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• W2067353642 abstract "Inducible nitric-oxide synthase (iNOS) has been identified as a marker and mediator of disease in human colonic inflammation and carcinogenesis. Accordingly, identification of mediators that trigger iNOS in colon carcinoma/epithelial cells is an important topic of current research. Here we demonstrate that interleukin (IL)-22, a newly described member of the IL-10 cytokine family, potently synergizes with interferon (IFN)-γ for iNOS expression in human DLD-1 colon carcinoma cells. Detection of both IL-22 receptor chains and STAT3 phosphorylation proved robust IL-22 responsiveness of these cells. Short interfering RNA technology identified STAT3 as being crucial for up-regulation of iNOS. Compared with IFNγ, STAT1 phosphorylation by IL-22 was insufficient. IL-22 did not stabilize IL-1β/tumor necrosis factor-α/IFNγ-induced iNOS mRNA. IL-22 also failed to amplify expression of the prototypic IFNγ-inducible parameters IL-18-binding protein and CXCL-10, indicating that IL-22 is not a general amplifier of IFNγ functions. This assumption is furthermore supported by the observation that IL-22 was unable to enhance cellular activation of the pro-inflammatory transcription factor nuclear factor-κB. In contrast, IL-22 increased iNOS promoter activation as detected by using DLD-1 cells stably transfected with a corresponding 16-kb promoter construct (pNOS2(16)-Luc). IL-22 likewise enhanced iNOS in Caco-2 colon carcinoma cells. With IL-22 we introduce a novel potent determinant of iNOS expression in human colon carcinoma/epithelial cells. Considering the eminent functions of STAT3 and iNOS in inflammation and carcinogenesis, IL-22 may represent a novel target for immunotherapeutic intervention. Inducible nitric-oxide synthase (iNOS) has been identified as a marker and mediator of disease in human colonic inflammation and carcinogenesis. Accordingly, identification of mediators that trigger iNOS in colon carcinoma/epithelial cells is an important topic of current research. Here we demonstrate that interleukin (IL)-22, a newly described member of the IL-10 cytokine family, potently synergizes with interferon (IFN)-γ for iNOS expression in human DLD-1 colon carcinoma cells. Detection of both IL-22 receptor chains and STAT3 phosphorylation proved robust IL-22 responsiveness of these cells. Short interfering RNA technology identified STAT3 as being crucial for up-regulation of iNOS. Compared with IFNγ, STAT1 phosphorylation by IL-22 was insufficient. IL-22 did not stabilize IL-1β/tumor necrosis factor-α/IFNγ-induced iNOS mRNA. IL-22 also failed to amplify expression of the prototypic IFNγ-inducible parameters IL-18-binding protein and CXCL-10, indicating that IL-22 is not a general amplifier of IFNγ functions. This assumption is furthermore supported by the observation that IL-22 was unable to enhance cellular activation of the pro-inflammatory transcription factor nuclear factor-κB. In contrast, IL-22 increased iNOS promoter activation as detected by using DLD-1 cells stably transfected with a corresponding 16-kb promoter construct (pNOS2(16)-Luc). IL-22 likewise enhanced iNOS in Caco-2 colon carcinoma cells. With IL-22 we introduce a novel potent determinant of iNOS expression in human colon carcinoma/epithelial cells. Considering the eminent functions of STAT3 and iNOS in inflammation and carcinogenesis, IL-22 may represent a novel target for immunotherapeutic intervention. Interleukin (IL) 2The abbreviations used are: IL, interleukin; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFNγ, interferon-γ; IL-18BPa, interleukin-18-binding protein a; iNOS, inducible NO synthase; NF-κB, nuclear factor-κB; NO, nitric oxide; RT, reverse transcription; TNFα, tumor necrosis factor-α; siRNA, short interfering RNA; STAT, signal transducer and activator of transcription.2The abbreviations used are: IL, interleukin; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFNγ, interferon-γ; IL-18BPa, interleukin-18-binding protein a; iNOS, inducible NO synthase; NF-κB, nuclear factor-κB; NO, nitric oxide; RT, reverse transcription; TNFα, tumor necrosis factor-α; siRNA, short interfering RNA; STAT, signal transducer and activator of transcription.