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, 174 (3), 829-41

Cyclooxygenase-2 Is Involved in the Up-Regulation of Matrix metalloproteinase-9 in Cholangiocarcinoma Induced by Tumor Necrosis Factor-Alpha

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Cyclooxygenase-2 Is Involved in the Up-Regulation of Matrix metalloproteinase-9 in Cholangiocarcinoma Induced by Tumor Necrosis Factor-Alpha

Keita Itatsu et al. Am J Pathol.

Abstract

Matrix metalloproteinase-9 (MMP-9) is an important enzyme in tumor invasion and metastasis in malignant tumors, including cholangiocarcinoma (CC). Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, was recently reported to induce the up-regulation of MMP-9 in cultured CC cells. We examined whether cyclooxygenase-2 (COX-2) and prostaglandin-E2 (PGE2), another endogenous tumor promoter, are involved in the up-regulation of MMP-9 in CC using CC tissue specimens and a CC cell line, HuCCT-1. MMP-9 and COX-2 were immunohistochemically expressed in 58% and 89% of 110 CC cases, respectively; the expression of MMP-9 and COX-2 was correlated (r = 0.32, P = 0.00072). Using zymography, latent MMP-9 was detectable in all cases and active MMP-9 was detected in 24% of cases of the CC specimens. The TNF-alpha/TNF-receptor 1 (TNF-R1) interaction induced MMP-9 production and activation, as well as COX-2 overexpression and PGE2 production, and increased the migration of CC cells. MMP-9 up-regulation was inhibited by COX inhibitors, antagonists of EP2/4 (receptors of PGE2), and COX-1 and COX-2 siRNAs. Inhibitors of both MMP-9 and MMP-9 siRNA treatment abrogated the increase in the migration of CC cells induced by TNF-alpha. In conclusion, we propose a novel signaling pathway of MMP-9 up-regulation in CC cells such that TNF-alpha induces the activation of COX-2 and PGE2 via TNF-R1 followed by the up-regulation of MMP-9 via the PGE2 (EP2/4) receptor.

