Anthrax Lethal Toxin Enhances IκB Kinase Activation and Differentially Regulates Pro-inflammatory Genes in Human Endothelium
2009; Elsevier BV; Volume: 284; Issue: 38 Linguagem: Inglês
10.1074/jbc.m109.036970
ISSN1083-351X
AutoresJason M. Warfel, Felice D’Agnillo,
Tópico(s)Yersinia bacterium, plague, ectoparasites research
ResumoAnthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-κB in tumor necrosis factor-α-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-κB activation is associated with the enhanced degradation of the inhibitory proteins IκBα and IκBβ but not IκBϵ. Moreover, this was accompanied by enhanced activation of the IκB kinase complex (IKK), which is responsible for targeting IκB proteins for degradation. Importantly, LT enhancement of IκBα degradation was completely blocked by a selective IKKβ inhibitor, whereas IκBβ degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IκBα and p65, both of which were blocked by incubation with the IKKβ inhibitor. Consistent with NF-κB activation, LT increased transcription of the NF-κB regulated gene CD40. Conversely, LT inhibited transcription of another NF-κB-regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-κB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax. Anthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-κB in tumor necrosis factor-α-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-κB activation is associated with the enhanced degradation of the inhibitory proteins IκBα and IκBβ but not IκBϵ. Moreover, this was accompanied by enhanced activation of the IκB kinase complex (IKK), which is responsible for targeting IκB proteins for degradation. Importantly, LT enhancement of IκBα degradation was completely blocked by a selective IKKβ inhibitor, whereas IκBβ degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IκBα and p65, both of which were blocked by incubation with the IKKβ inhibitor. Consistent with NF-κB activation, LT increased transcription of the NF-κB regulated gene CD40. Conversely, LT inhibited transcription of another NF-κB-regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-κB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax. Anthrax is a disease caused by the Gram-positive spore-forming bacterium Bacillus anthracis. Many of the symptoms of systemic anthrax can be attributed to the action of anthrax toxin, which is made up of three secreted proteins, protective antigen (PA), 2The abbreviations used are: PAprotective antigenEFedema factorGAPDHglyceraldehyde-3-phosphate dehydrogenaseIKKIκB kinaseILinterleukinIRF-1interferon regulatory factor-1JNKc-Jun N-terminal kinaseLFlethal factorLTlethal toxinMAPKmitogen-activated protein kinaseSTAT-1signal transducers and activators of transcription-1TNFtumor necrosis factor-αVCAM-1vascular cell adhesion molecule-1ELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMOPS4-morpholinepropanesulfonic acid. 2The abbreviations used are: PAprotective antigenEFedema factorGAPDHglyceraldehyde-3-phosphate dehydrogenaseIKKIκB kinaseILinterleukinIRF-1interferon regulatory factor-1JNKc-Jun N-terminal kinaseLFlethal factorLTlethal toxinMAPKmitogen-activated protein kinaseSTAT-1signal transducers and activators of transcription-1TNFtumor necrosis factor-αVCAM-1vascular cell adhesion molecule-1ELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMOPS4-morpholinepropanesulfonic acid. and lethal factor (LF), which combine to form lethal toxin (LT), and edema factor (EF), which combines with PA to form edema toxin (1Moayeri M. Leppla S.H. Curr. Opin. Microbiol. 2004; 7: 19-24Crossref PubMed Scopus (217) Google Scholar, 2Mourez M. Rev. Physiol. Biochem. Pharmacol. 2004; 152: 135-164Crossref PubMed Scopus (82) Google Scholar). PA binds to the cell-surface receptors ANTXR1 and ANTXR2, leading to endocytosis of the enzymatic moieties EF and LF (3Scobie H.M. Young J.A. Curr. Opin. Microbiol. 