-22 is a newly described member of the IL-10 family of cytokines that is produced by T and NK cells under conditions of immunoactivation. Initiation of the Jak1/Tyk2/signal transducer and activator of transcription (STAT) 3 pathway appears to be the major mode of IL-22 signal transduction (1.Xie M.H. Aggarwal S. Ho W.H. Foster J. Zhang Z. Stinson J. Wood W.I. Goddard A.D. Gurney A.L. J. Biol. Chem. 2000; 275: 31335-31339Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 2.Donnelly R.P. Sheikh F. Kotenko S.V. Dickensheets H. J. Leukocyte Biol. 2004; 76: 314-321Crossref PubMed Scopus (237) Google Scholar, 3.Wolk K. Sabat R. Cytokine Growth Factor Rev. 2006; 17: 367-380Crossref PubMed Scopus (251) Google Scholar, 4.Liang S.C. Tan X.Y. Luxenberg D.P. Karim R. Dunussi-Joannopoulos K. Collins M. Fouser L.A. J. Exp. Med. 2006; 203: 2271-2279Crossref PubMed Scopus (1796) Google Scholar), although activation of STAT1 (5.Lejeune D. Dumoutier L. Constantinescu S. Kruijer W. Schuringa J.J. Renauld J.C. J. Biol. Chem. 2002; 277: 33676-33682Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar), mitogen-activated protein kinases (5.Lejeune D. Dumoutier L. Constantinescu S. Kruijer W. Schuringa J.J. Renauld J.C. J. Biol. Chem. 2002; 277: 33676-33682Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar, 7.Ikeuchi H. Kuroiwa T. Hiramatsu N. Kaneko Y. Hiromura K. Ueki K. Nojima Y. Arthritis Rheum. 2005; 52: 1037-1046Crossref PubMed Scopus (331) Google Scholar), nuclear factor κB (NF-κB) (8.Andoh A. Zhang Z. Inatomi O. Fujino S. Deguchi Y. Araki Y. Tsujikawa T. Kitoh K. Kim-Mitsuyama S. Takayanagi A. Shimizu N. Fujiyama Y. Gastroenterology. 2005; 129: 969-984Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), activator protein-1 (8.Andoh A. Zhang Z. Inatomi O. Fujino S. Deguchi Y. Araki Y. Tsujikawa T. Kitoh K. Kim-Mitsuyama S. Takayanagi A. Shimizu N. Fujiyama Y. Gastroenterology. 2005; 129: 969-984Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), and protein kinase B (6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar) has been related to this cytokine under specific conditions. IL-22 signaling is established by binding of the cytokine to its heterodimeric receptor complex consisting of IL-22R1 and IL-10R2 (2.Donnelly R.P. Sheikh F. Kotenko S.V. Dickensheets H. J. Leukocyte Biol. 2004; 76: 314-321Crossref PubMed Scopus (237) Google Scholar, 3.Wolk K. Sabat R. Cytokine Growth Factor Rev. 2006; 17: 367-380Crossref PubMed Scopus (251) Google Scholar). Because IL-10R2 is a ubiquitous protein, cellular IL-22 responsiveness is mainly determined by expression of the IL-22R1 receptor chain. Interestingly, IL-22R1 expression is restricted to nonleukocytic cells (9.Wolk K. Kunz S. Asadullah K. Sabat R. J. Immunol. 2002; 168: 5397-5402Crossref PubMed Scopus (499) Google Scholar, 10.Wolk K. Kunz S. Witte E. Friedrich M. Asadullah K. Sabat R. Immunity. 2004; 21: 241-254Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar, 11.Gurney A.L. Int. Immunopharmacol. 2004; 4: 669-677Crossref PubMed Scopus (98) Google Scholar). Therefore, IL-22 appears to be unique among a vast array of cytokines in that this protein is incapable of mediating autocrine or paracrine functions between leukocytes but is rather specialized to transmit information between leukocytes and the nonleukocytic cell compartment. This distinctive biological characteristic essentially discriminates IL-22 from another major activator of the STAT3 signaling system, namely IL-6 (12.Hodge D.R. Hurt E.M. Farrar W.L. Eur. J. Cancer. 