Cyclooxygenase-2 Inhibitor Enhances Whereas Prostaglandin E2Inhibits the Production of Interferon-Induced Protein of 10 kDa in Epidermoid Carcinoma A431
2002; Elsevier BV; Volume: 119; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2002.19510.x
ISSN1523-1747
AutoresNaoko Kanda, Shinichi Watanabe,
Tópico(s)Estrogen and related hormone effects
ResumoInterferon-induced protein of 10 kDa (IP-10) induces antitumor immunity. Cyclooxygenase-2 and its metabolite prostaglandin E2 (PGE2) are overexpressed in tumor cells, which may suppress antitumor immunity. We examined the in vitro effects of cyclooxygenase-2 inhibitor NS398 on IP-10 production in human epidermoid carcinoma A431. NS398 enhanced interferon-γ-induced IP-10 secretion, mRNA expression, and promoter activation in A431, and exogenous PGE2 antagonized the enhancement. Interferon-stimulated response element (ISRE) on IP-10 promoter was responsible for the transcriptional regulation by NS398 and PGE2. NS398 enhanced interferon-γ-induced transcription through ISRE and binding of signal transducer and activator of transcription 1α (STAT1α to ISRE in A431, and PGE2 antagonized the enhancement. NS398 enhanced interferon-γ-induced tyrosine phosphorylation of STAT1α, Janus tyrosine kinase 1, and Janus tyrosine kinase 2, and PGE2 antagonized the enhancement. PGE2-mediated suppression of IP-10 synthesis was counteracted by adenylate cyclase inhibitor SQ22536 and protein kinase A inhibitor H-89, and PGE2 receptor EP4 antagonist AH23848B. AH23848B, SQ22536, and H-89 counteracted the PGE2-mediated suppression of ISRE-dependent transcription, STAT1α binding to ISRE, and tyrosine phosphorylation of STAT1α, Janus tyrosine kinase 1, and Janus tyrosine kinase 2. PGE2 increased intracellular cAMP level and protein kinase A activity in A431 pretreated with NS398, and AH23848B blocked the effects of PGE2. These results suggest that A431-derived PGE2 may generate cAMP signal via EP4 in A431, which may activate protein kinase A, and may resultantly inhibit interferon-γ-induced STAT1α activation and IP-10 synthesis. The results also suggest that NS398 may restore IP-10 synthesis by preventing PGE2 production in A431 and thus may be therapeutically useful for skin cancer. Interferon-induced protein of 10 kDa (IP-10) induces antitumor immunity. Cyclooxygenase-2 and its metabolite prostaglandin E2 (PGE2) are overexpressed in tumor cells, which may suppress antitumor immunity. We examined the in vitro effects of cyclooxygenase-2 inhibitor NS398 on IP-10 production in human epidermoid carcinoma A431. NS398 enhanced interferon-γ-induced IP-10 secretion, mRNA expression, and promoter activation in A431, and exogenous PGE2 antagonized the enhancement. Interferon-stimulated response element (ISRE) on IP-10 promoter was responsible for the transcriptional regulation by NS398 and PGE2. NS398 enhanced interferon-γ-induced transcription through ISRE and binding of signal transducer and activator of transcription 1α (STAT1α to ISRE in A431, and PGE2 antagonized the enhancement. NS398 enhanced interferon-γ-induced tyrosine phosphorylation of STAT1α, Janus tyrosine kinase 1, and Janus tyrosine kinase 2, and PGE2 antagonized the enhancement. PGE2-mediated suppression of IP-10 synthesis was counteracted by adenylate cyclase inhibitor SQ22536 and protein kinase A inhibitor H-89, and PGE2 receptor EP4 antagonist AH23848B. AH23848B, SQ22536, and H-89 counteracted the PGE2-mediated suppression of ISRE-dependent transcription, STAT1α binding to ISRE, and tyrosine phosphorylation of STAT1α, Janus tyrosine kinase 1, and Janus tyrosine kinase 2. PGE2 increased intracellular cAMP level and protein kinase A activity in A431 pretreated with NS398, and AH23848B blocked the effects of PGE2. These results suggest that A431-derived PGE2 may generate cAMP signal via EP4 in A431, which may activate protein kinase A, and may resultantly inhibit interferon-γ-induced STAT1α activation and IP-10 synthesis. The results also suggest that NS398 may restore IP-10 synthesis by preventing PGE2 production in A431 and thus may be therapeutically useful for skin