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Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

2010; Elsevier BV; Volume: 285; Issue: 10 Linguagem: Inglês

10.1074/jbc.m109.056614

ISSN

1083-351X

Autores

Martin McLaughlin, Iraide Alloza, Hùng Phạm Quốc, Christopher J. Scott, Yasuhiko Hirabayashi, Koen Vandenbroeck,

Tópico(s)

Toxin Mechanisms and Immunotoxins

Resumo

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5–3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions. Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5–3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions. IntroductionThe interleukin-12 (IL-12) 2The abbreviations used are: ILinterleukinCEcelecoxibCHXcycloheximideCRTcalreticulinDSPdithiobis[succinimidylpropionate]ERendoplasmic reticulumERADendoplasmic reticulum-associated protein degradationERQCendoplasmic reticulum-associated quality controlHERPhomocysteine-induced endoplasmic reticulum proteinTFM-C4-[5-(4- trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (short name: 4-trifluoromethyl-celecoxib)UPRunfolded protein responsePBSphosphate-buffered salineNTAnitrilotriacetic acidANOVAanalysis of varianceMOPS4-morpholinepropanesulfonic acidCOXcyclooxygenaseGAPDHglyceraldehyde-3-phosphate dehydrogenase. subfamily of cytokines is structurally based around a shared p40 subunit, covalently linked to p35 in IL-12 and to p19 in IL-23. In addition, p40 can be secreted both as a monomer and as a homodimer, named p80. Further members of this family include IL-27, which is a heterodimer consisting of the p40-related protein "Epstein-Barr virus-induced gene 3" (EBI3) and the p35-related subunit p28 and IL-35 (EBI3/p35) (1.Collison L.W. Vignali D.A. Immunol. Rev. 2008; 226: 248-262Crossref PubMed Scopus (215) Google Scholar). IL-12 and IL-23 have been identified at elevated levels in a number of autoimmune diseases, including multiple sclerosis (MS) and its animal model EAE (2.Kroenke M.A. Carlson T.J. Andjelkovic A.V. Segal B.M. J. Exp. Med. 2008; 205: 1535-1541Crossref PubMed Scopus (482) Google Scholar, 3.Steinman L. J. Exp. Med. 2008; 205: 1517-1522Crossref PubMed Scopus (150) Google Scholar), psoriasis (4.Torti D.C. Feldman S.R. J. Am. Acad. Dermatol. 2007; 57: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and inflammatory bowel disease (5.Mannon P.J. Fuss I.J. Mayer L. Elson C.O. Sandborn W.J. Present D. Dolin B. Goodman N. Groden C. Hornung R.L. Quezado M. Yang Z. Neurath M.F. Salfeld J. Veldman G.M. Schwertschlag U. Strober W. N. Engl. J. Med. 2004; 351: 2069-2079Crossref PubMed Scopus (742) Google Scholar). p80 has recently been shown to control macrophage chemotaxis and dendritic cell migration in lung inflammatory disease pathogenesis (1.Collison L.W. Vignali D.A. Immunol. Rev. 2008; 226: 248-262Crossref PubMed Scopus (215) Google Scholar, 6.Cooper A.M. Khader S.A. Trends Immunol. 2007; 28: 33-38Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7.Mikols C.L. Yan L. Norris J.Y. Russell T.D. Khalifah A.P. Hachem R.R. Chakinala M.M. Yusen R.D. Castro M. Kuo E. Patterson G.A. Mohanakumar T. Trulock E.P. Walter M.J. Am. J. Respir. Crit. Care Med. 2006; 174: 461-470Crossref PubMed Scopus (29) Google Scholar, 8.Russell T.D. Yan Q. Fan G. Khalifah A.P. Bishop D.K. Brody S.L. Walter M.J. J. Immunol. 2003; 171: 6866-6874Crossref PubMed Scopus (62) Google Scholar). There is widespread interest in the assessment of IL-12-type cytokines as therapeutic targets for preventing or attenuation of disease. Monoclonal p40 antibodies (9.Segal B.M. Constantinescu C.S. Raychaudhuri A. Kim L. Fidelus-Gort R. Kasper L.H. Lancet Neurol. 2008; 7: 796-804Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 10.Leonardi C.L. Kimball A.B. Papp K.A. Yeilding N. Guzzo C. Wang Y. Li S. Dooley L.T. Gordon K.B. Lancet. 2008; 371: 1665-1674Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar) have shown promise in limiting inflammatory disease severity. Various small molecule drugs have been reported that are capable of blocking IL-12, IL-23, and p80 at distinct stages of their biological pathways (transcription, assembly, receptor binding, and signal transduction) (for review see Ref. 11.Vandenbroeck K. Alloza I. Gadina M. Matthys P. J. Pharm. Pharmacol. 