Figures

Figure 1
Figure 1
Expression of MMP-9, COX-1, and COX-2 in human CC tissues. A: Representative histology of CC (a) and the immunohistochemical staining of MMP-9 (b and e), COX-1 (c and f), and COX-2 (d and g). a: A representative histology of CC. H&E. b: MMP-9 is strongly and diffusely expressed in the cytoplasm of almost all CC cells. Immunohistochemistry of MMP-9. c: COX-1 is expressed diffusely in the cytoplasm of CC cells. Immunohistochemistry of COX-1. d: COX-2 is expressed strongly and diffusely in the cytoplasm and membrane of almost all CC cells. Immunohistochemistry of COX-2. e: MMP-9 is not expressed in the nonneoplastic bile ducts in the adjacent normal tissue. f: COX-1 is faintly expressed in the nonneoplastic bile ducts in the adjacent normal tissue. g: COX-2 is faintly expressed in the nonneoplastic bile ducts in the adjacent normal tissue. B: Frequency and degree of immunohistochemical expression of MMP-9, COX-1, and COX-2 in CC tissues (110 cases of CC). C: Double-immunofluorescent staining of MMP-9 (a) (Alexa Fluor 594, red) and COX-2 (b) (Alexa Fluor 488, green) and their merged images (c) and that of MMP-9 (d) (Alexa Fluor 594, red) and COX-1 (e) (Alexa Fluor 488, green) and their merged images (f) in CC. A subset of CC cells are positive for COX-1 alone, whereas another subset of CC cells are positive for both MMP-9 and COX-1. The majority of CC cells are positive for MMP-9 and COX-2, and the merging of double-positive tumor cells gives a yellow color (c), whereas the expression of MMP-9 and COX-1 are dissimilar in CC; some cells expressed MMP-P and the others expressed both proteins. Original magnifications, ×400 (A).
Figure 2
Figure 2
Detection of MMP-9, TNF-α, COX-1, and COX-2 protein and mRNA in frozen tissue specimens of ICC. A: Gelatin zymography of MMP-9 in 21 cases of ICC and 5 normal control samples. Latent MMP-9 (92 kDa) and active MMP-9 (82 kDa) were observed in all cases (1 to 21) and in 5 cases (1 to 5) of ICC, respectively. Latent MMP-9 (92 kDa) was observed faintly in all of normal control samples (N1 to N5). HT1080 (human fibrosarcoma cell line) was used as a positive control of latent and active MMP-9. B: TNF-α, MMP-9, COX-1, and COX-2 mRNAs were detected in all 21 cases of ICC examined. TNF-α, MMP-9, COX-1, and COX-2 mRNA were also detected in one, four, five, and four of five frozen control normal samples, respectively. GAPDH was the internal control. RT-PCR study. C: The correlation of the mRNA expression of TNF-α, MMP-9, COX-1, and COX-2 in 21 CC tissues and 5 control tissues. TNF-α mRNA level was correlated positively with the mRNA level of MMP-9, COX-1, and COX-2, respectively. MMP-9 was correlated with COX-1. CC case, closed diamond; normal control case, half-tone triangle. Expression of mRNA was quantified with real-time PCR. The expression was normalized as a ratio using GAPDH as an internal control.
Figure 3
Figure 3
TNF-α-induced MMP-9 up-regulation was mediated by TNF-receptor 1 (TNF-R1) in cultured CC cells of HuCCT-1. A: TNF-α (24 hours) induced latent MMP-9 in the cell lysate and conditioned medium in a dose-dependent manner and induced active MMP-9 in the cell lysate of cultured CC cells of HuCCT-1. HGF and epidermal growth factor mildly increased only latent MMP-9 in the cell lysate, whereas SDF-1α, IL-1β, and IL-6 did not induce MMP-9. MMP-2 used as an internal control was not affected by any of these stimulations. Gelatin zymography of MMP-9 and MMP-2. B: TNF-R1, TNF-R2, and MMP-9 mRNA were detected in the cultured CC cells of HuCCT-1. GAPDH was used as the internal control. PCR study. C: Inhibition study using neutralizing antibodies against TNF-α and its receptors in TNF-α-induced MMP-9 up-regulation. A PCR study for MMP-9 mRNA expression (a) and gelatin zymography of MMP-9 and MMP-2 (b). a: Anti-TNF-R1 neutralizing antibodies (AR1, 20 μg/ml) clearly inhibited the TNF-α (100 ng/ml, 12 hours)-induced up-regulation of MMP-9 mRNA expression, whereas anti-TNF-R2 neutralizing antibody alone (AR2, 20 μg/ml) failed to inhibit such TNF-α-induced MMP-9 up-regulation. Experiments were performed in triplicate and the data are the mean ± SD. *P < 0.05 versus control group; **P < 0.05 versus TNF-α group. b: Anti-TNF-α neutralizing antibodies (AT, 4 μg/ml), anti-TNF-R1 neutralizing antibodies (AR1, 20 μg/ml), and anti-TNF-R1 neutralizing antibody (20 μg/ml) + anti-TNF-R2 neutralizing antibody (AR2, 20 μg/ml), clearly inhibited TNF-α (100 ng/ml, 24 hours)-induced up-regulation of MMP-9 production and activation, whereas anti-TNF-R2 neutralizing antibody alone (20 μg/ml) failed to inhibit such TNF-α-induced MMP-9 up-regulation. MMP-2 used as an internal control was not affected by any of these stimulations. Gelatin zymography of MMP-9 and MMP-2.
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.
Figure 5
Figure 5
Migration study of cultured CC cells of HuCTT-1 after TNF-α treatment with respect to MMP-9 up-regulation. A: Migration assay of cultured CC cells. TNF-α (100 ng/ml, 24 hours) increased tumor cell migration 5.8-fold in cultured CC cells. Pretreatment by anti-TNF-α neutralization antibodies (4 μg/ml), GM6001 (nonselective MMP inhibitor; 10 μmol/L), and MMP-9 inhibitor I (50 nmol/L) significantly inhibited such increased tumor cell migration. The migration index of CC cells by treatment with control IgG and vehicle DMSO (D) was similar to that by TNF-α treatment alone. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.005 versus control, **P < 0.005 versus TNF-α (100 ng/ml) alone. AT, anti-TNF-α neutralization antibody; GM, GM 6001; M9I, MMP-9 inhibitor I; IgG, IgG; D, vehicle DMSO. B: Migration assay of cultured CC cells. Indomethacin (nonselective COX inhibitor), SC560 (selective COX1 inhibitor), NS398 (selective COX-2 inhibitor) inhibited TNF-α-induced migration of cultured CC cells, significantly. The migration index of CC cells by vehicle DMSO (D) was similar to that by TNF-α treatment alone. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.01 versus control group; **P < 0.005 versus TNF-α group.
Figure 6
Figure 6
The knockdown of MMP-9, COX-1, and COX-2 by siRNA inhibited TNF-α-induced MMP-9 up-regulation and cell migration. A: Gelatin zymography (a) and a quantification by an LI-COR Odyssey IR imaging system (b) for latent and active MMP-9 after treatment by TNF-α alone or TNF-α + either of MMP-9, COX-1, and COX-2 siRNA. MMP-9 and COX-1 siRNA treatment inhibited TNF-α-induced MMP-9 up-regulation effectively in the cell lysate of HuCCT-1. COX-2 siRNA also showed a partial inhibitory effect on TNF-α-induced MMP-9 up-regulation. The experiments were performed in triplicate and the data are the mean ± SD. *P < 0.01 versus control. B: COX-1 and COX-2 siRNA inhibit the protein expression of COX-1 and COX-2, respectively, and MMP-9 inhibition did not affect on the expression of COX-1 and COX-2 in HuCCT-1. β-Actin was used as the internal control. Western blot study. C: A migration assay of HuCTT-1. TNF-α (100 ng/ml, 24 hours) increased tumor cell migration 5.2-fold in cultured CC cells. Pretreatment by MMP-9, COX-1, and COX-2 siRNA significantly inhibited TNF-α-induced increase of tumor cell migration. The experiments were performed in triplicates and the data are the mean ± SD. *P < 0.05 versus control, **P < 0.05 versus TNF-α (100 ng/ml) alone.
Figure 7
Figure 7
Schema of TNF-α-induced up-regulation of COX-2 and MMP-9, and their interaction. Activated MMP-9 induced the migration of CC cells. In addition to active participation of COX-2, COX-1 may be also partly involved in this process. TNF-R1, TNF receptor 1; EP2/4, PGE2 receptors EP2 and EP4; MMP-9.

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