Signal Transduction by the Chemokine Receptor CXCR3
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m010303200
ISSN1083-351X
AutoresAndrea Bonacchi, Paola Romagnani, Roberto Giulio Romanelli, Eva Efsen, Francesco Annunziato, Laura Lasagni, Michela Francalanci, Mario Serio, Giacomo Laffi, Massimo Pinzani, Paolo Geñtilini, Fabio Marra,
Tópico(s)interferon and immune responses
ResumoHepatic stellate cells (HSC) and glomerular mesangial cells (MC) are tissue-specific pericytes involved in tissue repair, a process that is regulated by members of the chemokine family. In this study, we explored the signal transduction pathways activated by the chemokine receptor CXCR3 in vascular pericytes. In HSC, interaction of CXCR3 with its ligands resulted in increased chemotaxis and activation of the Ras/ERK cascade. Activation of CXCR3 also stimulated Src phosphorylation and kinase activity and increased the activity of phosphatidylinositol 3-kinase and its downstream pathway, Akt. The increase in ERK activity was inhibited by genistein and PP1, but not by wortmannin, indicating that Src activation is necessary for the activation of the Ras/ERK pathway by CXCR3. Inhibition of ERK activation resulted in a decreased chemotactic and mitogenic effect of CXCR3 ligands. In MC, which respond to CXCR3 ligands with increased DNA synthesis, CXCR3 activation resulted in a biphasic stimulation of ERK activation, a pattern similar to the one observed in HSC exposed to platelet-derived growth factor, indicating that this type of response is related to the stimulation of cell proliferation. These data characterize CXCR3 signaling in pericytes and clarify the relevance of downstream pathways in the modulation of different biologic responses. Hepatic stellate cells (HSC) and glomerular mesangial cells (MC) are tissue-specific pericytes involved in tissue repair, a process that is regulated by members of the chemokine family. In this study, we explored the signal transduction pathways activated by the chemokine receptor CXCR3 in vascular pericytes. In HSC, interaction of CXCR3 with its ligands resulted in increased chemotaxis and activation of the Ras/ERK cascade. Activation of CXCR3 also stimulated Src phosphorylation and kinase activity and increased the activity of phosphatidylinositol 3-kinase and its downstream pathway, Akt. The increase in ERK activity was inhibited by genistein and PP1, but not by wortmannin, indicating that Src activation is necessary for the activation of the Ras/ERK pathway by CXCR3. Inhibition of ERK activation resulted in a decreased chemotactic and mitogenic effect of CXCR3 ligands. In MC, which respond to CXCR3 ligands with increased DNA synthesis, CXCR3 activation resulted in a biphasic stimulation of ERK activation, a pattern similar to the one observed in HSC exposed to platelet-derived growth factor, indicating that this type of response is related to the stimulation of cell proliferation. These data characterize CXCR3 signaling in pericytes and clarify the relevance of downstream pathways in the modulation of different biologic responses. hepatic stellate cells epidermal growth factor extracellular signal-regulated kinase G protein-coupled receptors glutathione S-transferase-Ras-binding domain interferon-inducible protein-10 mitogen-activated protein kinase MAPK/ERK kinase glomerular mesangial cells monokine activated by interferon-γ platelet-derived growth factor phosphatidylinositol 3-kinase polyacrylamide gel electrophoresis In different tissues, the wound healing response shares many similarities, involving the recruitment of inflammatory cells and the deposition of extracellular matrix, to fill the gap created by the dying cells. The concurrent presence of inflammation and extracellular matrix deposition is a characteristic of chronic tissue injury, where the persistence of a wound healing response may lead to permanent scarring and end-stage organ failure, such as in the case of glomerulosclerosis in the kidney, cirrhosis of the liver, atherosclerosis, or pulmonary fibrosis (1Collins T. Robbin's Pathological Basis of Disease. 6th Ed. W. B. Saunders, Philadelphia1999: 50-88Google Scholar). The pivotal role played by vascular pericytes of different tissues in the process of wound healing has been clearly recognized in recent years. These cells become activated in the presence of damage to the specific tissue, proliferate, migrate, and acquire a myofibroblast-like phenotype, resulting in the production of extracellular matrix as part of the healing process. Hepatic stellate cells (HSC)1 and renal glomerular mesangial cells (MC) represent tissue-specific pericytes, which are deeply involved in the development of the wound healing response and in the pathogenesis of tissue fibrosis in the setting of a chronic damage (2Abboud H.E. Annu. Rev. Physiol. 