2004; 56: 145-160Crossref PubMed Scopus (41) Google Scholar). One such compound is celecoxib (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar).Celecoxib is a non-steroidal anti-inflammatory drug originally designed to specifically inhibit cyclooxygenase-2 (COX-2), up-regulated in cancer and sites of inflammation (13.Lou J. Fatima N. Xiao Z. Stauffer S. Smythers G. Greenwald P. Ali I.U. Cancer Epidemiol. Biomarkers Prev. 2006; 15: 1598-1606Crossref PubMed Scopus (38) Google Scholar). However, COX-2 inhibitors have been subject to critical analysis because of cardiovascular problems, and a number of functions related to celecoxib are now known to be attributable to COX-2-independent mechanisms (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 13.Lou J. Fatima N. Xiao Z. Stauffer S. Smythers G. Greenwald P. Ali I.U. Cancer Epidemiol. Biomarkers Prev. 2006; 15: 1598-1606Crossref PubMed Scopus (38) Google Scholar, 14.Grösch S. Maier T.J. Schiffmann S. Geisslinger G. J. Natl. Cancer Inst. 2006; 98: 736-747Crossref PubMed Scopus (397) Google Scholar, 15.Schönthal A.H. Br. J. Cancer. 2007; 97: 1465-1468Crossref PubMed Scopus (89) Google Scholar). One such effect is the ability of celecoxib to increase cytoplasmic calcium concentrations through inhibition of ER Ca2+-ATPases (SERCA; Refs. 12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 16.Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar, 17.Pyrko P. Kardosh A. Liu Y.T. Soriano N. Xiong W. Chow R.H. Uddin J. Petasis N.A. Mircheff A.K. Farley R.A. Louie S.G. Chen T.C. Schönthal A.H. Mol. Cancer Ther. 2007; 6: 1262-1275Crossref PubMed Scopus (112) Google Scholar, 18.Tsutsumi S. Namba T. Tanaka K. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (113) Google Scholar). Experiments with other COX-2 inhibitors such as rofecoxib, ibuprofen, aspirin, naproxen, DuP697, and NS298 have all shown that this calcium-mobilizing property is unique to celecoxib (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 16.Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar).We have shown that both celecoxib and a celecoxib analogue from which COX-2 inhibitory properties have been knocked out, i.e. 4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide, referred to hereafter as 4-trifluoromethyl-celecoxib (TFM-C), block IL-12 and p80 secretion through a post-transcriptional mechanism involving retention of the cytokines in the ER (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). TFM-C shares the effect of celecoxib in depleting ER Ca2+ stores in noncancerous HEK293 cells, i.e. the recombinant cytokine producer cells used in our study (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). For p80, this secretion block is accompanied with altered interaction with the ER chaperones calreticulin (CRT) and ERp44 (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). In the present study, we show that celecoxib and TFM-C block secretion of a third dimeric member of the IL-12 subfamily, namely IL-23, concomitant with intracellular retention of the IL-23 subunit p19. In an attempt to identify the ER factors crucially involved in this process we analyzed the effect of TFM-C on expression of 23 ER chaperones and cofactors. We identified homocysteine-inducible endoplasmic reticulum protein (HERP) as the gene transcript most dramatically up-regulated by TFM-C. HERP is a 54 kDa ubiquitously expressed ER membrane protein that is up-regulated as part of the ER-unfolded protein response (UPR) (19.