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Naxin Guo, Steven E. Feldon, Richard P. Phipps; A PPAR Endogenous Ligand 15-deoxy-12,14-Prostaglandin J2 Inhibits TGF-β-mediated Responses In Human Orbital Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5103.
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© ARVO (1962-2015); The Authors (2016-present)
Infiltration of white blood cells and accumulation of extracellular matrix, especially hyaluronan (HA) in orbital tissue are characteristic of Thyroid eye disease (TED). Transforming growth factor beta (TGF-β) is up-regulated in orbital tissue from patients with TED where it acts as a key inducer of fibrosis by enhancing extracellular matrix production. 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is an endogenous Peroxisome Proliferator-Activated Receptor gamma (PPARγ) ligand that exhibits diverse biological effects, including anti-inflammatory and anti-fibrogenic activities. In this study, we report that 15d-PGJ2 inhibits TGF-β-mediated responses in orbital fibroblasts.
Primary orbital fibroblasts were isolated from Graves’ disease patients undergoing orbital decompression surgery. The cells were grown in RPMI 1640 media containing 10% FBS. The amount of HA in the cell culture supernatant and pericellular extraction was measured by ELISA. Activated human T cell expression of CD44 was measured by flow cytometry. T cell adhesion to orbital fibroblasts was detected by measuring fluorescent intensity of bound calcein-AM labeled T cells. A fibrotic marker, alpha smooth muscle actin (α-SMA), was detected using western blot.
TGF-β strongly induced HA levels in conditioned medium and pericellular extracts of human orbital fibroblasts after 24 hour treatment compared to untreated control. TGF-β also enhanced fibroblast-T cell adhesion and this cell-cell adhesion was inhibited by orbital fibroblast pre-incubation with hyaluronidase or HA synthase 2 siRNA, indicating that newly synthesized HA mediated the adhesion, probably through the interaction with its cellular receptor CD44 on the T cell surface. As a key inducer of fibrosis, TGF-β significantly up-regulated α-SMA protein levels in orbital fibroblasts. Remarkably, addition of the PPARγ ligand 15d-PGJ2 to TGF-β treated orbital fibroblasts blocked HA synthesis and T cell-fibroblast adhesion in a dose-dependent manner. In addition, 15d-PGJ2 also inhibited TGF-β induced α-SMA expression.
Our data reveal that 15d-PGJ2 is a potent inhibitor of TGF-β mediated pro-inflammatory and fibrogenic activities in orbital fibroblasts. Newly synthesized HA plays an impotent role in T cell-fibroblast adhesion. The ability to block HA synthesis with inhibitors such as 15d-PGJ2, therefore, presents a potential for therapeutic application.
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