1995; 57: 297-309Crossref PubMed Scopus (105) Google Scholar, 3Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar). Understanding the biology of these cells may help to devise novel strategies for the treatment of chronic liver and kidney diseases. The chemokine system has received considerable attention for its involvement in a number of biologic processes. Although members of this family have been initially recognized for their ability to recruit leukocytes to sites of injury, more recent investigation has shown that this system participates in the regulation of a wide number of conditions, including physiologic leukocyte homeostasis, development, angiogenesis, cancer, and the response to infection (4Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar, 5Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2109) Google Scholar). Activation of the chemokine system has been reported in the presence of chronic inflammation and fibrosis, two processes that are part of the wound healing response (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar, 7Marra F. DeFranco R. Grappone C. Milani S. Pastacaldi S. Pinzani M. Romanelli R.G. Laffi G. Gentilini P. Am. J. Pathol. 1998; 152: 423-430PubMed Google Scholar). In addition, several studies have shown that the pericytes responsible for tissue fibrosis may both express chemokines and be targets of the action of chemokines (8Gharaee-Kermani M. Denholm E.M. Phan S.H. J. Biol. Chem. 1996; 271: 17779-17784Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 9Wang X. Yue T.L. Ohlstein E.H. Sung C.P. Feuerstein G.Z. J. Biol. Chem. 1996; 271: 24286-24293Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 10Schecter A.D. Rollins B.J. Zhang Y.J. Charo I.F. Fallon J.T. Rossikhina M. Giesen P.L. Nemerson Y. Taubman M.B. J. Biol. Chem. 1997; 272: 28568-28573Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Marra F. Romanelli R.G. Giannini C. Failli P. Pastacaldi S. Arrighi M.C. Pinzani M. Laffi G. Montalto P. Gentilini P. Hepatology. 1999; 29: 140-148Crossref PubMed Scopus (227) Google Scholar). In fact, pericytes express chemokine receptors, which, upon activation, elicit biologic actions that favor the wound healing process, including proliferation, migration, and extracellular matrix synthesis (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar, 12Banas B. Luckow B. Moller M. Klier C. Nelson P.J. Schadde E. Brigl M. Halevy D. Holthofer H. Reinhart B. Schlondorff D. J. Am. Soc. Nephrol. 1999; 10: 2314-2322Crossref PubMed Google Scholar,13Schecter A.D. Calderon T.M. Berman A.B. McManus C.M. Fallon J.T. Rossikhina M. Zhao W. Christ G. Berman J.W. Taubman M.B. J. Biol. Chem. 2000; 275: 5466-5471Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The chemokine receptor CXCR3 is bound with high affinity by the chemokines interferon-inducible protein-10 (IP-10), monokine activated by interferon-γ (Mig), and interferon-inducible T cell α chemoattractant (14Piali L. Weber C. LaRosa G. Mackay C.R. Springer T.A. Clark-Lewis I. Moser B. Eur. J. Immunol. 1998; 28: 961-972Crossref PubMed Scopus (199) Google Scholar, 15Cole K.E. Strick C.A. Paradis T.J. Ogborne K.T. Loetscher M. Gladue R.P. Lin W. Boyd J.G. Moser B. Wood D.E. Sahagan B.G. Neote K. J. Exp. Med. 1998; 187: 2009-2021Crossref PubMed Scopus (734) Google Scholar). CXCR3 has been identified on several cells of hematopoietic lineage, including T and B lymphocytes, and natural killer cells (4Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar, 16Loetscher M. Gerber B. Loetscher P. Jones S.A. Piali L. Clark-Lewis I. Baggiolini M. Moser B. J. Exp. Med. 1996; 184: 963-969Crossref PubMed Scopus (1066) Google Scholar). In addition, expression of CXCR3 has been indicated as a marker of polarization of the T helper subset of T cells toward a Th1 phenotype (17Bonecchi R. Bianchi G. Bordignon P.P. D'Ambrosio D. Lang R. Borsatti A. Sozzani S. Allavena P. Gray P.A. Mantovani A. Sinigaglia F. J. Exp. Med. 1998; 187: 129-134Crossref PubMed Scopus (1845) Google Scholar). We have recently reported that CXCR3 is expressed by human MC in culture, where CXCR3 agonists induce an increase in cell proliferation, and the expression of CXCR3 on MC is up-regulated in conditions of chronic glomerular damage, indicating a possible involvement in wound healing and repair (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar). Despite the relevance of this system in a number of pathophysiologic conditions, little information is available on the signal transduction pathways activated by CXCR3 and their