Skip to main content
. 2009 Mar;174(3):829–841. doi: 10.2353/ajpath.2009.080012

Figure 4.

Figure 4

Induction of mRNA and protein of COX-2 and PGE2 by TNF-α treatment with respect to MMP-9 up-regulation in cultured CC cells of HuCCT-1. Participation of COX-1, COX-2, and PGE2, and PGE2 receptor (EP1, EP2, EP3, and EP4) in this process was examined. A: COX-1, COX-2, and receptors of PGE2 (EP1, EP2, and EP) mRNA were detected in cultured CC cells of HuCCT-1, although EP3 mRNA was not detectable. GAPDH was used as an internal control. RT-PCR study. B: TNF-α treatment (100 ng/ml, 6 hours) increased COX-2 mRNA 5.6-fold in cultured CC cells of HuCCT-1, although no such increment was evident in COX-1 mRNA expression. This increase was dose-dependent. Experiments were performed in triplicate and the data are the mean ± SD. *P < 0.005 versus control. Real-time PCR study. C: TNF-α treatment (100 ng/ml, 24 hours) increased COX-2 protein concentration in the supernatant of cultured CC cells of HuCCT-1, but not COX-2 protein. β-Actin was used as the internal control. Western blot study. D: Gelatin zymography for latent and active MMP-9 and MMP-2 after treatment by TNF-α alone or TNF-α + either COX inhibitor or either EP blocking peptide. Indomethacin (Indo, nonselective COX inhibitor) and NS398 (NS, selective COX-2 inhibitor) inhibited the TNF-α-induced MMP-9 production and activation in the cell lysate of cultured CC cells of HuCCT-1, whereas SC560 (SC, selective COX1 inhibitor) partially inhibited the production in HuCCT-1. AH6809 (BP1, blocking peptide of EP1) and SC19220 (BP1/2, blocking peptide of EP1/2) also inhibited completely, whereas AH23848 (BP4 blocking peptide of EP4) did not suppress it. MMP-2 used as an internal control was not affected by any of these stimulations. E: MMP-9 mRNA expression was increased by TNF-α treatment (100 ng/ml, 24 hours). Indomethacin (Indo, nonselective COX inhibitor; 100 μmol/L), SC560 (SC, selective COX1 inhibitor; 100 μmol/L), and NS398 (NC, selective COX-2 inhibitor; 100 μmol/L) reduced TNF-α (100 ng/ml, 24 hours)-induced increased MMP-9 promoter activity in cultured CC cells of HuCCT-1. Expression of mRNA was quantified with real-time PCR. The expression was normalized as a ratio using GAPDH as an internal control. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.01 versus control group; **P < 0.01 versus TNF-α group. F: TNF-α treatment (25 ng/ml and 100 ng/ml) significantly enhanced PGE2 protein production in the conditioned medium in a dose- and time-dependent manner in both cultured CC cells of HuCCT-1. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.05 versus control. G: Indomethacin (Indo, nonselective COX inhibitor), SC560 (C, selective COX1 inhibitor), and NS398 (NS, selective COX-2 inhibitor) inhibited TNF-α-induced PGE2 production. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.05 versus control, TNF-α + indomethacin, TNF-α + SC560, TNF-α + NS398. H: Gelatin zymography for latent and active MMP-9 and MMP-2 after treatment by PGE2 alone or PGE2 + TNF-α. PGE2 alone did not induce MMP-9 production and activation and the administration of PGE2 in addition to TNF-α (100 ng/ml) did not affect TNF-α-induced MMP-9 production and activation. MMP-2 used as an internal control was not affected by any of these stimulations.