2005; 41: 2502-2512Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). Cell types identified to be responsive to IL-22 include synoviocytes (7.Ikeuchi H. Kuroiwa T. Hiramatsu N. Kaneko Y. Hiromura K. Ueki K. Nojima Y. Arthritis Rheum. 2005; 52: 1037-1046Crossref PubMed Scopus (331) Google Scholar), pancreatic acinar cells (11.Gurney A.L. Int. Immunopharmacol. 2004; 4: 669-677Crossref PubMed Scopus (98) Google Scholar), hepatocytes (5.Lejeune D. Dumoutier L. Constantinescu S. Kruijer W. Schuringa J.J. Renauld J.C. J. Biol. Chem. 2002; 277: 33676-33682Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 13.Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (320) Google Scholar, 14.Radaeva S. Sun R. Pan H.N. Hong F. Gao B. Hepatology. 2004; 39: 1332-1342Crossref PubMed Scopus (477) Google Scholar), colonic epithelial myofibroblasts (8.Andoh A. Zhang Z. Inatomi O. Fujino S. Deguchi Y. Araki Y. Tsujikawa T. Kitoh K. Kim-Mitsuyama S. Takayanagi A. Shimizu N. Fujiyama Y. Gastroenterology. 2005; 129: 969-984Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), and in particular cells of epithelial origin such as keratinocytes (10.Wolk K. Kunz S. Witte E. Friedrich M. Asadullah K. Sabat R. Immunity. 2004; 21: 241-254Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar, 15.Wolk K. Witte E. Wallace E. Docke W.D. Kunz S. Asadullah K. Volk H.D. Sterry W. Sabat R. Eur. J. Immunol. 2006; 36: 1309-1323Crossref PubMed Scopus (767) Google Scholar), lung carcinoma cells (16.Whittington H.A. Armstrong L. Uppington K.M. Millar A.B. Am. J. Respir. Cell Mol. Biol. 2004; 31: 220-226Crossref PubMed Scopus (95) Google Scholar), and colon carcinoma cells (6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar, 17.Nagalakshmi M.L. Rascle A. Zurawski S. Menon S. de Waal Malefyt R. Int. Immunopharmacol. 2004; 4: 679-691Crossref PubMed Scopus (170) Google Scholar). Proteins that have been reported to be inducible by IL-22 include pro-inflammatory and pro-angiogenic mediators such as IL-8 and enzymes that are involved in cell migration and tissue remodeling such as matrix metalloprotease-1 and -3 (6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar, 8.Andoh A. Zhang Z. Inatomi O. Fujino S. Deguchi Y. Araki Y. Tsujikawa T. Kitoh K. Kim-Mitsuyama S. Takayanagi A. Shimizu N. Fujiyama Y. Gastroenterology. 2005; 129: 969-984Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), effector molecules of innate immunity such as β-defensins (10.Wolk K. Kunz S. Witte E. Friedrich M. Asadullah K. Sabat R. Immunity. 2004; 21: 241-254Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar), and immunosuppressive modulators such as IL-10 (17.Nagalakshmi M.L. Rascle A. Zurawski S. Menon S. de Waal Malefyt R. Int. Immunopharmacol. 2004; 4: 679-691Crossref PubMed Scopus (170) Google Scholar) and SOCS proteins (17.Nagalakshmi M.L. Rascle A. Zurawski S. Menon S. de Waal Malefyt R. Int. Immunopharmacol. 2004; 4: 679-691Crossref PubMed Scopus (170) Google Scholar, 18.Kotenko S.V. Izotova L.S. Mirochnitchenko O.V. Esterova E. Dickensheets H. Donnelly R.P. Pestka S. J. Immunol. 2001; 166: 7096-7103Crossref PubMed Scopus (213) Google Scholar). IL-22-induced STAT3 has been associated with induction of the acute phase response (13.Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (320) Google Scholar), with proliferation, and with protection from cell death (6.Brand S. Beigel F. Olszak T. Zitzmann K. Eichhorst S.T. Otte J.M. Diepolder H. Marquardt A. Jagla W. Popp A. Leclair S. Herrmann K. Seiderer J. Ochsenkuhn T. Goke B. Auernhammer C.J. Dambacher J. Am. J. Physiol. 2006; 290: G827-G838Crossref PubMed Scopus (458) Google Scholar, 14.Radaeva S. Sun R. Pan H.N. Hong F. Gao B. Hepatology. 