possible correlation with the biologic actions elicited by its agonists. In this study we have characterized CXCR3's signaling in HSC and in MC, as paradigm of vascular pericytes belonging to different tissues. We report that CXCR3 activates multiple signaling pathways, including the Ras/ERK pathway, Src, and the PI3K/Akt pathway, which correlate with the ability to induce biologic actions in target cells. Human recombinant IP-10, Mig, EGF, and PDGF-BB were purchased from Peprotech (Rocky Hill, NJ). Monoclonal antibodies against CXCR3 were from R&D Systems (Minneapolis, MN). Phospho-specific antibodies against the activated form of ERK, MEK, Raf-1, and Akt (Ser-473), and anti-MEK antibodies were from New England BioLabs (Beverly, MA). Polyclonal Anti-ERK antibodies used for Western blotting, polyclonal anti-Akt antibodies, and rat monoclonal anti-Ras antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Agarose-conjugated anti-phosphotyrosine antibodies were from Oncogene Science (Uniondale, NY). Polyclonal anti-ERK antibodies used for immune complex kinase assay, phospho-specific antibodies against activated Src (Y416), and monoclonal anti-Src antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY). For anti-phosphotyrosine immunoblotting, a mixture of PY99 (Santa Cruz Biotechnology) and 4G10 (Upstate Biotechnology Inc.) monoclonal antibodies was used. PD98059, AG1478, wortmannin, and genistein were purchased from Calbiochem (La Jolla, CA). PP1 was from Biomol (Plymouth Meeting, PA). Radionuclides were purchased from ICN (Costa Mesa, CA). All other reagent were of analytical grade. Human HSC were isolated from wedge sections of liver tissue unsuitable for transplantation by collagenase/pronase digestion and centrifugation on Stractan gradients. Procedures used for cell isolation and characterization have been extensively described elsewhere (18Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar). All the experiments were conducted on cells cultured on uncoated plastic dishes (passage 3–6), showing an "activated" or "myofibroblast-like" phenotype. Cultures of MC were obtained from macroscopically normal kidneys of patients with localized renal tumors undergoing nephrectomy, as described previously (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar, 19Pupilli C. Lasagni L. Romagnani P. Bellini F. Mannelli M. Misciglia N. Mavilia C. Vellei U. Villari D. Serio M. J. Am. Soc. Nephrol. 1999; 10: 245-255PubMed Google Scholar). The cortex was separated from the medulla and minced; glomeruli were isolated by a standard sieving technique through graded mesh size screens (60, 80, 150 mesh). The glomerular suspension was collected, washed with RPMI 1640 (Sigma), and incubated with 750 units/ml collagenase type IV at 37 °C for 30 min. The glomeruli were then cultured in RPMI 1640 with 17% fetal calf serum and other supplements to obtain MC (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar). Cultured glomeruli were maintained in a humidified environment of 5% CO2/95% air at 37 °C, and the medium was changed three times a week. MC were used between passages 4 and 7. After saturation of nonspecific binding sites with total rabbit IgG, cells were incubated for 20 min on ice with specific or isotype control antibody. In the indirect staining, this step was followed by a second incubation on ice with an appropriate anti-isotype-conjugated antibody (Southern Biotechnology Associates, Birmingham, AL). Finally, cells were washed and analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson, San Jose, CA). In all cytofluorimetric analyses, a total of 104 events for each sample was acquired. Confluent HSC or MC were washed with phosphate-buffered saline and incubated in serum-free medium for 48 h. The cells were incubated with agonists for 24 or 36 h and then pulsed with [3H]thymidine. DNA synthesis was measured as the incorporation of tritiated thymidine, as described elsewhere (20Pinzani M. Gesualdo L. Sabbah G.M. Abboud H.E. J. Clin. Invest. 1989; 84: 1786-1793Crossref PubMed Scopus (427) Google Scholar). Confluent HSC or MC were serum-starved for 48 h and then washed, trypsinized, and resuspended in serum-free medium containing 1% albumin at a concentration of 3 × 105 cells/ml. Chemotaxis was measured in modified Boyden chambers equipped with 8-μm pore filters (Poretics, Livermore, CA) coated with rat tail collagen (Collaborative Biomedical Products, Bedford, MA), as previously described (21Marra F. Efsen E. Romanelli R.G. Caligiuri A. Pastacaldi S. Batignani G. Bonacchi A. Caporale R. Laffi G. Pinzani M. Gentilini P. Gastroenterology. 