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 20.Lenz B. Bleich S. Beutler S. Schlierf B. Schwager K. Reulbach U. Kornhuber J. Bönsch D. Exp. Cell Res. 2006; 312: 4049-4055Crossref PubMed Scopus (30) Google Scholar, 21.Ma Y. Hendershot L.M. J. Biol. Chem. 2004; 279: 13792-13799Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22.Liang G. Audas T.E. Li Y. Cockram G.P. Dean J.D. Martyn A.C. Kokame K. Lu R. Mol. Cell Biol. 2006; 26: 7999-8010Crossref PubMed Scopus (112) Google Scholar, 23.Sai X. Kokame K. Shiraishi H. Kawamura Y. Miyata T. Yanagisawa K. Komano H. FEBS Lett. 2003; 553: 151-156Crossref PubMed Scopus (36) Google Scholar). HERP is required for destruction of a number of proteins via the ER-associated degradation (ERAD) pathway, such as CD3-delta, connexin 43, non-secreted Igκ LC, mutant Igγ LC, and truncated Igγ HC (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar, 25.Okuda-Shimizu Y. Hendershot L.M. Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar). HERP is a component of a larger ERAD retrotranslocation complex comprising the E3 ligase HRD1, Derlin-1, VIMP, and the ATPase p97 (26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar, 27.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar). HERP knock-out also results in increased susceptibility to ER stress-related cell death by thapsigargin, tunicamycin, or A23187 (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar). The proteasome inhibitor lactacystin accelerates cell death in HERP knock-out cells and blocks the degradation of connexin 43, Igκ LC, Igγ LC, and Igγ HC, indicating that HERP is involved in delivering ERAD-targeted proteins to the proteasome (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar, 25.Okuda-Shimizu Y. Hendershot L.M. Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In this study, we show that TFM-C induces protein interaction of HERP with intracellularly retained p80 and IL-23, and that HERP is functionally required for the degradation of intracellularly retained dimeric p80.DISCUSSIONIn this study we have demonstrated the ability of celecoxib and the non-coxib analogue TFM-C to inhibit the secretion of IL-23, as well as of IL-12 (p35/p40) and p80 (p40/p40) (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar), whereas leaving secretion of monomeric p40 largely unaffected. TFM-C strongly up-regulates the expression of the ER transmembrane protein HERP, and by use of co-immunoprecipitation, CHX chase, and siRNA/overexpression assays, HERP is involved in delivery of p80 and IL-23 for degradation in TFM-C-treated cells.In conjunction with our earlier work (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar), we can now present a more detailed rationale for the putative mechanism by which TFM-C inhibits folding and secretion, and induces degradation of IL-12-type cytokines. Whereas TFM-C was designed to have no COX-2 inhibitory activity (IC50 for inhibition of COX-2 of 8.2 μm for TFM-C versus 0.04 μm for celecoxib; see Ref. 12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar), both celecoxib and TFM-C perturb Ca2+ homeostasis, most likely through blockage of ER Ca2+- ATPases (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 16.Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar). TFM-C selectively inhibits secretion of dimeric polypeptide conformers of the IL-12 family (IL-12, p80, and IL-23), and to a much lesser extent of p40 monomers. Analysis of IgG1 secretion (Fig. 1) demonstrates that TFM-C-mediated perturbation of ER function does not induce a generic secretion block of oligomeric proteins and that TFM-C has a certain degree of selectivity for an as yet undefined group of susceptible secreted proteins that includes IL-12/p80/IL-23. Proteomic analysis of the secretome of cells treated with TFM-C is currently being undertaken to address the diversity and nature of susceptible secretory proteins. ER-targeting compounds, such as tunicamycin, have previously been shown to have little effect on the successful secretion of monoclonal antibodies (35.Barnabé N. Butler M. J. Biotechnol. 