2005; 8: 106-112Crossref PubMed Scopus (88) Google Scholar). Once in the cytosol, EF is a calcium-calmodulin-dependent adenylate cyclase, causing accumulation of the secondary signaling molecule cAMP (4Leppla S.H. Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 3162-3166Crossref PubMed Scopus (756) Google Scholar). LF is a zinc metalloprotease that cleaves proteins of the MEK family, disrupting MAPK signaling (5Turk B.E. Biochem. J. 2007; 402: 405-417Crossref PubMed Scopus (124) Google Scholar, 6Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Crossref PubMed Scopus (885) Google Scholar).The high mortality resulting from systemic anthrax infection is generally associated with profound vascular pathologies, including vascular leakage, edema, hemorrhage, vasculitis, and a poor immune response (7Tournier J.N. Quesnel-Hellmann A. Cleret A. Vidal D.R. Cell. Microbiol. 2007; 9: 555-565Crossref PubMed Scopus (64) Google Scholar, 8Guarner J. Jernigan J.A. Shieh W.J. Tatti K. Flannagan L.M. Stephens D.S. Popovic T. Ashford D.A. Perkins B.A. Zaki S.R. Am. J. Pathol. 2003; 163: 701-709Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 9Grinberg L.M. Abramova F.A. Yampolskaya O.V. Walker D.H. Smith J.H. Mod. Pathol. 2001; 14: 482-495Crossref PubMed Scopus (193) Google Scholar). Importantly, many of these symptoms are also observed in animals treated with purified LT (10Culley N.C. Pinson D.M. Chakrabarty A. Mayo M.S. Levine S.M. Infect. Immun. 2005; 73: 7006-7010Crossref PubMed Scopus (39) Google Scholar, 11Cui X. Moayeri M. Li Y. Li X. Haley M. Fitz Y. Correa-Araujo R. Banks S.M. Leppla S.H. Eichacker P.Q. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004; 286: R699-R709Crossref PubMed Scopus (110) Google Scholar, 12Moayeri M. Haines D. Young H.A. Leppla S.H. J. Clin. Invest. 2003; 112: 670-682Crossref PubMed Scopus (257) Google Scholar, 13Kuo S.R. Willingham M.C. Bour S.H. Andreas E.A. Park S.K. Jackson C. Duesbery N.S. Leppla S.H. Tang W.J. Frankel A.E. Microb. Pathog. 2008; 44: 467-472Crossref PubMed Scopus (45) Google Scholar, 14Bolcome 3rd, R.E. Sullivan S.E. Zeller R. Barker A.P. Collier R.J. Chan J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2439-2444Crossref PubMed Scopus (47) Google Scholar). In addition, toxin receptor expression appears to be enriched on the endothelium (15Deshpande A. Hammon R.J. Sanders C.K. Graves S.W. FEBS Lett. 2006; 580: 4172-4175Crossref PubMed Scopus (11) Google Scholar). These findings have supported the idea that LT may directly target the endothelium during systemic anthrax infection, when serum levels of LF and PA can exceed 200 and 1000 ng/ml respectively (7Tournier J.N. Quesnel-Hellmann A. Cleret A. Vidal D.R. Cell. Microbiol. 2007; 9: 555-565Crossref PubMed Scopus (64) Google Scholar, 16Walsh J.J. Pesik N. Quinn C.P. Urdaneta V. Dykewicz C.A. Boyer A.E. Guarner J. Wilkins P. Norville K.J. Barr J.R. Zaki S.R. Patel J.B. Reagan S.P. Pirkle J.L. Treadwell T.A. Messonnier N.R. Rotz L.D. Meyer R.F. Stephens D.S. Clin. Infect. Dis. 2007; 44: 968-971Crossref PubMed Scopus (99) Google Scholar, 17Mabry R. Brasky K. Geiger R. Carrion Jr., R. Hubbard G.B. Leppla S. Patterson J.L. Georgiou G. Iverson B.L. Clin. Vaccine Immunol. 2006; 13: 671-677Crossref PubMed Scopus (99) Google Scholar, 18Shoop W.L. Xiong Y. Wiltsie J. Woods A. Guo J. Pivnichny J.V. Felcetto T. Michael B.F. Bansal A. Cummings R.T. Cunningham B.R. Friedlander A.M. Douglas C.M. Patel S.B. Wisniewski D. Scapin G. Salowe S.P. Zaller D.M. Chapman K.T. Scolnick E.M. Schmatz D.M. Bartizal K. MacCoss M. Hermes J.D. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7958-7963Crossref PubMed Scopus (144) Google Scholar, 19Boyer A.E. Quinn C.P. Woolfitt A.R. Pirkle J.L. McWilliams L.G. Stamey K.L. Bagarozzi D.A. Hart Jr., J.C. Barr J.R. Anal. Chem. 2007; 79: 8463-8470Crossref PubMed Scopus (78) Google Scholar, 20Molin F.D. Fasanella A. Simonato M. Garofolo G. Montecucco C. Tonello F. Toxicon. 