cancer. adenylate cyclase 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester 3′,5′-adenosine cyclic monophosphate cyclooxygenase guanine nucleotide-binding protein G-protein inhibitory subunit G-protein stimulatory subunit N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide interferon-induced protein of 10 kDa interferon-stimulated response element Janus tyrosine kinase nuclear factor-κB N-(2-cyclohexyloxyloxy-4-nitrophenyl)methanesulfonamide 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole 9-(tetrahydro-2′-fluryl) adenine signal transducer and activator of transcription 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione The production of prostaglandin E2 (PGE2) is enhanced in a variety of human malignancies (Muller-Decker et al., 1995Muller-Decker K. Scholz K. Marks F. Furstenberger G. Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis.Mol Carcinog. 1995; 12: 31-41Crossref PubMed Scopus (135) Google Scholar). Tumor-derived PGE2 inhibits antitumor immunity; PGE2 enhances the production of immunosuppressive interleukin-10 by macrophages (Strassmann et al., 1994Strassmann G. Patil-Koota V. Finkelman F. Fong M. Kambayashi T. Evidence for the involvement of interleukin-10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2.J Exp Med. 1994; 180: 2365-2370Crossref PubMed Scopus (347) Google Scholar) and inhibits their production of interleukin-12, which enhances cytotoxic activity of natural killer cells and cytotoxic T cells (Tineke et al, 1995). PGE2 inhibits interleukin-2 and interferon-γ (IFN-γ) production in T cells and natural killer cells (Anastassiou et al., 1992Anastassiou E.D. Paliogianni F. Balow J.P. Yamada H. Boumpas T. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2Rα gene expression at multiple levels.J Immunol. 1992; 148: 2845-2852PubMed Google Scholar), and inhibits their cytotoxic activity (Uoita, 1996). Four subtypes of PGE2 receptor have been identified, EP1, EP2, EP3, and EP4, and the immunosuppressive effects of PGE2 are mainly mediated by EP2 or EP4 (Uoita, 1996). EP2 and EP4 are linked to adenylate cyclase (AC) via guanine nucleotide-binding protein (G-protein) stimulatory subunit (Gs). The stimulation of EP2 or EP4 generates 3′,5′-adenosine cyclic monophosphate (cAMP), the AC product, and activates cAMP-dependent protein kinase (protein kinase A; PKA) (Uoita, 1996), which leads to the repression of tumor-suppressive cytokine or chemokine production (Uoita, 1996). The synthesis of PGs is dependent on the activity of cyclooxygenase (COX), which converts arachidonic acid to PGH2. There are two isoforms of COX: COX-1 and COX-2. COX-1 is constitutively expressed whereas COX-2 is induced by phorbol esters, endotoxins, or growth factors (Tang et al., 2001Tang Q. Chen W. Gonzales M.S. Finch J. Inoue H. Bowden G.T. Role of cyclic AMP responsive element in the UVB induction of cyclooxygenase-2 transcription in human keratinocytes.Oncogene. 2001; 20: 5164-5172Crossref PubMed Scopus (72) Google Scholar). COX-2 expression is enhanced in malignant epithelial tumors (Muller-Decker et al., 1999Muller-Decker K. Reinerth G. Krieg P. et al.Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin.Int J Cancer. 1999; 82: 648-656Crossref PubMed Google Scholar;Gallo et al., 2001Gallo O. Franchi A. Magnelli L. et al.Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis.Neoplasia. 2001; 3: 53-61Abstract Full Text PDF PubMed Scopus (273) Google Scholar), which may result in PGE2 overproduction (Muller-Decker et al., 1995Muller-Decker K. Scholz K. Marks F. Furstenberger G. Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis.Mol Carcinog. 1995; 12: 31-41Crossref PubMed Scopus (135) Google Scholar). A recent study reported that COX-2-inhibiting agents in vivo restored interleukin-12 production and inhibited interleukin-10 production in macrophages or lymphocytes by preventing PGE2 production and inhibited the progression of murine lung carcinoma (Stolina et al., 2000Stolina M. Sharma S. Lin Y. et al.Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis.J Immunol. 