1998; 60: 67-80Crossref PubMed Scopus (14) Google Scholar).At the lowest concentration of celecoxib and TFM-C required for inducing IL-12 and p80 retention, there are no cytotoxic effects or significant alterations in the level of p40 or p35 subunit transcription (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). The comparison between intracellular and secreted levels of p19 (Fig. 1D) indicates that in celecoxib- and TFM-C-treated cells, p19 is not secreted as subunit of IL-23, yet it is still present intracellularly at levels similar to untreated control. In TFM-C-treated cells, p80 shows increased interaction with calreticulin, a luminal ER chaperone involved in ER quality control (ERQC) of folding and retention of misfolded proteins, as well as decreased interaction with the chaperone ERp44 (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). Thus, TFM-C seems to induce, either through ER Ca2+ perturbation or an as yet unknown mechanism, compositional changes in the ER environment making it more hostile to sustenance of folding of IL-12-type proteins, thus leading to abrogation of ensuing secretion. Misfolded or non-native proteins are normally intercepted by the ERQC system prior to retrotranslocation and delivery for ERAD (36.Vembar S.S. Brodsky J.L. Nat. Rev. Mol. Cell Biol. 2008; 9: 944-957Crossref PubMed Scopus (1011) Google Scholar). We wondered whether this holds true for the non-secreted p80 dimers produced in TFM-C-treated cells. We addressed this question in first instance by analyzing the effect of TFM-C on transcriptional regulation of 23 ER-resident proteins. Out of 23 ER chaperones and associated factors analyzed, HERP was identified as the factor showing the most dramatic, i.e. 7-fold, up-regulation by TFM-C. To a lesser extent, the mRNA levels of CRT, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29 showed statistically significant differences as well. Of these, celecoxib has already been shown to increase the expression of the ER chaperones GRP78 and GRP94 in a wider proteomics analysis of its effects on intracellular proteins (13.Lou J. Fatima N. Xiao Z. Stauffer S. Smythers G. Greenwald P. Ali I.U. Cancer Epidemiol. Biomarkers Prev. 2006; 15: 1598-1606Crossref PubMed Scopus (38) Google Scholar), and of ORP150/GRP170, GRP78, ERdj3, and ERdj4 in human gastric carcinoma cell lines (18.Tsutsumi S. Namba T. Tanaka K. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (113) Google Scholar, 37.Namba T. Hoshino T. Tanaka K. Tsutsumi S. Ishihara T. Mima S. Suzuki K. Ogawa S. Mizushima T. Mol. Pharmacol. 2007; 71: 860-870Crossref PubMed Scopus (53) Google Scholar). The celecoxib analogue 2,5-dimethylcelecoxib (DMC) has also been shown to up-regulate GRP78 in glioblastoma cell lines (38.Kardosh A. Golden E.B. Pyrko P. Uddin J. Hofman F.M. Chen T.C. Louie S.G. Petasis N.A. Schönthal A.H. Cancer Res. 2008; 68: 843-851Crossref PubMed Scopus (109) Google Scholar). Thus, the modulation of ER chaperone expression in response to celecoxib is shared with DMC and TFM-C despite the structural modifications in the latter compounds, and is reminiscent of the unfolded protein response (UPR) (39.Lai E. Teodoro T. Volchuk A. Physiology. 2007; 22: 193-201Crossref PubMed Scopus (391) Google Scholar). Part of the induction of the UPR is a decrease in general protein synthesis as biological means toward decreasing the protein folding load within the ER (40.Malhotra J.D. Kaufman R.J. Semin. Cell Dev. Biol. 2007; 18: 716-731Crossref PubMed Scopus (767) Google Scholar). This has shown to be the case with celecoxib treatment, but only at higher concentrations approaching 100 μm, whereas general protein synthesis remains relatively unaffected below this concentration (41.Pyrko P. Kardosh A. Schönthal A.H. Biochem. Pharmacol. 2008; 75: 395-404Crossref PubMed Scopus (25) Google Scholar). It would appear that 50 μm TFM-C induces a pre-apoptotic state in the ER, which causes retention of IL-12 family cytokines. Mild TFM-C-induced ER calcium perturbation may result in ineffective Ca2+-dependent chaperone function. A link between GRP78 and GRP94 up-regulation because of ER calcium depletion has long been identified (42.Li W.W. Alexandre S. Cao X. Lee A.S. J. Biol. Chem. 1993; 268: 12003-12009Abstract Full Text PDF PubMed Google Scholar). The low cytotoxicity of both celecoxib and TFM-C in EcR293p40His cells (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar), coupled to little apoptosis in 50 μm celecoxib-treated cells (18.Tsutsumi S. Namba T. Tanaka K. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (113) Google Scholar) suggests at this level, that cells can successfully compensate for calcium perturbation through increased chaperone expression. This appears sufficient to prevent terminal protein misfolding on a scale, which would lead to overwhelming of the unfolded protein response and apoptosis, a tipping point which seems to be reached at concentrations around 100 μm celecoxib or TFM-C, and much less so at 50 μm.Mechanistically of relevance to the cellular tolerance for 50 μm TFM-C, was the discovery that HERP was by some distance the ER factor showing the highest level of inducibility. HERP is ubiquitously expressed (19.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), whereas analysis in HT29, LNCaP, and EcR293 (HEK293) cells (Fig. 2B) suggests that TFM-C-induced up-regulation is also ubiquitous. HERP is also strongly up-regulated by thapsigargin, but only marginally by the selective COX-2 inhibitor rofecoxib or the geldanamycin analogue 17AAG (Fig. 2C), in line with an earlier report showing HERP up-regulation by thapsigargin and the Ca2+ ionophore A23187 (19.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). HERP resides normally in the trans-Golgi network, but relocates mainly to the ER upon calcium perturbation with thapsigargin (43.Tuvia S. Taglicht D. Erez O. Alroy I. Alchanati I. Bicoviski V. Dori-Bachash M. Ben-Avraham D. Reiss Y. J. Cell Biol. 2007; 177: 51-61Crossref PubMed Scopus (37) Google Scholar). In neuronal cells, HERP is capable of preventing ER stress-induced apoptosis by stabilizing ER Ca2+ levels (33.Chan S.L. Fu W. Zhang P. Cheng A. Lee J. Kokame K. Mattson M.P. J. Biol. Chem. 2004; 279: 28733-28743Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Taken together with the data presented here, it can be inferred with some degree of certainty that the HERP-inducing properties of TFM-C are a COX-2-independent function of celecoxib and are likely associated with perturbation of ER calcium levels.HERP is part of high molecular mass protein complex involved in ERAD (26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar) and is regulated by the ER stress-specific branch of the UPR (21.Ma Y. Hendershot L.M. J. Biol. Chem. 2004; 279: 13792-13799Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22.Liang G. Audas T.E. Li Y. Cockram G.P. Dean J.D. Martyn A.C. Kokame K. Lu R. Mol. Cell Biol. 2006; 26: 7999-8010Crossref PubMed Scopus (112) Google Scholar, 23.Sai X. Kokame K. Shiraishi H. Kawamura Y. Miyata T. Yanagisawa K. Komano H. FEBS Lett. 2003; 553: 151-156Crossref PubMed Scopus (36) Google Scholar), making it a prime candidate for a role in degradation of secretion-incompetent IL-12-type cytokines retained within TFM-C-treated cells. In immunoprecipitation experiments under DSP cross-linking conditions, HERP was co-captured with p80 and IL-23 in TFM-C-treated cells (Fig. 4). Because of the reducing nature of the experiment, it cannot conclusively be proven that HERP is co-captured with the cytokine subunits in monomeric or dimeric form, or indeed both. Because TFM-C and celecoxib have consistently been shown to leave p40 monomer secretion largely intact (Fig. 1, A and B), it can be inferred that the co-captured HERP is likely to be associated with retained p80 or IL-23.A role for HERP in degradation of secretion-incompetent p80 was further substantiated in CHX chase experiments using cells transfected with HERP siRNA and treated with TFM-C (Fig. 6). Whereas in cells transfected with control siRNA, p80 is virtually completely degraded at 2 h of CHX chase, a dramatic accumulation of p80 was evident in cells treated with HERP siRNA at this time point. In a complementary experiment, ectopic expression of HERP effectuated rapid and enhanced clearance of p80 in TFM-C-stressed cells compared with cells transfected with empty vector, while no such effect was apparent in untreated cells (Fig. 7). Thus, unlike other proteins degraded in a HERP-dependent manner, such as connexin 43 (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar) and CD3-delta (26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar), both