2008; 52: 824-828Crossref PubMed Scopus (36) Google Scholar).Data from our laboratory further support the hypothesis that vascular endothelium is an important target of LT. We previously reported that LT induces endothelial barrier dysfunction consistent with vascular leakage associated with anthrax (21Warfel J.M. Steele A.D. D'Agnillo F. Am. J. Pathol. 2005; 166: 1871-1881Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In addition, we showed that LT enhances vascular cell adhesion molecule-1 (VCAM-1) expression and monocyte adhesion on the surface of tumor necrosis factor-α (TNF)-activated primary human endothelial cells, suggesting a possible link between LT and the vasculitis associated with anthrax (9Grinberg L.M. Abramova F.A. Yampolskaya O.V. Walker D.H. Smith J.H. Mod. Pathol. 2001; 14: 482-495Crossref PubMed Scopus (193) Google Scholar, 22Steele A.D. Warfel J.M. D'Agnillo F. Biochem. Biophys. Res. Commun. 2005; 337: 1249-1256Crossref PubMed Scopus (14) Google Scholar, 23Shieh W.J. Guarner J. Paddock C. Greer P. Tatti K. Fischer M. Layton M. Philips M. Bresnitz E. Quinn C.P. Popovic T. Perkins B.A. Zaki S.R. Am. J. Pathol. 2003; 163: 1901-1910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The enhanced expression of VCAM-1 was found to be transcriptionally driven by the cooperative activation of the VCAM1-regulating transcription factors, interferon regulatory factor-1 (IRF-1), and NF-κB (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar). Specifically, LT enhanced nuclear translocation of IRF-1 and NF-κB, which correlated with increased DNA binding of both transcription factors by electromobility shift assay. Considering the critical role of NF-κB in regulating the endothelial inflammatory response, we investigated the mechanisms underlying the enhancement of this pathway by LT.In this study, we show that LT enhancement of NF-κB correlates temporally with the delayed reaccumulation of the inhibitory molecules IκBα and IκBβ. We also provide evidence that LT enhances activation and phosphorylation of the IκB kinase (IKK) complex that is responsible for initiating and maintaining NF-κB activity via phosphorylation of the IκB proteins and the p65 subunit of NF-κB (25Kishore N. Sommers C. Mathialagan S. Guzova J. Yao M. Hauser S. Huynh K. Bonar S. Mielke C. Albee L. Weier R. Graneto M. Hanau C. Perry T. Tripp C.S. J. Biol. Chem. 2003; 278: 32861-32871Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 26Sakurai H. Suzuki S. Kawasaki N. Nakano H. Okazaki T. Chino A. Doi T. Saiki I. J. Biol. Chem. 2003; 278: 36916-36923Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 27Perkins N.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 49-62Crossref PubMed Scopus (1902) Google Scholar). In addition to these findings, we tested our previously reported postulate that LT may differentially regulate NF-κB genes in a promoter-dependent manner (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar). We show that LT enhances transcription and expression of the surface receptor CD40 but significantly decreases the expression of MCP-1 (monocyte chemotactic protein-1). The inhibitory effect on this latter gene is driven by LT-mediated inhibition of AP-1 (activator protein-1) activity. Together, these findings provide new mechanistic insight into how LT may alter immune and vascular function and contribute to the poor host response to anthrax infection.DISCUSSIONVascular pathologies associated with anthrax may be the result of the direct interaction of LT with the endothelium. We have shown in vitro that LT disrupts endothelial barrier function and has both enhancing and suppressive effects on endothelial inflammatory responses (21Warfel J.M. Steele A.D. D'Agnillo F. Am. J. Pathol. 2005; 166: 1871-1881Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 22Steele A.D. Warfel J.M. D'Agnillo F. Biochem. Biophys. Res. Commun. 