Suppression of TGF-β-Induced Upregulation of Connective Tissue Growth Factor by Ceramide in Human RPE Cells

Abstract

Purpose: : Connective tissue growth factor (CTGF) is a matricellular protein found in the extracellular matrix and is reported to be induced in response to TGF-β in fibroblasts and RPE. CTGF plays an important role in the pathogenesis of proliferative vitreoretinopathy. The aim of the present study was to investigate the effect of oxidative stress from the lipid messenger ceramide on CTGF secretion from human RPE cells.

Methods: : Early passage fetal human RPE cells were cultured in DMEM containing 10% fetal bovine serum (FBS). After overnight incubation with DMEM containing 0.5% FBS, cells were treated with 8 ng/ml recombinant human (rh) TGF-β for 24 h. The effect of shortchain ceramide was studied by pretreatment for 1 hour with or without 25 µM C2-ceramide prior to rh-TGF-β exposure. Incubations with C2-dihydroceramide served as negative controls. CTGF protein secreted in the culture medium and its gene expression were analyzed by ELISA and real-time PCR respectively.

Results: : TGF-β increased CTGF levels in the medium significantly from 185±32 pg/ml in non-treated controls to 592±82 pg/ml with TGF-β (p<0.01). Pretreatment with C2 ceramide inhibited this response significantly by decreasing CTGF (285±63 pg/ml vs 592±82 pg/ml in TGF-β alone group; p<0.01). Real-time PCR analysis revealed that CTGF mRNA expression in untreated and TGF-β-treated conditions was lower than that in the presence of C2 ceramide. The doses of TGF-β and C2 ceramide used in the present study had no appreciable effect on the viability of RPE cells by MTTassay.

Conclusions: : Our results show that C2 ceramide suppresses the action of TGF-β and downregulates CTGF secretion in human RPE. Investigating the mechanism of this phenomenon would be valuable in identifying drug targets for ocular disease.

Keywords: retinal pigment epithelium • proliferative vitreoretinopathy • oxidation/oxidative or free radical damage