Signaling by the Cysteinyl-Leukotriene Receptor 2

Abstract

Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-κB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-κB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IκBα prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-κB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCϵ prevented p65 phosphorylation, whereas dnPKCδ prevented AP-1 binding. Moreover, dnPKCδ, dnPKCϵ, and dnPKCζ prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-κB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription. Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-κB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-κB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IκBα prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-κB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCϵ prevented p65 phosphorylation, whereas dnPKCδ prevented AP-1 binding. Moreover, dnPKCδ, dnPKCϵ, and dnPKCζ prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-κB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription. Cysteinyl-leukotrienes (cysLTs) 2The abbreviations used are: CysLTcysteinyl-leukotrieneLTC4leukotriene C4LTD4leukotriene D4LTE4leukotriene E4IL-8interleukin-8MCP-1monocyte chemotactic protein 1COX-2cyclooxygenase 2HEK293human embryonic kidney 293AP-1activator protein-1NFnuclear factorHUVEChuman umbilical vein endothelial cellsdndominant negativeCMVcytomegalovirusPKCprotein kinase CEMSAelectrophoretic mobility shift assayJNKc-Jun N-terminal kinaseC/EBPCCAAT enhancer-binding protein. 2The abbreviations used are: CysLTcysteinyl-leukotrieneLTC4leukotriene C4LTD4leukotriene D4LTE4leukotriene E4IL-8interleukin-8MCP-1monocyte chemotactic protein 1COX-2cyclooxygenase 2HEK293human embryonic kidney 293AP-1activator protein-1NFnuclear factorHUVEChuman umbilical vein endothelial cellsdndominant negativeCMVcytomegalovirusPKCprotein kinase CEMSAelectrophoretic mobility shift assayJNKc-Jun N-terminal kinaseC/EBPCCAAT enhancer-binding protein. are lipid mediators implicated in several inflammatory processes, including allergy and asthma. Leukotriene (LT) C4, LTD4, and LTE4 act on two G-protein-coupled receptors, CysLT1 and CysLT2 (1Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (886) Google Scholar, 2Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (304) Google Scholar, 3Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. 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Selective antagonists for CysLT1 are in clinical use for the treatment of asthma. Conversely, the function of the CysLT2 receptor is, as yet, poorly defined. cysteinyl-leukotriene leukotriene C4 leukotriene D4 leukotriene E4 interleukin-8 monocyte chemotactic protein 1 cyclooxygenase 2 human embryonic kidney 293 activator protein-1 nuclear factor human umbilical vein endothelial cells dominant negative cytomegalovirus protein kinase C electrophoretic mobility shift assay c-Jun N-terminal kinase CCAAT enhancer-binding protein. cysteinyl-leukotriene leukotriene C4 leukotriene D4 leukotriene E4 interleukin-8 monocyte chemotactic protein 1 cyclooxygenase 2 human embryonic kidney 293 activator protein-1 nuclear factor human umbilical vein endothelial cells dominant negative cytomegalovirus protein kinase C electrophoretic mobility shift assay c-Jun N-terminal kinase CCAAT enhancer-binding protein. Various reports have suggested a role for CysLT2 in a number of pathologies. The CysLT2 gene is localized on chromosome 13q14, a region linked to atopic asthma (3Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 14Cookson W. Nature. 1999; 402: B5-B11Crossref PubMed Scopus (349) Google Scholar). Interestingly, a coding polymorphism in the CysLT2 receptor, which decreases responsiveness to LTD4, has been associated with resistance to asthma (15Pillai S.G. Cousens D.J. Barnes A.A. Buckley P.T. Chiano M.N. Hosking L.K. Cameron L.A. Fling M.E. Foley J.J. Green A. Sarau H.M. Schmidt D.B. Sprankle C.S. Blumenthal M.N. Vestbo J. Kennedy-Wilson K. Wixted W.E. Wagner M.J. Anderson W.H. Ignar D.M. Pharmacogenetics. 2004; 14: 627-633Crossref PubMed Scopus (51) Google Scholar). Furthermore, another report established an association of a CysLT2 variant with atopy in the Tristan da Cunha population (16Thompson M.D. Storm van's Gravesande K. Galczenski H. Burnham W.M. Siminovitch K.A. Zamel N. Slutsky A. Drazen J.M. George S.R. Evans J.F. O'Dowd B.F. Pharmacogenetics. 2003; 13: 641-649Crossref PubMed Scopus (48) Google Scholar). An implication of this receptor in vascular permeability and bleomycin-induced pulmonary fibrosis has been suggested, using cyslt2 receptor-null mice (17Beller T.C. A. D.S. Austen K.F. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and K. R. M. A. R. E. Galczenski H. Evans J.F. A.J. Biol. 2003; PubMed Google an of the CysLT2 receptor in inflammation and this and H. A. M. A. J. G. S. A. PubMed Scopus Google the of CysLT2 receptor in of mice to of CysLT2 a in and (3Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 4Nothacker H.P. Wang Z. Zhu Y. Reinscheid R.K. Lin S.H. Civelli O. Mol. Pharmacol. 2000; 58: 1601-1608Crossref PubMed Scopus (164) Google Scholar). the of the CysLT2 receptor in the human in (3Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Moreover, of CysLT2 has been on mast and cells in the of patients D.J. L. G. Austin C.P. Evans J.F. Clin. Exp. 2003; PubMed Scopus Google Scholar). of cysLTs for the CysLT2 receptor is LTC4 as by binding (3Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 4Nothacker H.P. Wang Z. Zhu Y. Reinscheid R.K. Lin S.H. Civelli O. Mol. Pharmacol. 