2004; 39: 1332-1342Crossref PubMed Scopus (477) Google Scholar). Interestingly, constitutive activation of the STAT3 pathway is characteristic for numerous human malignancies. Based on the capabilities of this transcription factor to inhibit apoptosis and to promote cell proliferation, STAT3 is actually considered an oncogenic protein (12.Hodge D.R. Hurt E.M. Farrar W.L. Eur. J. Cancer. 2005; 41: 2502-2512Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar, 19.Bromberg J. J. Clin. Invest. 2002; 109: 1139-1142Crossref PubMed Scopus (740) Google Scholar, 20.Haura E.B. Turkson J. Jove R. Nat. Clin. Pract. Oncol. 2005; 2: 315-324Crossref PubMed Scopus (360) Google Scholar).Inducible nitric-oxide synthase (iNOS) and its volatile enzymatic product nitric oxide (NO) have been identified as potential promoters of tumor growth in a variety of human neoplasia, among other colorectal cancers (20.Haura E.B. Turkson J. Jove R. Nat. Clin. Pract. Oncol. 2005; 2: 315-324Crossref PubMed Scopus (360) Google Scholar, 21.Beck K.F. Eberhardt W. Frank S. 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A. 1995; 92: 4392-4396Crossref PubMed Scopus (744) Google Scholar, 25.Cianchi F. Cortesini C. Fantappie O. Messerini L. Schiavone N. Vannacci A. Nistri S. Sardi I. Baroni G. Marzocca C. Perna F. Mazzanti R. Bechi P. Masini E. Am. J. Pathol. 2003; 162: 793-801Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 26.Van Buren 2nd, G. Camp E.R. Yang A.D. Gray M.J. Fan F. Somcio R. Ellis L.M. Expert. Opin. Ther. Targets. 2006; 10: 689-701Crossref PubMed Scopus (13) Google Scholar, 27.Mühl H. Chang J.H. Huwiler A. Bosmann M. Paulukat J. Ninic R. Nold M. Hellmuth M. Pfeilschifter J. Free Radic. Biol. Med. 2000; 29: 969-980Crossref PubMed Scopus (28) Google Scholar, 28.Hofseth L.J. Hussain S.P. Wogan G.N. Harris C.C. Free Radic. Biol. Med. 2003; 34: 955-968Crossref PubMed Scopus (197) Google Scholar, 29.Lala P.K. Chakraborty C. Lancet Oncol. 2001; 2: 149-156Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Because iNOS and STAT3 (23.Ambs S. Merriam W.G. Bennett W.P. Felley-Bosco E. Ogunfusika M.O. Oser S.M. Klein S. Shields P.G. Billiar T.R. Harris C.C. Cancer Res. 1998; 58: 334-341PubMed Google Scholar, 30.Szaleczky E. Pronai L. Nakazawa H. Tulassay Z. J. Clin. Gastroenterol. 2000; 30: 47-51Crossref PubMed Scopus (44) Google Scholar, 31.Ma X.T. Wang S. Ye Y.J. Du R.Y. Cui Z.R. Somsouk M. World J. Gastroenterol. 2004; 10: 1569-1573Crossref PubMed Scopus (104) Google Scholar, 32.Corvinus F.M. Orth C. Moriggl R. Tsareva S.A. Wagner S. Pfitzner E.B. Baus D. Kaufmann R. Huber L.A. Zatloukal K. Beug H. Ohlschlager P. Schutz A. Halbhuber K.J. Friedrich K. Neoplasia. 2005; 7: 545-555Crossref PubMed Scopus (318) Google Scholar) are both activated in cancerous tissues of the colon, we sought to investigate herein whether IL-22 has the potential to regulate iNOS expression in colon carcinoma/epithelial cells.MATERIALS AND METHODSReagents−Human IFNγ, IL-6, and IL-22 were from PeproTech Inc. (Frankfurt, Germany). IL-1β was from BIOSOURCE. TNFα was kindly provided by the Knoll AG (Ludwigshafen, Germany). Actinomycin D was purchased from Sigma.Cultivation of Human DLD-1 and Caco-2 Colon Carcinoma Cells−Human DLD-1 and Caco-2 colon carcinoma/epithelial cells were obtained from the Centre for Applied Microbiology and Research (Salisbury, UK) and the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), respectively. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Invitrogen). For experiments with DLD-1 cells, confluent cells grown on polystyrene plates (Greiner, Frickenhausen, Germany) were washed with phosphate-buffered saline and incubated with the indicated agents in the aforementioned medium. DLD-1 cells stably transfected with a 16-kb iNOS promoter construct (pNOS2(16)-Luc) (33.Witteck A. Yao Y. Fechir M. Forstermann U. Kleinert H. Exp. Cell Res. 2003; 287: 106-115Crossref PubMed Scopus (41) Google Scholar) were cultivated in the aforementioned culture medium with the addition of 0.5 mg/ml G418 (Invitrogen). For experiments, confluent cells grown on polystyrene plates (Greiner) were washed with phosphate-buffered saline and incubated using this same culture medium without the addition of G418. For experiments with Caco-2 cells, stimulations were performed in the state of postconfluency. It has been reported previously that postconfluent Caco-2 cells gain responsiveness toward IFNγ (34.Chavez A.M. Morin M.J. Unno N. Fink M.P. Hodin R.A. Gut. 1999; 44: 659-665Crossref PubMed Scopus (35) Google Scholar). For that purpose, already confluent cells were further grown on polystyrene plates for an additional 14-day period. Thereafter, experiments were performed as indicated.Detection of Human IL-22RA1, IL-10R2, and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) mRNA by Standard PCR−After RNA isolation using peqGold TriFast (Peqlab, Erlangen, Germany), 1 μg of total RNA was transcribed using random hexameric primers and Moloney virus reverse transcriptase (RT) (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s instructions. The following sequence was performed for each PCR: GAPDH, 94 °C for 10 min (1 cycle) followed by 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min (25 cycles); IL-22RA1 and IL-10R2, 95 °C for 10 min (1 cycle) followed by 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min (35 cycles); followed by a final extension phase at 72 °C for 7 min. The following primers were used: IL-22R1, forward 5′-GTATAAGACGTACGGAGA-3′ and reverse 5′-TCCAAGGTGCATTTGGTA-3′; IL-10R2, forward 5′-CATTGGGAATGGTACCAC-3′ and reverse 5′-CCAATAATGGTGTCATCCAC-3′; and GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The possibility of amplification of contaminating genomic DNA was eliminated by selecting amplicons that cross exon/intron boundaries. PCR products (IL-22RA1, 372 bp; IL-10R2, 292 bp; GAPDH, 452 bp) were run on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The identity of amplicons was confirmed by sequencing (AbiPrism 310 Genetic Analyzer, Applied Biosystems).Evaluation of Human iNOS mRNA by RNase Protection Assay−Total RNAs (20 μg) were used for RNase protection assay, performed as described previously (35.Paulukat J. Bosmann M. Nold M. Garkisch S. Kämpfer H. Frank S. Raedle J. Zeuzem S. Pfeilschifter J. Mühl H. J. Immunol. 2001; 167: 7038-7043Crossref PubMed Scopus (117) Google Scholar). Briefly, DNA probes were cloned into the transcription vector pBluescript II KS(+) (Stratagene, Heidelberg, Germany). After linearization, an antisense transcript was synthesized in vitro with T7 RNA polymerase (Roche Diagnostics) and [α-32P]UTP (800 Ci/mmol; Amersham Biosciences). RNA samples were hybridized at 42 °C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested with RNase A (Roche Diagnostics) and T1 (Roche Diagnostics) for 1 h at 30 °C. Under these conditions every single mismatch was recognized by the RNases. Protected fragments were separated on 5% polyacrylamide, 8 m urea gels and analyzed using a PhosphorImager (Fuji, Straubenhardt, Germany). The individual gene expression of iNOS was evaluated on the basis of the GAPDH housekeeping gene expression. The cDNAs correspond to nucleotides 3724–3469 and 3607–3352, respectively (iNOS; transcript variant 1, GenBank™ accession number NM000625; transcript variant 2, GenBank™ accession number NM153292) and nucleotides 961–1071 (human GAPDH; GenBank™ accession number AC M33197) of the published sequences.Determination of human IL-18-binding Protein a (IL-18BPa) mRNA by Quantitative Real Time PCR−Real time PCR was performed to assess expression of IL-18BPa and GAPDH. Changes in fluorescence are caused by the Taq polymerase degrading the probe that contains a fluorescent dye (6-carboxyfluorescein for IL-18BPa, VIC for GAPDH) and a quencher (6-carboxytetramethylrhodamine). Primers and probe for IL-18BPa were designed using Primer Express (Applied Biosystems) according to the published sequence (GenBank™ accession number XM035063.1): forward 5′-ACCTCCCAGGCCGACTG-3′ and reverse 5′-CCTTGCACAGCTGCGTACC-3′; probe 5′-CACCAGCCGGGAACGTGGGA-3′. The possibility of amplification of contaminating genomic DNA was eliminated by selecting an amplicon that crosses an exon/intron boundary. For GAPDH, pre-developed assay reagents were used (Applied Biosystems). Specificity of PCR products was tested by classic PCR using the aforementioned primers. 