2000; 119: 466-478Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). When inhibitors were used, cultured cells were incubated with the drugs to be tested or with their vehicle for 15 min before trypsinization, and equal concentrations were added in the Boyden chamber. Confluent, serum-starved HSC or MC were treated with the appropriate conditions, quickly placed on ice, and washed with ice-cold phosphate-buffered saline. Except for the analysis of Ras activation (see below), the monolayer was lysed in radioimmune precipitation buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 0.05% (w/v) aprotinin). Insoluble proteins were discarded by high speed centrifugation at 4 °C. Protein concentration in the supernatant was measured in triplicate using a commercially available assay (Pierce, Rockford, IL). Equal amounts of total cellular proteins were separated by SDS-PAGE and analyzed by Western blot as previously described (21Marra F. Efsen E. Romanelli R.G. Caligiuri A. Pastacaldi S. Batignani G. Bonacchi A. Caporale R. Laffi G. Pinzani M. Gentilini P. Gastroenterology. 2000; 119: 466-478Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Immunoblot analysis of EGF receptor tyrosine phosphorylation was conducted after immunoprecipitation. Briefly, 150 μg of total cellular proteins were incubated with anti-EGF receptor antibodies and protein A-Sepharose for 2 h at 4 °C. The Immunobeads were washed twice in lysis buffer and once in 20 mm Tris-HCl, pH 7.4, 1 mmNa3VO4, resuspended in Laemmli buffer, and analyzed by Western blot as described above. ERK was immunoprecipitated from 25 μg of total cell lysate using polyclonal anti-ERK antibodies and protein A-Sepharose. After washing, the immunobeads were incubated in a buffer containing 10 mm HEPES, pH 7.4, 10 mmMgCl2, 0.5 mm dithiothreitol, 0.5 mm Na3VO4, 25 μm ATP, 1 μCi of [γ-32P]ATP, and 0.4 mg/ml myelin basic protein for 30 min at 30 °C. At the end of the incubation, the reaction was stopped by addition of Laemmli buffer and run on 15% SDS-PAGE. After electrophoresis, the gel was dried and autoradiographed. This assay was performed after immunoprecipitation with anti-phosphotyrosine antibodies, as described elsewhere (22Choudhury G.G. Wang L.M. Pierce J. Harvey S.A. Sakaguchi A.Y. J. Biol. Chem. 1991; 266: 8068-8072Abstract Full Text PDF PubMed Google Scholar, 23Marra F. Choudhury G.G. Abboud H.E. J. Clin. Invest. 1996; 98: 1218-1230Crossref PubMed Scopus (71) Google Scholar). Radioactive lipids were separated by thin-layer chromatography, using chloroform/methanol/30% ammonium hydroxide/water (46/41/5/8, v/v). After drying, the plates were autoradiographed. The radioactive spots were then scraped and counted in a β-counter. Activation of Ras in response to CXCR3 ligands was determined exactly as described by deRooij and Bos (24deRooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). The plasmid encoding for the GST-RBD fusion protein (kindly provided by Dr. J. L. Bos) was expressed in bacteria and used to obtain recombinant GST-RBD, as described elsewhere (24deRooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). Serum-deprived HSC exposed to different conditions were lysed in a buffer containing 50 mm Tris, 150 mm NaCl, 0.5% deoxycholase, 1% Nonidet P-40, 0.1% SDS, and protease inhibitors. One milligram of protein was incubated with recombinant GST-RBD and glutathione-agarose beads (Sigma), washed, and analyzed by 15% SDS-PAGE followed by anti-Ras immunoblotting. Activation of Src was analyzed by immune complex kinase assay as described by Ishida et al. (25Ishida M. Marrero M.B. Schieffer B. Ishida T. Bernstein K.E. Berk B.C. Circ. Res. 1995; 77: 1053-1059Crossref PubMed Scopus (167) Google Scholar), with minor modifications. Protein (200 μg) in radioimmune precipitation buffer was incubated with monoclonal anti-Src antibodies and precipitated by the addition of rabbit anti-mouse IgG and protein A-Sepharose. After washing with radioimmune precipitation buffer, the immunobeads were incubated with a buffer containing 20 mmHepes, pH 7.0, 10 mm MnCl2, 20 μg of enolase (Sigma), and 5 μCi of [γ-32P]ATP for 10 min at 30 °C. At the end of the incubation, the reaction was stopped with Laemmli buffer and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie Blue, and the band corresponding to enolase was cut and counted in a β-counter. An immune complex kinase assay of Akt activity was performed as described elsewhere (26Robino G. Parola M. Marra F. Caligiuri A. DeFranco R.M. Zamara E. Bellomo G. Gentilini P. Pinzani M. Dianzani M.U. J. Biol. Chem. 2000; 275: 40561-40567Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, 100 μg of protein was immunoprecipitated using anti-Akt antibodies and protein G-agarose. The Immunobeads were washed