Nephron. Experimental nephrology · 2005
Differential effects of transforming growth factor-beta on the synthesis of connective tissue growth factor and vascular endothelial growth factor by peritoneal mesothelial cell.
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Szeto Cheuk-Chun, Lai Ka-Bik, Chow Kai-Ming, Szeto Carol Yi-Ki, Wong Teresa Yuk-Hwa, Li Philip Kam-Tao
Аннотация
BACKGROUND: Previous studies found that transforming growth factor-beta (TGF-beta) plays a conflicting role in peritoneal fibrosis. We hypothesise that TGF-beta acts on peritoneal mesothelial cells (PMC) via VEGF and CTGF as downstream mediators.
METHODS: The effect of TGF-beta in primary culture of rat PMC was studied. VEGF and CTGF mRNA expression was examined by real time quantitative polymerase chain reaction (RT-QPCR), and VEGF antigen level in cell supernatant by ELISA.
RESULTS: Incubation of rat PMC with TGF-beta resulted in a time- (3-72 h) and concentration- (0-50 pg/ml) dependent increase in VEGF mRNA expression, and VEGF protein level in the cell supernatant. When stimulated with TGF-beta 100 pg/ml, there was a 20-fold up-regulation of VEGF mRNA expression (p < 0.001). The CTGF mRNA expression and protein level of PMC was slightly increased at low concentration of TGF-beta (50 pg/ml) but decreased at a higher concentration (100 pg/ml or above). The effect of TGF-beta on PMC CTGF, but not VEGF, gene expression was inhibited by Smad decoy oligodeoxynucleotide. The effect of TGF-beta on PMC VEGF gene expression and protein synthesis was inhibited by PD98059 (a specific MAP kinase inhibitor) and chelerythrine (a specific protein kinase C inhibitor), but not cholera toxin (activator of cyclic AMP) or herbimycin A (inhibitor of protein tyrosine kinase). The up-regulation of CTGF mRNA expression was inhibited by PD98059, but not chelerythrine, cholera toxin or herbimycin A. Furthermore, CTGF gene expression in TGF-beta-stimulated PMC was inhibited by co-administration of recombinant VEGF.
CONCLUSIONS: Our data demonstrate that TGF-beta induces PMC production of VEGF and CTGF via different signalling pathways. At high concentration of TGF-beta, VEGF production predominates and CTGF production was inhibited. Since CTGF and VEGF have different biologic effects, our results may explain the complex activity of TGF-beta in peritoneal physiology.
METHODS: The effect of TGF-beta in primary culture of rat PMC was studied. VEGF and CTGF mRNA expression was examined by real time quantitative polymerase chain reaction (RT-QPCR), and VEGF antigen level in cell supernatant by ELISA.
RESULTS: Incubation of rat PMC with TGF-beta resulted in a time- (3-72 h) and concentration- (0-50 pg/ml) dependent increase in VEGF mRNA expression, and VEGF protein level in the cell supernatant. When stimulated with TGF-beta 100 pg/ml, there was a 20-fold up-regulation of VEGF mRNA expression (p < 0.001). The CTGF mRNA expression and protein level of PMC was slightly increased at low concentration of TGF-beta (50 pg/ml) but decreased at a higher concentration (100 pg/ml or above). The effect of TGF-beta on PMC CTGF, but not VEGF, gene expression was inhibited by Smad decoy oligodeoxynucleotide. The effect of TGF-beta on PMC VEGF gene expression and protein synthesis was inhibited by PD98059 (a specific MAP kinase inhibitor) and chelerythrine (a specific protein kinase C inhibitor), but not cholera toxin (activator of cyclic AMP) or herbimycin A (inhibitor of protein tyrosine kinase). The up-regulation of CTGF mRNA expression was inhibited by PD98059, but not chelerythrine, cholera toxin or herbimycin A. Furthermore, CTGF gene expression in TGF-beta-stimulated PMC was inhibited by co-administration of recombinant VEGF.
CONCLUSIONS: Our data demonstrate that TGF-beta induces PMC production of VEGF and CTGF via different signalling pathways. At high concentration of TGF-beta, VEGF production predominates and CTGF production was inhibited. Since CTGF and VEGF have different biologic effects, our results may explain the complex activity of TGF-beta in peritoneal physiology.
Предметные рубрики MeSH
AnimalsBlotting, WesternCells, CulturedConnective Tissue Growth FactorDose-Response Relationship, DrugDrug InteractionsEnzyme-Linked Immunosorbent AssayEpithelial CellsImmediate-Early ProteinsIntercellular Signaling Peptides and ProteinsPeritoneumRatsRats, Sprague-DawleySignal TransductionSmad ProteinsTransforming Growth Factor betaUp-RegulationVascular Endothelial Growth Factor A