1 μg of total RNA was transcribed using random hexameric primers and Moloney virus RT (Applied Biosystems) according to the manufacturer’s instructions. Real time PCR was performed on the AbiPrism 7700 sequence detector (Applied Biosystems) as follows: one initial step at 50 °C for 2 min and 95 °C for 2 min was followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Detection of the dequenched probe, calculation of threshold cycles (Ct values), and further analysis of these data were performed by the Sequence Detector software. mRNA expression was quantified by use of cloned cDNA standards for IL-18BPa and GAPDH. All results for IL-18BPa expression were normalized to that of GAPDH.Detection of Human iNOS, pSTAT3/STAT3, pSTAT1, IL-18BPa, andβ-Tubulin by Immunoblot Analysis−For detection of intracellular proteins, cells were treated with lysis buffer (150 mm NaCl, 1 mm CaCl2, 25 mm Tris-Cl, pH 7.4, 1% Triton X-100, supplemented with protease inhibitor mixture (Roche Diagnostics) and dithiothreitol, Na3VO4, phenylmethylsulfonyl fluoride (each 1 mm), and NaF (20 mm). Routinely, 50 μg of total protein/lane were used. For detection of total STAT3 (Fig. 1, B and C) blots were stripped and reprobed. For detection of pSTAT1 or pSTAT3 and β-tubulin in Fig. 1, D and E, blots were cut in half. To detect iNOS, STAT3, and β-tubulin on the same blot, the blot was cut in three parts as shown in Fig. 4. Antibodies and SDS-PAGE conditions were as follows: iNOS (8/10% SDS-PAGE; mouse monoclonal antibody; BD Biosciences), pSTAT3 (10% SDS-PAGE; Y705; 58E12; rabbit monoclonal antibody; Cell Signaling, Frankfurt, Germany), STAT3 (10% SDS-PAGE; mouse monoclonal antibody; Cell Signaling), pSTAT1 (10% SDS-PAGE; Y701; rabbit polyclonal antibody; Cell Signaling), and β-tubulin (10% SDS-PAGE; mouse monoclonal antibody; Santa Cruz Biotechnology). For detection of IL-18BPa, cell-free supernatants (5 ml/PS-10 plate) were trichloroacetic acid-precipitated, as described previously (35.Paulukat J. Bosmann M. Nold M. Garkisch S. Kämpfer H. Frank S. Raedle J. Zeuzem S. Pfeilschifter J. Mühl H. J. Immunol. 2001; 167: 7038-7043Crossref PubMed Scopus (117) Google Scholar). Briefly, 1/10 volume of 70% trichloroacetic acid was added to cell-free supernatants. After 30 min on ice and a 30-min centrifugation step at 16,000 × g, pellets were washed in acetone and resuspended in Laemmli buffer. Trichloroacetic acid-precipitated IL-18BPa was separated by 10% SDS-PAGE and detected using a goat polyclonal antibody (R&D Systems, Wiesbaden, Germany).FIGURE 4Suppression of STAT3 by siRNA impairs induction of iNOS by IL-22/IFNγ. DLD-1 cells were transfected as outlined under “Materials and Methods” with either siRNA targeting STAT3 or with control-siRNA. In addition, cells were mock-transfected for control conditions or IL-22 (20 ng/ml)/IFNγ (10 ng/ml) stimulations that were performed in the absence of STAT3-siRNA or control siRNA, respectively. After a stimulation period of 24 h, cells were harvested, and expression of iNOS, STAT3, and β-tubulin was evaluated by Western blot analysis. To ensure detection of these proteins on the same blot, blots were cut in three parts. One representative of four independently performed experiments is displayed (A). B shows a densitometric quantification of iNOS and STAT3 protein expression (relative to that of β-tubulin) in this particular experiment. C, quantified data on iNOS and STAT3 protein expression from these four independent experiments are depicted in a scatter plot, and a linear regression was performed; r denotes regression coefficient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Determination of CXCL-10 (IP-10) in Cell Culture Supernatants by Enzyme-linked Immunosorbent Assay (ELISA)−Levels of CXCL-10 in cell-free culture supernatants obtained from DLD-1 cultures were determined by ELISA according to the manufacturer’s instruction (BD Biosciences).Suppression of STAT3 by siRNA Technology−For experiments, DLD-1 cells were seeded at a density of 2 × 105 cells 24 h prior to transfection in 6-well polystyrene plates (Greiner) using the aforementioned medium. 50 nm of either STAT3-directed siRNA (number 51320, Ambion, Cambridgeshire, UK or control siRNA (Silencer®Negative Control siRNA, number 4611, Ambion) were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s instruction. All cultures without siRNA or control siRNA were mock-transfected under the same conditions. After 72 h of incubation in culture medium, cells were stimulated as indicated and harvested thereafter.Analysis of the Human iNOS Promoter Activity in DLD-1/pXP2-16-kb Cells Stably Overexpressing a 16-kb iNOS Promoter and the Firefly Luciferase Gene−DLD-1/pXP2-16-kb cells that stably overexpress a 16-kb iNOS promoter and the firefly luciferase gene (33.Witteck A. Yao Y. Fechir M. Forstermann U. Kleinert H. Exp. Cell Res. 2003; 287: 106-115Crossref PubMed Scopus (41) Google Scholar) were seeded in 6-well polystyrene plates (Greiner) using the aforementioned medium. Stimulation was performed as indicated. Six independent experiments were performed in triplicate. Protein content of the extracts was used for normalization of the luciferase activity. Luciferase activities were measured with the luciferase assay system (Promega Corp., Madison, WI) according to the manufacturer’s instruction using an automated chemiluminescence detector (Berthold, Bad Wildbad, Germany).Electrophoretic Mobility Shift Assay (EMSA)−Preparation of nuclear extracts from DLD-1 cells was performed as described previously (36.Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3908) Google Scholar). Consensus oligonucleotides used in the binding reactions were obtained from Santa Cruz Biotechnology (Santa Cruz, Heidelberg, Germany). Sequences of the double-stranded oligonucleotides are as follows: NF-κB, WT 5′-AGTTGAGGGGACTTTCCCAGGC-3′. Complementary oligonucleotides were end-labeled by T4 polynucleotide kinase (MBI Fermentas, St. Leon-Rot, Germany) using [γ-32P]ATP (6000 Ci/mmol; Amersham Biosciences). Binding reactions were performed for 45 min on ice with 7.5 μg of protein in 20 μl of binding buffer containing 4% Ficoll, 20 mm HEPES, pH 7.9, 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 0.25 mg/ml bovine serum albumin, 1.25 μg of poly(dI-dC), and 50,000 cpm of 32P-labeled oligonucleotide. For NF-κB supershift analysis, nuclear proteins were preincubated for 30 min at room temperature with a polyclonal anti-p65 antibody (Santa Cruz Biotechnology). DNA-protein complexes were separated from unbound oligonucleotide by electrophoresis through a 4.5% polyacrylamide gel using 0.5× TBE buffer. Thereafter, gels were fixed and analyzed by PhosphorImager analysis (Fuji).Analysis of Nitrite Production−To verify NO production, nitrite, a stable end product of NO metabolism, was measured in cell-free supernatant using the Griess reagent (Merck). Briefly, DLD-1 cells were seeded in" @default.
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• W2067353642 title "The Interleukin-22/STAT3 Pathway Potentiates Expression of Inducible Nitric-oxide Synthase in Human Colon Carcinoma Cells" @default.
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