of which are native substrates of ERAD, p80 does not appear to be targeted for degradation by HERP in the absence of an ER stress response. It is therefore possible that the p80 dimers intercepted by and degraded through HERP under conditions of TFM-C treatment occur in a non-native/misfolded conformation. Our earlier observation of enhanced interaction between p80 and the ERQC factor CRT in TFM-C-treated cells (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar), lends support to this hypothesis.At this point, it is unknown whether p80 and IL-23 interact directly with HERP or whether HERP is simply co-captured as part of the larger HERP-containing retrotranslocation complex, because of DSP cross-linking used in this experiment (Fig. 4). HERP has also been identified to bind directly to the ER transmembrane E3 ligase HRD1. HRD1 in turn binds to the ER membrane protein Derlin-1 and the cytosolic p97 (26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar, 27.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar). The membrane protein VIMP is subsequently recruited to the complex by p97. Studies of the GRP78 substrates, non-secreted Igκ LC, mutant Igγ LC and truncated Igγ HC, show all three interact with HERP (25.Okuda-Shimizu Y. Hendershot L.M. Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Furthermore, US11 from cytomegalovirus induces MHC I complex retrotranslocation resulting in a direct association with Derlin-1 and VIMP. Thus, HERP and p80/IL-23 may directly interact, but as Derlin-1 is the putative translocation pore (27.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar), it is also possible that these cytokines interact with Derlin-1 and that HERP is co-purified through its interaction with Derlin-1 via HRD1. Further elucidation of the composition of the cytokine-associated heteromeric HERP retrotranslocation complex is being addressed through diagonal electrophoresis.In conclusion, the intracellular retention of IL-12, IL-23, and p80 by celecoxib and TFM-C represents a novel anti-inflammatory property of celecoxib, one independent of COX-2 inhibition. This is likely to contribute significantly to the overall spectrum of anti-inflammatory properties of celecoxib, because of inhibition of differentiation of IL-12-based IFN-γ producing Th1 cells and IL-23-based IL-17 producing Th17 cells; as well as of the neutrophil chemo-attractant properties of p80 (44.Paunovic V. Carroll H.P. Vandenbroeck K. Gadina M. Rheumatology. 2008; 47: 771-776Crossref PubMed Scopus (43) Google Scholar). IntroductionThe interleukin-12 (IL-12) 2The abbreviations used are: ILinterleukinCEcelecoxibCHXcycloheximideCRTcalreticulinDSPdithiobis[succinimidylpropionate]ERendoplasmic reticulumERADendoplasmic reticulum-associated protein degradationERQCendoplasmic reticulum-associated quality controlHERPhomocysteine-induced endoplasmic reticulum proteinTFM-C4-[5-(4- trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (short name: 4-trifluoromethyl-celecoxib)UPRunfolded protein responsePBSphosphate-buffered salineNTAnitrilotriacetic acidANOVAanalysis of varianceMOPS4-morpholinepropanesulfonic acidCOXcyclooxygenaseGAPDHglyceraldehyde-3-phosphate dehydrogenase. subfamily of cytokines is structurally based around a shared p40 subunit, covalently linked to p35 in IL-12 and to p19 in IL-23. In addition, p40 can be secreted both as a monomer and as a homodimer, named p80. Further members of this family include IL-27, which is a heterodimer consisting of the p40-related protein "Epstein-Barr virus-induced gene 3" (EBI3) and the p35-related subunit p28 and IL-35 (EBI3/p35) (1.Collison L.W. Vignali D.A. Immunol. Rev. 2008; 226: 248-262Crossref PubMed Scopus (215) Google Scholar). IL-12 and IL-23 have been identified at elevated levels in a number of autoimmune diseases, including multiple sclerosis (MS) and its animal model EAE (2.Kroenke M.A. Carlson T.J. Andjelkovic A.V. Segal B.M. J. Exp. Med. 2008; 205: 1535-1541Crossref PubMed Scopus (482) Google Scholar, 3.Steinman L. J. Exp. Med. 2008; 205: 1517-1522Crossref PubMed Scopus (150) Google Scholar), psoriasis (4.Torti D.C. Feldman S.R. J. Am. Acad. Dermatol. 