three times with washing buffer (20 mm HEPES (pH 7.5), 40 mm NaCl, 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40, 20 mm β-glycerophosphate, 0.5 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin). The assay was performed by resuspending the beads in kinase buffer (50 mm HEPES (pH 7.5), 100 mm NaCl, 10 mm MgCl2, 10 mm MnCl2, 10 mmβ-glycerophosphate, and 0.5 mm sodium orthovanadate) in the presence of 1 μm protein kinase A inhibitor peptide, 50 μm unlabeled ATP, and 6 μCi of [γ-32P]ATP, using exogenous histone H2B (1.5 μg/assay tube) as the substrate and incubating for 20 min at room temperature. The proteins in the samples were resolved by 12% SDS-PAGE, and the gel was stained with Coomassie Blue and subjected to autoradiography. Data presented herein are representative of at least three experiments with comparable results. Unless otherwise indicated, bar graphs show the mean ± S.D. of data from a representative experiment. Because CXCR3 expression has been previously reported in MC (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar), we first investigated by flow cytometry whether human HSC, as liver-specific pericytes, express this chemokine receptor. As compared with the cells treated with isotype-specific control antibody, anti-CXCR3 antibodies induced a clear shift in fluorescence, indicating CXCR3 expression on the cell surface (Fig.1 A). Expression of the receptor by human HSC was also confirmed by Western blotting (data not shown). To test whether CXCR3 expressed by HSC is functional, we assessed the effects of IP-10 on HSC migration, because activation of chemokine receptors is often associated with the induction of chemotaxis. Exposure of HSC to increasing concentrations of IP-10 resulted in a dose-dependent increase in cell migration, which was 3- to 4-fold greater than that observed in unstimulated cells (Fig. 1 B). When IP-10 (100 ng/ml) was added to both the upper and lower chamber of the Boyden system, HSC migration was similar to that of unstimulated cells, indicating that the effects of IP-10 are dependent on chemotaxis rather than on chemokinesis (data not shown). CXCR3 may be activated by three ligands, including Mig, therefore, we compared the effects of IP-10 and Mig to confirm the role of CXCR3 in the IP-10-induced cell migration. Both agonists were equally effective in their ability to induce cell migration (Fig. 1 C). In addition, the chemotactic activity was shown to peak at a concentration of 100 ng/ml for both ligands (Fig. 1 C and data not shown). We and others have previously reported that ligands of CXCR3 stimulate proliferation of vascular pericytes, including MC (6Romagnani P. Beltrame C. Annunziato F. Lasagni L. Luconi M. Galli G. Cosmi L. Maggi E. Salvadori M. Pupilli C. Serio M. J. Am. Soc. Nephrol. 1999; 10: 2518-2526PubMed Google Scholar) and smooth muscle cells (9Wang X. Yue T.L. Ohlstein E.H. Sung C.P. Feuerstein G.Z. J. Biol. Chem. 1996; 271: 24286-24293Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Therefore, we examined the effects of CXCR3 ligands on DNA synthesis in HSC. However, neither IP-10 nor Mig, when tested at concentrations as high as 1 μg/ml, resulted in an increase in DNA synthesis, whereas the cells were clearly responsive to known mitogens, such as PDGF (Fig. 1 D). These results indicate that HSC express functional CXCR3 receptors on the cells surface and that activation of this chemokine receptor induces migration, but not proliferation, of this cell type. Despite the relevance of CXCR3 in many pathophysiologic processes, no information on the signal transduction pathways activated by this chemokine receptor is presently available. Using HSC as a model system of cells constitutively expressing this receptor, we explored the intracellular signaling pathways activated by CXCR3. We first focused on the Ras/ERK pathway, which is activated in response to many soluble factors, including chemokines (27Venkatakrishnan G. Salgia R. Groopman J. J. Biol. Chem. 2000; 275: 6868-6875Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), and regulates different cellular functions. Exposure of HSC to IP-10 for different periods of time resulted in increased phosphorylation of both ERK isoforms, p44ERK-1and p42ERK-2, as assessed by using antibodies that specifically recognize the phosphorylated forms of ERK (Fig.2 A). Peak activation was observed at 15–30 min after addition of IP-10 and returned to basal levels within 4 h after stimulation. At later time points, no ERK phosphorylation could be detected (Fig. 2 A, and data not shown). Concentrations of IP-10 higher than 100 ng/ml did not result in a further increase in ERK phosphorylation (Fig. 2 