2000; 164: 361-370Crossref PubMed Scopus (444) Google Scholar). Thus COX-2 inhibitors may be useful for the treatment of human malignancies with COX-2 and PGE2 overexpression. Interferon-induced protein of 10 kDa (IP-10) induces antitumor immune responses in synergy with interleukin-12 (Narvaiza et al., 2000Narvaiza I. Mazzolini G. Barajas M. et al.Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-γ-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy.Immunol. 2000; 164: 3112-3122Crossref Scopus (162) Google Scholar). Tumor cells can produce IP-10 in response to IFN-γ released by tumor-surrounding lymphocytes (Bukowski et al., 1999Bukowski R.M. Rayman P. Molto L. et al.Interferon-γ and CXC chemokine induction by interleukin 12 in renal cell carcinoma.Clin Cancer Res. 1999; 5: 2780-2789PubMed Google Scholar). The tumor-derived IP-10 chemoattracts activated CD4+ T helper 1 cells, which promote tumor killing by natural killer cells and macrophages or induce dendritic cells to prime cytotoxic T cells (Hung et al., 1998Hung K. Hayashi R. Lafond-Walker A. Lowenstein C. Pardoll D. Levitsky H. The central role of CD4+ T cells in the antitumor immune response.J Exp Med. 1998; 188: 2357-2368Crossref PubMed Scopus (1084) Google Scholar;Lanzavecchia, 1998Lanzavecchia A. Licence to kill.Nature. 1998; 393: 413-414Crossref PubMed Scopus (304) Google Scholar;Narvaiza et al., 2000Narvaiza I. Mazzolini G. Barajas M. et al.Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-γ-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy.Immunol. 2000; 164: 3112-3122Crossref Scopus (162) Google Scholar). In addition, IP-10 inhibits tumor angiogenesis (Arenberg et al., 1996Arenberg D.A. Kunkel S.L. Polverini P.J. et al.Interferon-γ-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases.J Exp Med. 1996; 184: 981-992Crossref PubMed Scopus (321) Google Scholar). The induction of IP-10 production in tumor cells themselves is thus therapeutically useful for malignancy. A recent study reported that exogenous PGE2 inhibited IP-10 secretion in macrophages via cAMP (Kuroda et al., 2001Kuroda E. Sugiura T. Okada K. Zeki K. Yamashita U. Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFN-inducible protein-10 production by APC.J Immunol. 2001; 166: 1650-1658Crossref PubMed Scopus (65) Google Scholar); however, the precise mechanism for the inhibition is not defined. Besides it has not been examined if tumor-derived PGE2 may suppress IP-10 production in tumor cells themselves or if COX-2 inhibitor may restore their IP-10 production by preventing PGE2 production. In this study, we investigated the in vitro effect of a selective COX-2 inhibitor, N-(2-cyclohexyloxyloxy-4-nitrophenyl)methanesulfonamide (NS398), on IFN-γ-induced IP-10 production in human epidermoid carcinoma A431. We found that NS398 enhanced IP-10 production and exogenous PGE2 reversed the enhancement. We further analyzed the mechanism for the suppression by PGE2 of IFN-γ-induced IP-10 production. 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), NS398, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560), 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), N- [2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89), and 9-(tetrahydro-2′-fluryl)adenine (SQ22536) were obtained from Calbiochem (La Jolla, CA). PGE2, 17-phenyl trinor PGE2, and butaprost were from Cayman Chemicals (Ann Arbor, MI). AH23848B was from Funakoshi Pharmaceuticals (Tokyo, Japan). Recombinant human IFN-γ was purchased from R&D Systems (Minneapolis, MN). Antibodies against COX-1, COX-2, β-actin, phosphotyrosine (PY20), signal transducer and activator of transcription 1α (STAT1α p91, 48 kDa IFN-stimulated response element (ISRE) binding protein (p48), Janus tyrosine kinase 1 (Jak1), and Jak2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human epidermoid carcinoma A431 was purchased from Dainippon Pharmaceuticals (Osaka, Japan), and was cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco-BRL), 1% nonessential amino acids, 1 mM sodium pyruvate (ICN Biomedicals, Aurora, OH), 100 U per ml penicillin G, and 100 mg per ml streptomycin (Gibco-BRL). A431 cells (5×104 per well) were