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Ram Kannan, Shozo Sonoda, Chandrasekharam N. Nagineni, Mizuki Kitamura, David R. Hinton; Connective Tissue Growth Factor (CTGF) Expression in Human RPE Cells is Regulated by Inflammatory Cytokines, TGF-β and Ceramide. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5303.
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CTGF is a matricellular protein secreted and sequestered in the extracellular matrix where it interacts with integrins, growth factors and cytokines. Our previous work reported that CTGF is secreted by human retinal pigment epithelial cells (HRPE) and is a major mediator of retinal fibrosis . The aim of the present study was to investigate the effects of inflammatory cytokines, TGF-β and ceramide-induced oxidative stress on CTGF expression and to characterize the nature of polarized secretion of CTGF by HRPE cultures.
Microarray analysis using Affymetrix GeneChip was performed using RNA extracted from HRPE cells treated with TGF-β1 or inflammatory cytokine mix (IFN-γ, TNF-α and IL-1β) for 8h. CTGF gene expression and protein secretion were studied in both confluent fetal human RPE cells and polarized RPE monolayers (TER 350 ohms.cm2) treated with TGF-β1 alone (4 ng/ml) or in the presence of 25 µM C2 ceramide. Quantitative ELISA assay was used to determine the levels of cellular and secreted CTGF.
Microarray analysis revealed that TGF-β treatment upregulated CTGF expression by 4.5 fold, while cytokine mix treatment downregulated CTGF gene expression by more than 10 fold. In confluent HRPE cells, ceramide inhibited both constitutive and TGF-β-enhanced secretion of CTGF significantly (p<0.05). Further, CTGF exhibited asymmetry of secretion in polarized HRPE cultures. CTGF secretion was predominantly apical (323 ± 25 pg/ml) and not basolateral (35 ± 5 pg/ml). Both apical and basolateral secretion of CTGF were augmented by TGF-β and were significantly attenuated by ceramide pretreatment. TGF-β and ceramide treatment had no significant effect on cell associated CTGF levels. Real time PCR analysis showed that CTGF mRNA levels were significantly upregulated by TGF-β and inhibited by ceramide suggesting transcriptional regulation of CTGF by TGF-β and ceramide.
Our data show that a) inflammatory cytokines and ceramide cause a marked inhibition of the expression of CTGF while TGF-β enhances CTGF expression by RPE, b) ceramide suppresses the action of TGF-β on CTGF secretion and c) CTGF secretion in HRPE cells is predominantly apical. Our results suggest that enhanced apical secretion of CTGF in the presence of TGF-β may be associated with retinal fibrosis.
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