2005; 337: 1249-1256Crossref PubMed Scopus (14) Google Scholar, 24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar). The latter is likely to play an important role in the poor immune response and vasculitis associated with anthrax and occurs in part due to enhancement and prolonging of NF-κB activity under varying inflammatory stimuli, including IL-1β and TNFα. The duration and intensity of NF-κB activity are regulated by the degradation and reaccumulation of the inhibitory proteins IκBα, IκBβ, and IκBϵ. In endothelial cells as well as other cell types exposed to prolonged TNF treatment, the differential rates of degradation and reaccumulation of these three proteins account for the two distinct phases or "oscillations" of NF-κB activity (34Hoffmann A. Levchenko A. Scott M.L. Baltimore D. Science. 2002; 298: 1241-1245Crossref PubMed Scopus (1469) Google Scholar, 35Spiecker M. Darius H. Liao J.K. J. Immunol. 2000; 164: 3316-3322Crossref PubMed Scopus (55) Google Scholar, 36Johnson D.R. Douglas I. Jahnke A. Ghosh S. Pober J.S. J. Biol. Chem. 1996; 271: 16317-16322Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). An early phase, which peaks within the first hour is characterized by the rapid degradation and resynthesis of IκBα. In endothelial cells, the late phase, (i.e. >2 h), has been attributed to the much slower degradation and reaccumulation of IκBβ (36Johnson D.R. Douglas I. Jahnke A. Ghosh S. Pober J.S. J. Biol. Chem. 1996; 271: 16317-16322Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Importantly, we show that although LT has no effect on IκB expression during time points associated with the early phase, LT cotreatment significantly delayed the reaccumulation of IκBα (at 2 and 6 h) and IκBβ (at 12 and 24 h), but not IκBϵ, during the late phase. The fact that the reduced expression of IκB proteins was most striking at 6 and 12 h suggests a mechanistic link with the enhanced NF-κB activity that was observed at those time points in LT-cotreated cells. Although the reduced IκBβ expression could be partially explained by reduction in gene transcription, LT conversely enhanced transcription of NFKBIA. Importantly, the reduced IκB protein expression in LT-cotreated cells was not because of alterations in proteasome activity or phosphatase activity, and in the case of IκBα, it was not because of impaired translation of NFKBIA mRNA. Together these data suggest the involvement of the IKK complex in the delayed IκB reaccumulation.The IKK complex, composed of two functionally distinct kinases, IKKα and IKKβ, and the structural subunit IKKγ, is activated in response to a variety of stimuli by phosphorylation as a triggering event in the NF-κB response. Both IKKα and IKKβ have been shown to phosphorylate IκB proteins, targeting them for ubiquitination and subsequent degradation by the chymotrypsin-like proteasome activity (39Häcker H. Karin M. Sci. STKE 2006. 2006; : re13Google Scholar, 45Solt L.A. May M.J. Immunol. Res. 2008; 42: 3-18Crossref PubMed Scopus (173) Google Scholar, 46Wu C. Ghosh S. J. Biol. Chem. 2003; 278: 31980-31987Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This results in the exposure of a nuclear localization sequence on the p50/p65 heterodimer and leads to nuclear import and subsequent transcription of NF-κB-responsive genes. IKKα and IKKβ have also been shown to phosphorylate p65, resulting in enhanced nuclear localization and transcriptional activity (27Perkins N.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 49-62Crossref PubMed Scopus (1902) Google Scholar, 39Häcker H. Karin M. Sci. STKE 2006. 2006; : re13Google Scholar, 47Luedde T. Heinrichsdorff J. de Lorenzi R. De Vos R. Roskams T. Pasparakis M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9733-9738Crossref PubMed Scopus (70) Google Scholar). Importantly, we show that LT cotreatment significantly enhanced IKKβ activity and phosphorylation. In addition, we observed phosphorylation of the IKK targets IκBα and p65 that correlated well with the IKK enhancement in terms of magnitude and kinetics. In addition, we showed that inhibition of IKKβ completely blocked the LT enhancement of p65 phosphorylation and IκBα phosphorylation and degradation. The