seeded in triplicate into 24-well plates, adhered overnight, washed, and then incubated with serum-free DMEM for 24 h. The cells were preincubated with NS398 for 2 h, and then incubated with PGE2 or several PGE2 analogs for 10 min and finally with 10 ng per ml IFN-γ for another 24 h. In some experiments, various signal inhibitors or EP4 antagonist AH23848B were added 10 min prior to the addition of PGE2. The culture supernatants were assayed for IP-10 by enzyme-linked immunosorbent assay (ELISA) (HyCult Biotechnology, Uden, The Netherlands) according to the manufacturer's instruction. The sensitivity of the assay was 20 pg per ml. A431 cells were harvested from subconfluent culture or after incubation with IFN-γ for 3 h in the presence or absence of NS398 or PGE2 as above. The cellular mRNA was extracted using an mRNA purification kit (Pharmacia, Uppsala, Sweden) according to the manufacturer's instruction. cDNA was made from RNA samples as described previously (Tjandrawinata et al., 1997Tjandrawinata R.R. Dahiya R. Hughes-Fulford M. Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells.Br J Cancer. 1997; 75: 1111-1118Crossref PubMed Scopus (209) Google Scholar). Primer sequences for COX-1, COX-2, IP-10, EP1, EP2, EP3, EP4, and β-actin were as described previously (Mukhopadhyay et al., 1997Mukhopadhyay P. Geoghegan T.E. Patil R.V. Bhattacherjee P. Paterson C.A. Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells.Biochem Pharmacol. 1997; 53: 1249-1255Crossref PubMed Scopus (52) Google Scholar;Boorsma et al., 1998Boorsma D.M. Flier J. Sampat S. et al.Chemokine IP-10 expression in cultured human keratinocytes.Arch Dermatol Res. 1998; 290: 335-341Crossref PubMed Scopus (54) Google Scholar;Kanda and Watanabe, 2001Kanda N. Watanabe S. Regulatory roles of adenylate cyclase and cyclic nucleotide phosphodiesterases 1 and 4 in interleukin-13 production by activated human T cells.Biochem Pharmacol. 2001; 62: 495-507Crossref PubMed Scopus (47) Google Scholar;Yoshida et al., 2001Yoshida T. Sakamoto H. Horiuchi T. Yamamoto S. Suematsu A. Oda H. Koshihara Y. Involvement of prostaglandin E2 in interleukin-1α-induced parathyroid hormone-related peptide production in synovial fibroslasts of patients with rheumatoid arthritis.J Clin Endocrinol Metab. 2001; 86: 3272-3278PubMed Google Scholar). PCR was performed by one denaturing cycle of 95°C for 3 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 3 min. The PCR products were analyzed by electrophoresis on 2.5% agarose gels and stained with ethidium bromide, viewed by ultraviolet light, and photographed. Densitometric analysis was performed by scanning the bands into Photoshop and performing densitometry with NIH Image Software. Results are expressed as the intensity ratio relative to that of β-actin product. The firefly luciferase reporter plasmids driven by human IP-10 promoter (–525/+97 bp relative to the transcriptional start site) were constructed by PCR and insertion into pGL3 basic vector (Promega, Madison, WI) as described previously (Majumder et al., 1998Majumder S. Zhou L.S.-H. Chaturvedi P. Babcock G. Aras S. Ransohoff R.M. p48/STAT-1α-containing complexes play a predominant role in induction of IFN-γ-inducible protein, 10 kDa (IP-10) by IFN-γ alone or in synergy with TNF-α.J Immunol. 1998; 161: 4736-4744PubMed Google Scholar). Site-specific mutation in ISRE and two nuclear factor-κB (NF-κB) binding sites of the human IP-10 promoter were created by PCR as described previously (Ohmori and Hamilton, 1993Ohmori Y. Hamilton T.A. Cooperative interaction between interferon (IFN) stimulus response element and κB sequence motifs controls IFNγ- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter.J Biol Chem. 1993; 268: 6677-6688Abstract Full Text PDF PubMed Google Scholar). p3xISRE-SV-luc was constructed by inserting three copies of ISRE (5′-CGCTTTGGAAAGTGAAACCTACCTC-3′ with consensus sequence underlined) from human IP-10 promoter in front of heterologous minimal SV40 promoter upstream of firefly luciferase reporter as described previously (Ohmori and Hamilton, 1993Ohmori Y. Hamilton T.A. Cooperative