2007; 57: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and inflammatory bowel disease (5.Mannon P.J. Fuss I.J. Mayer L. Elson C.O. Sandborn W.J. Present D. Dolin B. Goodman N. Groden C. Hornung R.L. Quezado M. Yang Z. Neurath M.F. Salfeld J. Veldman G.M. Schwertschlag U. Strober W. N. Engl. J. Med. 2004; 351: 2069-2079Crossref PubMed Scopus (742) Google Scholar). p80 has recently been shown to control macrophage chemotaxis and dendritic cell migration in lung inflammatory disease pathogenesis (1.Collison L.W. Vignali D.A. Immunol. Rev. 2008; 226: 248-262Crossref PubMed Scopus (215) Google Scholar, 6.Cooper A.M. Khader S.A. Trends Immunol. 2007; 28: 33-38Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 7.Mikols C.L. Yan L. Norris J.Y. Russell T.D. Khalifah A.P. Hachem R.R. Chakinala M.M. Yusen R.D. Castro M. Kuo E. Patterson G.A. Mohanakumar T. Trulock E.P. Walter M.J. Am. J. Respir. Crit. Care Med. 2006; 174: 461-470Crossref PubMed Scopus (29) Google Scholar, 8.Russell T.D. Yan Q. Fan G. Khalifah A.P. Bishop D.K. Brody S.L. Walter M.J. J. Immunol. 2003; 171: 6866-6874Crossref PubMed Scopus (62) Google Scholar). There is widespread interest in the assessment of IL-12-type cytokines as therapeutic targets for preventing or attenuation of disease. Monoclonal p40 antibodies (9.Segal B.M. Constantinescu C.S. Raychaudhuri A. Kim L. Fidelus-Gort R. Kasper L.H. Lancet Neurol. 2008; 7: 796-804Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 10.Leonardi C.L. Kimball A.B. Papp K.A. Yeilding N. Guzzo C. Wang Y. Li S. Dooley L.T. Gordon K.B. Lancet. 2008; 371: 1665-1674Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar) have shown promise in limiting inflammatory disease severity. Various small molecule drugs have been reported that are capable of blocking IL-12, IL-23, and p80 at distinct stages of their biological pathways (transcription, assembly, receptor binding, and signal transduction) (for review see Ref. 11.Vandenbroeck K. Alloza I. Gadina M. Matthys P. J. Pharm. Pharmacol. 2004; 56: 145-160Crossref PubMed Scopus (41) Google Scholar). One such compound is celecoxib (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar).Celecoxib is a non-steroidal anti-inflammatory drug originally designed to specifically inhibit cyclooxygenase-2 (COX-2), up-regulated in cancer and sites of inflammation (13.Lou J. Fatima N. Xiao Z. Stauffer S. Smythers G. Greenwald P. Ali I.U. Cancer Epidemiol. Biomarkers Prev. 2006; 15: 1598-1606Crossref PubMed Scopus (38) Google Scholar). However, COX-2 inhibitors have been subject to critical analysis because of cardiovascular problems, and a number of functions related to celecoxib are now known to be attributable to COX-2-independent mechanisms (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 13.Lou J. Fatima N. Xiao Z. Stauffer S. Smythers G. Greenwald P. Ali I.U. Cancer Epidemiol. Biomarkers Prev. 2006; 15: 1598-1606Crossref PubMed Scopus (38) Google Scholar, 14.Grösch S. Maier T.J. Schiffmann S. Geisslinger G. J. Natl. Cancer Inst. 2006; 98: 736-747Crossref PubMed Scopus (397) Google Scholar, 15.Schönthal A.H. Br. J. Cancer. 2007; 97: 1465-1468Crossref PubMed Scopus (89) Google Scholar). One such effect is the ability of celecoxib to increase cytoplasmic calcium concentrations through inhibition of ER Ca2+-ATPases (SERCA; Refs. 12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 16.Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar, 17.Pyrko P. Kardosh A. Liu Y.T. Soriano N. Xiong W. Chow R.H. Uddin J. Petasis N.A. Mircheff A.K. Farley R.A. Louie S.G. Chen T.C. Schönthal A.H. Mol. Cancer Ther. 2007; 6: 1262-1275Crossref PubMed Scopus (112) Google Scholar, 18.Tsutsumi S. Namba T. Tanaka K. