the PI3K inhibitor. Secretion of Chi3L1 was induced in a dosage-dependent manner by TGF-ß1, TGF-ß2, and TNF-alpha. No significant changes in the levels of secreted Chi3L1 were observed following incubation with IL-6, IL-8, and VEGF. Induction of Chi3L1 by TNF-alpha was accompanied by increased levels of mRNA (3 fold). TGF-ß1 and TGF-ß2 did not have a significant effect on the levels of mRNA. In addition, serum deprivation dramatically induced in a time-dependent manner the levels of secreted Chi3L1, which was inhibited by lactacystin
Our results demonstrated high levels of production of Chi3L1 protein by TM cells. Its constitutive production appeared to be modulated by stress-related pathways, suggesting a potential role of Chi3L1 in the response to stress. Since Chi3L1 has been associated in a number of pathological conditions and inflammatory diseases, its presence at high levels in the outflow pathway could be relevant to understand both the normal physiology of the TM and the alterations of this tissue in glaucoma.
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