LT-enhanced degradation of IκBβ was partially rescued by the IKKβ inhibitor. Together, these data suggest an active role for IKK in the LT enhancement of NF-κB activation observed in this study and as reported previously (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar).With regard to the mechanism leading to enhanced IKK activation, we provide evidence that LT cotreatment enhances expression of MEKK2, an upstream kinase that has been linked to IKK activation (38Zhao Q. Lee F.S. J. Biol. Chem. 1999; 274: 8355-8358Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 48Winsauer G. Resch U. Hofer-Warbinek R. Schichl Y.M. de Martin R. Cell. Signal. 2008; 20: 2107-2112Crossref PubMed Scopus (32) Google Scholar). Indeed, MEKK2 is thought to specifically control the late phase of NF-κB activity through assembly of a complex with IKK and IκB proteins (37Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The mechanisms underlying LT enhancement of MEKK2 expression are not yet clear, although one hypothesis is that this may represent a feedback response triggered by the LT-mediated cleavage of downstream MEK proteins. Significant attention has focused on LT-mediated MEK cleavage and the resulting downstream inhibition of MAPK signaling; however, to our knowledge, the present findings represent the first demonstration that LT can elicit increased expression of signaling components directly upstream of MEKs. However, it should be noted that MEKK2 expression only appeared enhanced in LT-cotreated cells and was not detectable in cells treated with LT alone. Besides pathways that stimulate IKK, another possibility that warrants further investigation is whether LT may inhibit endogenous endothelial mechanisms that are activated for resolution of the NF-κB response (49Winsauer G. de Martin R. Thromb. Haemost. 2007; 97: 364-369Crossref PubMed Scopus (27) Google Scholar). Potential targets include the COX-2 produced cyclopentenone prostaglandins, anti-inflammatory molecules that are exclusively synthesized late in the inflammatory response and have been shown to inhibit IKK activity (50Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google Scholar). Other molecules of interest include members of the ovarian tumor (OTU) family of deubiquitinating cysteine proteases that have been shown to be induced by TNF and IL-1 in endothelial cells and are believed to play a role in NF-κB resolution by deubiquitinating receptor-associated signaling intermediaries and inhibition of IKK activity (51Enesa K. Zakkar M. Chaudhury H. Luong le, A. Rawlinson L. Mason J.C. Haskard D.O. Dean J.L. Evans P.C. J. Biol. Chem. 2008; 283: 7036-7045Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar).Until recently, the NF-κB dogma has held that IKKβ and IKKα are functionally distinct kinases with the former being solely responsible for the canonical signaling pathway and the latter controlling the noncanonical pathway. However, it is known that both kinases are capable of phosphorylating IκB proteins, and recently it has been shown that, under certain conditions, IKKα can activate the canonical pathway in numerous cell types, including endothelial cells (47Luedde T. Heinrichsdorff J. de Lorenzi R. De Vos R. Roskams T. Pasparakis M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9733-9738Crossref PubMed Scopus (70) Google Scholar, 52DeBusk L.M. Massion P.P. Lin P.C. Cancer Res. 2008; 68: 10223-10228Crossref PubMed Scopus (18) Google Scholar). Indeed, IKKα may be the predominant kinase responsible for IκB degradation in IL-1-treated murine embryonic fibroblast, whereas IKKβ was shown to be generally dispensable (53Solt L.A. Madge L.A. Orange J.S. May M.J. J. Biol. Chem. 2007; 282: 8724-8733Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Interestingly, the present data show that pretreatment of TNF-treated cells with the selective IKKβ inhibitor TPCA-1 was unable to block p65 phosphorylation or IκBα phosphorylation and degradation at 6 h, a time