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A. Sethi, W. Mao, R. Krishnamoorthy, R. J. Wordinger, A. F. Clark; Transforming Growth Factor β Induces Extracellular Matrix Protein Crosslinking Enzyme Lysyl Oxidase and Other LOX Family Proteins: Implications for Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5720.
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Glaucoma is associated with elevated intraocular pressure (IOP) due to increased aqueous outflow resistance in the trabecular meshwork (TM). Increased deposition of extracellular matrix (ECM) material in the TM appears to be responsible for this glaucomatous IOP elevation. Lysyl oxidase (LOX) is a collagen and elastin polymer crosslinking enzyme, and recent genome wide association studies showed that SNPs in LOX Like 1 (LOXL1), a LOX family member, significantly increased the risk of developing exfoliation glaucoma. Elevated levels of TGF-β are observed in aqueous humor of glaucoma patients. TGFβ has been shown to increase ECM deposition and induce other families of cross linking enzymes like Transglutaminase-2 in human TM cells. The objective of the current study is to evaluate the effect and signaling mechanism of exogenous TGFβ (1, 2 and 3) on LOX and LOXL1-4 mRNA and protein induction in cultured human TM cells.
Primary glaucomatous and normal TM cells were cultured in DMEM containing 10% FBS and transferred to serum free medium for 24 hours prior to 48 hours of TGFβ treatment. Both cell associated and secreted LOX protein levels were analyzed using Western immunoblotting. mRNA levels were determined by RT-PCR. The nuclear extract after treatment was used for electrophoretic mobility shift assay (EMSA).
Cell associated LOX, LOXL2 and LOXL4 proteins and LOX, LOXL1, LOXL2 and LOXL3 mRNA were induced by the 3 isoforms of TGFβ. However, only TGFβ2 induced the expression of the secreted proteins. The induction of LOX, LOXL2 and LOXL4 proteins by the three isoforms of TGFβ was observed to be dose dependent. Abrogation of TGFβ signaling by small molecule inhibitors, LY-364947 and SB-431542, resulted in inhibition of LOX induction. TGFβ increased LOX enzyme activity, in that the LOX inhibitor β-aminopropionitrile increased levels of tropoelastin, the soluble precursor form of elastin. TGFβ activated JNK signaling in the TM, and EMSA showed upregulation of the downstream transcription factor AP-1. We are using chromatin immunoprecipitation assays to confirm the presence of AP-1 binding sites in the LOX gene promoters.
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