2000; 58: 1601-1608Crossref PubMed Scopus (164) Google and that CysLT1 CysLT2. CysLT2 is at this this receptor to is by by a IL-8 is an of the chemokine which a role in the and of inflammation A. N. T. T. N. K. J. Biol. 56: PubMed Scopus Google Scholar). including asthma and pulmonary are associated with of IL-8 Am. J. Respir. Crit. Care Med. 2000; 161: PubMed Scopus Google Scholar, J.L. N. 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PubMed Scopus Google Scholar). that LTC4 transcriptionally IL-8 and via the CysLT2 Furthermore, we the signaling LTC4 in the of IL-8 transcription. umbilical vein endothelial cells at first as well as cells stably transfected with CysLT2 were of the that were with transfected cells have been with endothelial cells and the we the first that the human in response to as well as the NF-κB and AP-1 transcription and that this in chemokine and were and were and and IκBα were and p65 p65 c-Jun and were and were and were for IκBα at and by of for human CysLT2 receptor and by a and the of a in The human IL-8 promoter IL-8 and were by R. the of S. I. A. 1999; PubMed Google Scholar). dominant negative (dn) and were a and G. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the were shown to of D.J. C. Z. S. J.F. M. J. 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The IL-8 IL-8 cells in were in for with LTC4 for the and in 1 1 1 1 2 for on were on and to were with with for 1 and with in and at and with an used for protein were by in and for at were with the and as cells were in cells were were with LTD4, or for the and were by an of and 1 were by and at for at The were in 1 and an 1 and of and a on an of the as well as a of were for at at The nuclear were with the in nuclear 1 and the a on were for at and nuclear were in and at were for protein The of the of the used for were as and binding were in the of to or at the of were on at in were to with at cells were in with of DNA using of to the The cells were stimulation with LTC4 or for were for as C. A. Y. M. P. J. M. Am. J. Respir. Mol. Biol. PubMed Scopus Google Scholar). cells were in and stimulation with LTC4 for the were in and at IL-8 concentrations were using the IL-8 assay using the for data or of as using were at LTC4 IL-8 and in several CysLT2 and CysLT1 and of a of CysLT2 we a that stably the CysLT2 receptor, to as as a to study the signaling of the CysLT2 of the that were with the cells have been with cells and the or HEK293 cells CysLT1 CysLT2 were to the of LTC4 on IL-8 and in cells. shown in LTC4 induced IL-8 1 of with a The of LTC4 on IL-8 as by a of of in response to LTC4 are shown in the of IL-8 protein in response to LTC4 at 2 and through of LTC4 stimulation HEK293 cells response to LTC4 stimulation LTC4 and IL-8 in a LTC4-induced IL-8 gene at the transcriptional cells were transfected with a gene by the IL-8 promoter and the promoter stimulation with LTC4 or shown in LTC4 IL-8 transactivation in a concentration-dependent with a at LTC4 for induced IL-8 with the induction Interestingly, LTC4 and induced IL-8 promoter at IL-8 promoter in response to stimulation induced in HEK293 cells LTC4-induced IL-8 on NF-κB, AP-1, and NF-IL-6 the transcription factor binding involved in the induction of IL-8 transactivation in response to mutants of the region of the IL-8 gene were used S. I. A. 1999; PubMed Google Scholar). shown in the NF-κB the IL-8 promoter induced by LTC4. the AP-1 resulted in a of the LTC4-induced IL-8 whereas the NF-IL-6 a LTC4 NF-κB we the of NF-κB, we the elements of this that by LTC4. the induction of IκBα LTC4 stimulation with a induction shown in LTC4 induced a of the p65 NF-κB on To IκBα for LTC4-induced IL-8 promoter cells were with a dominant-negative form of IκBα and the and with LTC4. in of dominant-negative IκBα IL-8 promoter by in response to LTC4. LTC4 NF-κB DNA on the we LTC4 NF-κB DNA binding. the of LTC4-induced NF-κB DNA binding, an induction that of LTC4 a in NF-κB binding of the DNA binding LTC4 stimulation using that the NF-κB binding and p65 LTC4 the DNA of LTC4 stimulation of the cells AP-1 DNA binding shown in cells to LTC4 for the of time a induction of AP-1 DNA binding which at a induction of AP-1 DNA binding by LTC4 by that the AP-1 binding of and LTC4 the of of of c-Jun is for of the AP-1 we the of the c-JUN·c-FOS in we stimulation with LTC4. shown in LTC4 induced a of c-Jun N-terminal kinase is a well for a LTC4-induced in concentrations of the induced by at The of the by its to induced by LTC4 to LTC4-induced IL-8 as shown in Interestingly, LTC4 to an of this is involved in LTC4-induced IL-8 C in IL-8 involved in LTC4-induced IL-8 we a role in IL-8 HEK293 cells are to and to a lesser PKCϵ and M. T. Y. A. A. Saito N. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). of cells with the or the LTC4-induced IL-8 promoter LTC4 to in a Furthermore, of the potential for using we to dominant negative of in dominant-negative for and to a lesser LTC4-induced IL-8 the implication of in LTC4-induced IL-8 gene transcription. the dominant-negative a on LTC4-induced IL-8 transcription for LTC4-induced AP-1 LTC4-induced of by that for in LTC4-induced AP-1 DNA binding with with and with the Furthermore, prevented LTC4-induced of cells were transfected with of LTC4-induced AP-1 DNA binding were to c-Jun using of the NF-κB to p65 in a and a by that a involved in LTC4-induced NF-κB DNA binding However, cells transfected with dominant-negative of or revealed that LTC4-induced of p65 on PKCϵ of the dominant-negative of LTC4-induced NF-κB DNA binding LTC4 IL-8 in the CysLT2 receptor, with of The of cysLTs on have been shown to the of CysLT2 CysLT1 antagonists stimulation K. R. M. A. R. E. Galczenski H. Evans J.F. A.J. 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