point associated with the NF-κB late phase (Fig. 5, B and C, and Fig. 6B). This observation is intriguing given that post-treatment with TPCA-1 (90 min after TNF) produced no net IκBα degradation in 6-h TNF-treated cells (Fig. 4C). It is tempting to speculate whether IKKα may compensate for IKKβ in the cells that were pretreated with TPCA-1. Further investigation will be required to address this interesting finding.Endothelial activation and subsequent gene expression in response to inflammatory stimuli are critical for coordinating the innate immune response to infection (54Pober J.S. Sessa W.C. Nat. Rev. Immunol. 2007; 7: 803-815Crossref PubMed Scopus (1164) Google Scholar). The vast majority of inflammatory products produced by endothelium are regulated by the IKK-NF-κB pathway (54Pober J.S. Sessa W.C. Nat. Rev. Immunol. 2007; 7: 803-815Crossref PubMed Scopus (1164) Google Scholar, 55Viemann D. Goebeler M. Schmid S. Klimmek K. Sorg C. Ludwig S. Roth J. Blood. 2004; 103: 3365-3373Crossref PubMed Scopus (124) Google Scholar, 56Denk A. Goebeler M. Schmid S. Berberich I. Ritz O. Lindemann D. Ludwig S. Wirth T. J. Biol. Chem. 2001; 276: 28451-28458Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 57Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1559) Google Scholar). In addition, recent studies have shown that abnormal or enhanced activity of the IKK-NF-κB pathway correlates with an unfavorable outcome in many diseases, including heart disease, cancer, and sepsis (58Moss N.C. Stansfield W.E. Willis M.S. Tang R.H. Selzman C.H. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: H2248-H2253Crossref PubMed Scopus (95) Google Scholar, 59Karin M. Nature. 2006; 441: 431-436Crossref PubMed Scopus (2942) Google Scholar, 60Liu S.F. Malik A.B. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006; 290: L622-L645Crossref PubMed Scopus (612) Google Scholar). Indeed, IKK inhibitor therapies are currently being developed as a promising treatment for many inflammatory disorders (61Strnad J. Burke J.R. Trends Pharmacol. Sci. 2007; 28: 142-148Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Therefore, the fact that LT alters IKK activity could suggest an important link between LT and certain anthrax-associated pathologies, including vasculitis and the poor immune response. To further investigate the consequences of the enhanced IKK-NF-κB pathway, we examined the effect of LT on the expression of several genes that are regulated by one or varying combinations of inflammatory transcription factors. In the case of the previously discussed NFKBIA, the IκBα-encoding gene regulated solely by NF-κB, LT was shown to enhance mRNA expression. Transcription of NFKBIB, which unlike NFKBIA is only minimally responsive to NF-κB (62Budde L.M. Wu C. Tilman C. Douglas I. Ghosh S. Mol. Biol. Cell. 2002; 13: 4179-4194Crossref PubMed Scopus (37) Google Scholar), was significantly decreased by LT cotreatment. The expression of CD40, which is regulated by NF-κB, IRF-1, and STAT-1, was likewise increased in LT-cotreated cells. However, transcription of CCL2 was significantly reduced by LT cotreatment and this was likely due to LT inhibition of AP-1. These observations may have pathogenic implications in that LT enhancement of CD40 expression in the presence of CD40L-expressing leukocytes could lead to a synergistic expansion of the inflammatory response through further NF-κB activation and endothelial adhesion molecule expression, suggesting a potential contribution to the vasculitic pathology associated with anthrax (63Chakrabarti S. Blair P. Freedman J.E. J. Biol. Chem. 2007; 282: 18307-18317Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 64Henn V. Slupsky J.R. Gräfe M. Anagnostopoulos I. Förster R. Müller-Berghaus G. Kroczek R.A. Nature. 