Activation of telomerase and cyclooxygenase-2 in PDGF and FGF inhibition of C₂-ceramide-induced apoptosis

In the present study, the roles of telomerase and prostaglandin E ₂ (PGE₂) in platelet-derived growth factor (PDGF's) and fibroblast growth factor-2 (FGF-2's) effects against C₂-ceramide- induced cell death were investigated. C₂-ceramide reduced the viability of NIH3T3 cells in a condition without calf serum (CS) in accordance with decreasing telomerase activity according to the TRAP assay. The addition of CS significantly protected cells from C₂-ceramide-induced apoptosis through increased telomerase activity, and the phosphorylations of PDGF and the FGF-2-like receptor in NIH3T3 cells were detected. Adding PDGF and FGF-2 decreased the cytotoxic effect elicited by C₂-ceramide through stimulating telomerase activity, which was blocked by adding a telomerase inhibitor (TI). Activations of ERKs and JNKs were detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities induced by PDGF and FGF were respectively inhibited by the addition of the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125. Accordingly, induction of cyclooxygenase-2 (COX-2) protein expression and PGE₂ production was detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities stimulated by PDGF and FGF were reduced by adding a specific COX-2 inhibitor, NS398, through a decrease in PGE₂ production. Incubation of cells with PGE₂ or the EP1 agonist, 17-PT, but not the EP2 agonist, sulprostone, the EP3 agonist, butaprost, or the EP4 agonist, PGE₁ alcohol, significantly enhanced the telomerase activity of NIH3T3 cells. PGE₂ protection of NIH3T3 cells against C₂-ceramide-induced cell death was identified by the MTT and LDH-release assays, and it was inhibited by adding the EP1 antagonist, SC-19220. Ceramide metabolites including ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), and a standard control of exogenous ceramide C₂-dihydroceramide show no effect on the telomerase activity and viability of NIH3T3 cells. The involvement of COX-2/PGE₂-mediated telomerase activation by PDGF and FGF-2 against C₂-ceramide-induced cell death is first demonstrated herein.