B). We also tested whether increased phosphorylation of ERK was indeed associated with increased catalytic activity using an immune complex kinase assay. Increased phosphorylation of the substrate myelin basic protein was observed at the same time points where increased ERK phosphorylation was detected (Fig. 2 C). To demonstrate that the effects of IP-10 on ERK are due to CXCR3 activation, similar experiments were conducted using Mig as an agonist. Also in this case, a transient activation of ERK was observed, with a similar time course as that observed in cells exposed to IP-10 (Fig. 2 D). Taken together, these data indicate that interaction between CXCR3 and its ligands leads to activation of ERK. Similar to other members of the mitogen-activated protein kinase (MAPK) family, ERK activation results from the sequential activation of a small G protein of the Ras superfamily, and a kinase cascade involving a MAPK kinase kinase and a MAPK kinase (28Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar). MEK-1/2 is the MAPK kinase responsible for ERK activation. Therefore we explored the effects of IP-10 on the phosphorylation of MEK. At the same time points in which activation of ERK was observed, IP-10 caused a clear increase in the amount of phosphorylated MEK, similarly to PDGF, a known activator of the Ras/ERK pathway (Fig. 3 A). Of note, activation of MEK was associated with activation of the upstream kinase Raf-1, which acts as a MAPK kinase kinase in the Ras/ERK cascade (Fig. 3 B). Finally, we tested whether exposure of HSC to a CXCR3 ligand was associated with increased activation of Ras. We used a fusion protein (GST-RBD) comprising the Ras-binding domain of Raf-1 to selectively precipitate active Ras, which physically interacts with this domain of Raf-1 (24deRooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). In unstimulated cells, active Ras was barely detectable as a doublet migrating at 21 kDa (Fig. 3 C). Exposure of the cells either to IP-10 or to PDGF, used as a positive control, resulted in a greater amount of precipitated Ras, indicating increased activation. Upon activation of G protein-coupled receptors, several mechanisms may be involved in the activation of the Ras/ERK pathway, including nonreceptor tyrosine kinases and phosphatidylinositol 3-kinase (PI3K). Because the nonreceptor tyrosine kinase Src has been implicated in the downstream signaling of different G protein-coupled receptors, we explored the ability of IP-10 to activate Src. CXCR3 activation by IP-10 was accompanied by a marked increase in Src phosphorylation on the activation-specific tyrosine 416 (Fig. 4 A) (29Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar), indicating that this receptor activates Src. To confirm that increased phosphorylation of Src was associated with enhanced kinase activity, we performed immune complex kinase assays using immunoprecipitated Src and enolase as a substrate. Also in this condition, an increase in Src activity was observed, which was of the same magnitude as that induced by PDGF, a known activator of this pathway (Fig. 4 B). Because cell migration may be critically regulated by activation of PI3K, we also tested whether the activity of this kinase could be modified by CXCR3 ligands. PI3K activity associated with anti-phosphotyrosine immunoprecipitates was increased by 3-fold in cells exposed to IP-10 (Fig.5 A) and declined thereafter. Activation of c-Akt, also known as protein kinase B, has been recognized as a pathway that lies downstream of PI3K activation (30Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar). Akt phosphorylation on the activation-specific residue Thr-473 (30Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar), was markedly increased in HSC exposed to IP-10, similarly to cells treated with PDGF, a known activator of Akt (Fig. 5 B). Moreover, activation of CXCR3 increased the catalytic activity of Akt, as assessed by immune complex kinase assay using PDGF as a positive control (Fig. 5 C) (31Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1670) Google Scholar). Taken together, these data indicate that CXCR3 activation leads to increased activity of Src and of the PI3K/Akt pathway. To establish which pathways are critical for the CXCR3-mediated activation of ERK, we treated HSC with specific inhibitors of different signaling pathways before adding IP-10. Tyrosine kinases, including Src, and PI3K have both been shown to be implicated in the activation of Ras and the ERK cascade by GPCR (32Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 33Hawes B.E. Luttrell L.M. van Biesen T. Lefkowitz R.J. J. Biol. 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