1998; 391: 591-594Crossref PubMed Scopus (1731) Google Scholar). In addition, the inhibition of MCP-1 release combined with the reduced expression of the chemokines interleukin-8 and CCL5 may severely alter lymphocyte diapedesis, suggesting an important link between the effect of LT on the endothelium and the poor immune response observed in anthrax (22Steele A.D. Warfel J.M. D'Agnillo F. Biochem. Biophys. Res. Commun. 2005; 337: 1249-1256Crossref PubMed Scopus (14) Google Scholar, 65Batty S. Chow E.M. Kassam A. Der S.D. Mogridge J. Cell. Microbiol. 2006; 8: 130-138Crossref PubMed Scopus (35) Google Scholar). Indeed, in vitro studies from our laboratory have shown that, despite enhanced binding of human monocytes and neutrophils (22Steele A.D. Warfel J.M. D'Agnillo F. Biochem. Biophys. Res. Commun. 2005; 337: 1249-1256Crossref PubMed Scopus (14) Google Scholar), LT-cotreated endothelial cells exhibit severe deficiencies in coordinating transmigration compared with cells treated with TNF alone (data not shown).As mentioned above, the reduction in CCL2 transcription was linked to a dramatic reduction in basal and TNF-induced AP-1 binding activity in LT-treated endothelial cells. This is consistent with previous studies using other cell types and is likely due to the widely reported inhibition of JNK, which stabilizes c-Jun through phosphorylation (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar, 66Musti A.M. Treier M. Bohmann D. Science. 1997; 275: 400-402Crossref PubMed Scopus (407) Google Scholar, 67Fang H. Cordoba-Rodriguez R. Lankford C.S. Frucht D.M. J. Immunol. 2005; 174: 4966-4971Crossref PubMed Scopus (59) Google Scholar, 68Paccani S.R. Tonello F. Ghittoni R. Natale M. Muraro L. D'Elios M.M. Tang W.J. Montecucco C. Baldari C.T. J. Exp. Med. 2005; 201: 325-331Crossref PubMed Scopus (138) Google Scholar, 69Kang Z. Webster Marketon J.I. Johnson A. Sternberg E.M. J. Mol. Biol. 2009; 389: 595-605Crossref PubMed Scopus (2) Google Scholar). Importantly, the MAPK family regulates other transcription factors involved in immune and inflammatory responses, as well as controlling the activity of additional components of the transcriptional machinery (70Turjanski A.G. Vaqué J.P. Gutkind J.S. Oncogene. 2007; 26: 3240-3253Crossref PubMed Scopus (340) Google Scholar). In addition, p38 and JNK have been shown to augment expression of certain genes by enhancing post-transcriptional mRNA stability (71Eberhardt W. Doller A. Akool el-S. Pfeilschifter J. Pharmacol. Ther. 2007; 114: 56-73Crossref PubMed Scopus (136) Google Scholar). By disrupting the above MAPK-regulated pathways, LT may influence the expression of many genes in cells exposed to the toxin.The finding that LT exerts opposing effects on important pro-inflammatory transcription factors appears to lead to an interesting dynamic where pro-inflammatory genes are differentially regulated in LT-cotreated endothelial cells. Whether a particular gene is up- or down-regulated by LT depends on what transcription factors regulate its expression. For example, for some genes, including CD40 and NFKBIA discussed here as well as previously reported VCAM1 and IRF1 (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar), the increased NF-κB and IRF-1 activity outweighs the reduction in AP-1 activity and results in enhanced transcription of these genes in LT-cotreated cells. However, for another subset of genes, including CCL2 discussed here and also SELE as reported previously (24Warfel J.M. D'Agnillo F. J. Immunol. 2008; 180: 7516-7524Crossref PubMed Scopus (27) Google Scholar), the reduction in AP-1 activity outweighs the increased NF-κB and IRF-1 activity and leads to reduced transcription in LT-cotreated cells.In conclusion, LT-mediated enhancement of TNF-induced NF-κB activation is because of increased activation of the IKK complex. When combined with the inhibition of AP-1 activity by LT, this leads to potentially important downstream transcriptional up- or down-regulation driven by the cumulative effect of LT on the particular set of transcription factors that control a given promoter. Given the important role of the IKK-NF-κB pathway and AP-1 in immune and inflammatory s
Referência(s)