interaction between interferon (IFN) stimulus response element and κB sequence motifs controls IFNγ- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter.J Biol Chem. 1993; 268: 6677-6688Abstract Full Text PDF PubMed Google Scholar;Ohmori et al., 1994Ohmori Y. Tebo J. Nedospasov S. Hamilton T.A. κB binding activity in a murine macrophage-like cell line. Sequence-specific differences in κB binding and transcriptional activation functions.J Biol Chem. 1994; 269: 17684-17690Abstract Full Text PDF PubMed Google Scholar). Transient transfection was performed using Lipofectamine Plus reagent (Gibco-BRL) according to the manufacturer's protocol. Briefly, A431 cells were plated in six-well plates, grown to about 80% confluence, and then serum-deprived for 24 h. The cells were incubated with 1.8 μg of luciferase construct and 0.2 μg of Rous sarcoma virus β-galactosidase vector per well in a mixture of serum-free DMEM, Plus reagent, and Lipofectamine for 5 h, then washed, and incubated in serum-free DMEM for 18 h. Cells were pretreated with NS398 for 2 h and then incubated with PGE2 or several PGE2 analogs for 10 min, and finally with IFN-γ for another 6 h. In some experiments, various signal inhibitors or AH23848B 20 μM were added 10 min prior to the addition of PGE2. The cells were lyzed and luciferase activities were quantified using a luciferase assay system (Promega). The same cell extracts were assayed for β-galactosidase activity using chemiluminescent Galacto-Light kit (Tropix, Bedford, MA). The results obtained in each transfection were normalized for β-galactosidase activity and expressed as relative luciferase activity. The probe used was annealed double-stranded DNA containing ISRE (sequence described above) from the human IP-10 promoter. The probe was labeled by [32P]dCTP with Klenow DNA polymerase. For gel-shift assays, 2–5 μg of nuclear protein extracts were incubated at room temperature for 5 min with a mixture containing 6 mM HEPES (pH 7.9), 0.4 mM ethylenediamine tetraacetic acid (EDTA), 125 mM KCl, 10% glycerol, 0.05 μg per μl poly(dI-dC), 1 mM dithiothreitol, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 50 μg per ml aprotinin, and 50 μg per ml leupeptin. Approximately 1 ng labeled probe was added and the reactions were incubated at room temperature for another 20 min. In antibody supershift experiments, the nuclear extracts were preincubated with various antibodies on ice for 30 min before the addition of the probe. Reactions were then fractionated on a nondenaturing 5% polyacrylamide gel in 0.5×TBE. The gels were dried and visualized with phosphorimager (Molecular Dynamics, Sunnyvale, CA). A431 cells from subconfluent culture were lyzed in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 50 μg per ml aprotinin, and 50 μg per ml leupeptin, and centrifuged at 14,000 rpm for 10 min. Protein concentration in the supernatant was determined by Bio-Rad DC reagent (Bio-Rad Laboratories, Hercules, CA). For Western analysis, 20 μg of proteins were resolved on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane, and then blocked in 5% nonfat dry milk TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 1 h. The membrane was incubated with anti-COX-2, anti-COX-1, or anti-β-actin antibodies for 2–3 h, and with horseradish-peroxidase-conjugated rabbit antigoat IgG for 1 h, and was then developed by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The phosphorylation status of Jak1/Jak2 and STAT1α was assessed by immunoprecipitation followed by immunoblotting as described previously (Delgado and Ganea, 2000Delgado M. Ganea D. Inhibition of IFN-γ-induced Janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide.J Immunol. 