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (113) Google Scholar). Experiments with other COX-2 inhibitors such as rofecoxib, ibuprofen, aspirin, naproxen, DuP697, and NS298 have all shown that this calcium-mobilizing property is unique to celecoxib (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar, 16.Johnson A.J. Hsu A.L. Lin H.P. Song X. Chen C.S. Biochem. J. 2002; 366: 831-837Crossref PubMed Google Scholar).We have shown that both celecoxib and a celecoxib analogue from which COX-2 inhibitory properties have been knocked out, i.e. 4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide, referred to hereafter as 4-trifluoromethyl-celecoxib (TFM-C), block IL-12 and p80 secretion through a post-transcriptional mechanism involving retention of the cytokines in the ER (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). TFM-C shares the effect of celecoxib in depleting ER Ca2+ stores in noncancerous HEK293 cells, i.e. the recombinant cytokine producer cells used in our study (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). For p80, this secretion block is accompanied with altered interaction with the ER chaperones calreticulin (CRT) and ERp44 (12.Alloza I. Baxter A. Chen Q. Matthiesen R. Vandenbroeck K. Mol. Pharmacol. 2006; 69: 1579-1587Crossref PubMed Scopus (37) Google Scholar). In the present study, we show that celecoxib and TFM-C block secretion of a third dimeric member of the IL-12 subfamily, namely IL-23, concomitant with intracellular retention of the IL-23 subunit p19. In an attempt to identify the ER factors crucially involved in this process we analyzed the effect of TFM-C on expression of 23 ER chaperones and cofactors. We identified homocysteine-inducible endoplasmic reticulum protein (HERP) as the gene transcript most dramatically up-regulated by TFM-C. HERP is a 54 kDa ubiquitously expressed ER membrane protein that is up-regulated as part of the ER-unfolded protein response (UPR) (19.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 20.Lenz B. Bleich S. Beutler S. Schlierf B. Schwager K. Reulbach U. Kornhuber J. Bönsch D. Exp. Cell Res. 2006; 312: 4049-4055Crossref PubMed Scopus (30) Google Scholar, 21.Ma Y. Hendershot L.M. J. Biol. Chem. 2004; 279: 13792-13799Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22.Liang G. Audas T.E. Li Y. Cockram G.P. Dean J.D. Martyn A.C. Kokame K. Lu R. Mol. Cell Biol. 2006; 26: 7999-8010Crossref PubMed Scopus (112) Google Scholar, 23.Sai X. Kokame K. Shiraishi H. Kawamura Y. Miyata T. Yanagisawa K. Komano H. FEBS Lett. 2003; 553: 151-156Crossref PubMed Scopus (36) Google Scholar). HERP is required for destruction of a number of proteins via the ER-associated degradation (ERAD) pathway, such as CD3-delta, connexin 43, non-secreted Igκ LC, mutant Igγ LC, and truncated Igγ HC (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar, 25.Okuda-Shimizu Y. Hendershot L.M. Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar). HERP is a component of a larger ERAD retrotranslocation complex comprising the E3 ligase HRD1, Derlin-1, VIMP, and the ATPase p97 (26.Schulze A. Standera S. Buerger E. Kikkert M. van Voorden S. Wiertz E. Koning F. Kloetzel P.M. Seeger M. J. Mol. Biol. 2005; 354: 1021-1027Crossref PubMed Scopus (165) Google Scholar, 27.Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. Nature. 2004; 429: 841-847Crossref PubMed Scopus (792) Google Scholar). HERP knock-out also results in increased susceptibility to ER stress-related cell death by thapsigargin, tunicamycin, or A23187 (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar). The proteasome inhibitor lactacystin accelerates cell death in HERP knock-out cells and blocks the degradation of connexin 43, Igκ LC, Igγ LC, and Igγ HC, indicating that HERP is involved in delivering ERAD-targeted proteins to the proteasome (24.Hori O. Ichinoda F. Yamaguchi A. Tamatani T. Taniguchi M. Koyama Y. Katayama T. Tohyama M. Stern D.M. Ozawa K. Kitao Y. Ogawa S. Genes Cells. 2004; 9: 457-469Crossref PubMed Scopus (97) Google Scholar, 25.Okuda-Shimizu Y. Hendershot L.M. Mol. Cell. 2007; 28: 544-554Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In this study, we show that TFM-C induces protein interaction of HERP with intracellularly retained p80 and IL-23, and that HERP is functionally required for the degradation of intracellularly retained dimeric p80.

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