2000; 165: 3051-3057Crossref PubMed Scopus (73) Google Scholar). Cells were lyzed in ice-cold immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM β-glycerol phosphate, 1 mM phenylmethylsulfonyl fluoride, 1 μg per ml leupeptin, 2 μg per ml aprotinin, 1 μg per ml pepstatin, 1% Nonidet p-40, 0.25% deoxycholate, and 0.1% SDS). STAT1α and Jak1/Jak2 molecules were immunoprecipitated by incubation with 10 μg per ml of the respective antibodies. The immune complexes were captured on protein G-Sepharose beads (Pharmacia, Piscataway, NJ) for 1 h at 4°C. Precipitated proteins were washed by immunoprecipitation buffer and were separated by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked and incubated with 1 μg per ml of antiphosphotyrosine antibody (PY20), and then incubated with horseradish-peroxidase-conjugated goat antimouse IgG and developed. Following the analysis of phosphorylated molecules, the blots were stripped and reprobed with antibodies against the respective proteins as described above. 5×104 per ml of A431 cells were plated in 24-well plates and adhered overnight, washed, and serum-deprived as described above. The cells were pretreated with serum-free DMEM alone or with NS398 or SC560 for 2 h and washed, and then 20 μM arachidonic acid (Sigma) was added and the cells were cultured in the presence or absence of NS398 or SC560 in serum-free DMEM. After 1 h, PGE2 levels of the supernatants were measured by ELISA (Cayman) according to the manufacturer's instruction. The sensitivity of the assay was 10 pg per ml. At the end of the culture the number of cells was counted, and the results are expressed as ng per 105 cells. A431 cells were plated at 5×104 cells per well in 24-well plates, adhered overnight, and then serum-deprived for 24 h. They were pretreated with NS398 for 2 h, and then incubated with PGE2 or PGE2 analogs for 10 min. The cells were lyzed with acetate buffer (pH 5.8) containing 0.25% dodecyltrimethylammonium bromide. The cAMP amount in the lysate was measured with an ELISA kit (Amersham) according to the manufacturer's instruction. The sensitivity of the assay was 12 fmol per assay well. The intracellular cAMP level was presented as pmol per 106 cells. A431 cells preincubated with NS398 were incubated with PGE2 or PGE2 analogs for 10 min as described above. The cells were lyzed in the buffer containing 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 μg per ml aprotinin, 1 μg per ml pepstatin, 1 μg per ml leupeptin, 15 mM benzamidine, and 3.75 mM β-mercaptoethanol. The cell lysate was assayed for the activity of PKA using an ELISA kit (Medical and Biological Laboratories, Nagoya, Japan) by examining the phosphorylation of plate-bound peptide substrate in the presence or absence of 2 μM cAMP for 10 min at room temperature. The plates were sequentially incubated with biotinylated antibody to the phosphorylated substrate, peroxidase-conjugated streptavidin, o-phenylenediamine, and then the optical density at 492 nm was read. The net optical density was calculated by subtracting the optical density in the presence of specific PKA inhibitor KT-5720 (Calbiochem) 1 μM from the total optical density. The PKA activity was expressed as an activity ratio, which is defined as the net optical density in the absence of exogenous cAMP divided by the net optical density in the presence of cAMP. As examined by RT-PCR or Western blotting (Figure 1a), the mRNAs and proteins for both COX-1 and COX-2 were constitutively expressed in A431 though the level of COX-1 expression, as examined by relative intensity, was slightly higher than that of COX-2: 1.8-fold and 1.2-fold higher by RT-PCR and Western blotting, respectively. We then analyzed the contribution of COX-1 or COX-2 to the PGE2 production. The constitutive PGE2 production in A431 was inhibited by 10 μM of selective COX-2 inhibitor NS398 (COX-1 IC50>100 μM; COX-2 IC50=3.8 μM;Futaki et al., 1994Futaki N. Takahashi S. Yokoyama M. Arai I. Higuchi S. Otomo S. NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro.Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (784) Google Scholar) by 95% whereas the percentage inhibition by 0.1 μM of selective COX-1 inhibitor SC560 (COX-1 IC50=0.009 μM; COX-2 IC50=6.3 μM;Smith et al., 1998Smith C.J. Zhang Y. Koboldt C.M. et al.Pharmacological analysis of cyclooxygenase-1 in inflammation.Proc Natl Acad Sci USA. 1998; 95: 13313-13318Crossref PubMed Scopus (710) Google Scholar) was only 6% (Figure 1b). An increased concentration of SC560, which may inhibit COX-2, was not used. Thus the constitutive PGE2 production in A431 was mainly dependent on COX-2. We then analyzed the mRNA expression for four subtypes of PGE2 receptors, EP1, EP2, EP3, and EP4, in A431. A431 expressed EP1, EP3, and EP4 mRNAs but did not that of EP2 (Figure 1c). The order of expression level was EP4>EP1>EP3; the band intensity normalized for that of β-actin was 0.61, 0.32, and 0.13, respectively. It is thus indicated that A431-derived PGE2 synthesized via COX-2 may act on A431 cells themselves via EP1, EP3, or EP4. To know if A431-derived PGE2, which is produced via COX-2, may suppress the IP-10 production in A431, we examined if COX-2 inhibitor NS398 may enhance the IP-10 secretion in A431, and if exogenous PGE2 may reverse the enhancement. The constitutive IP-10 secretion with medium alone was minimal in A431 (mean±SEM 51±6 pg per ml, n=5), and IFN-γ dose-dependently enhanced the IP-10 secretion: 203±22 pg per ml by IFN-γ 1 ng per ml, 1080±112 pg per ml by IFN-γ 10 ng per ml, 3523±321 pg per ml by IFN-γ 100 ng per ml; more than 100 ng per ml of IFN-γ did not further increase the IP-10 secretion. NS398 alone did not alter the IP-10 secretion; however, it increased IP-10 secretion induced by suboptimal concentrations of IFN-γ (1 and 10 ng per ml) (Figure 2a). IP-10 secretion induced by IFN-γ 1 or 10 ng per ml was potentiated by NS398 10 μM 2.6-fold or 2.1-fold of controls, respectively. On the other hand, NS398 did not further enhance the IP-10 secretion induced by a saturating concentration of IFN-γ (100 ng per ml). Thus NS398 increased IP-10 secretion in synergy with suboptimal concentrations of IFN-γ. In contrast, selective COX-1 inhibitor SC560 did not alter the constitutive or IFN-γ-induced IP-10 secretion. Thus the enhancement of IP-10 secretion by COX inhibitors paralleled their inhibition of PGE2 production (Figure 1b). Exogenous PGE2 reversed the enhancement by NS398 of IFN-γ-induced IP-10 secretion (Figure 2b), indicating that the enhancement by NS398 was mediated via preventing PGE2 production, and that PGE2 produced via COX-2 in A431 may suppress IFN-γ-induced IP-10 secretion. We then analyzed which of the receptors may be involved in the PGE2-induced inhibition of IP-10 secretion, using receptor agonists or antagonists. EP1 and EP3 agonist 17 phenyl trinor PGE2 or EP2 agonist butaprost did not reverse the enhancement by NS398 of IFN-γ-induced IP-10 secretion (Figure 2b), indicating that EP1, EP2, or EP3 may not be involved in the inhibition by PGE2 of IP-10 production. On the other hand, the PGE2-induced inhibition of IP-10 secretion was counteracted by EP4 antagonist AH23848B, indicating that the effect of PGE2 may be mediated via EP4. These results suggest that A431-derived PGE2, which is synthesized via COX-2, may latently suppress IFN-γ-induced IP-10 secretion via EP4 in A431 cells. We then examined if NS398 may enhance IP-10 mRNA expression induced by suboptimal concentrations of IFN-γ in A431 and if exogenous PGE2 may reverse the enhancement. Though NS398 alone did not alter IP-10 mRNA levels in unstimulated A431 cells, NS398 enhanced IP-10 mRNA expression induced by 10 ng per ml of IFN-γ 2.5-fold (Figure 3). The effect of NS398 was reversed by exogenous PGE2 but not by butaprost or 17 phenyl trinor PGE2 (data not shown), and the effect of PGE2 was counteracted by EP4 antagonist AH23848B. On the other hand, SC560 did not enhance the IFN-γ-induced IP-10 mRNA expression. IFN-γ 100 ng per ml increased IP-10 mRNA level 13.5-fold of controls, which was not further enhanced by NS398 (data not shown). Thus the results on IP-10 mRNA expression paralleled those for IP-10 secretion (Figure 2). The results suggest that NS398-induced stimulation and PGE2-induced inhibition of IFN-γ-induced IP-10 synthesis occurred at pretranslational level, and that the effect of PGE2 may be mediated via EP4. We then examined whether NS398 may enhance IP-10 promoter activity induced by IFN-γ in A431 and whether exogenous PGE2 may reverse the enhancement by NS398. We transiently transfected human
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