CXCR4-mediated Suppressor of Cytokine Signaling Up-regulation Inactivates Growth Hormone Function
2004; Elsevier BV; Volume: 279; Issue: 43 Linguagem: Inglês
10.1074/jbc.m408010200
ISSN1083-351X
AutoresRuth Garzón, Silvia F. Soriano, José Miguel Rodríguez‐Frade, Lucio Gómez, Ana Martı́n de Ana, Myriam Sánchez-Gómez, Carlos Martínez‐A, Mario Mellado,
Tópico(s)Immune Cell Function and Interaction
ResumoCoordinated action between cytokines and chemokines is required for effective endocrine and immune responses. Proteins of both families promote receptor oligomerization, activation of the Janus kinase (JAK)/STAT pathway, and transcription of many genes, including the suppressor of cytokine signaling (SOCS) family. In this study, we show that chemokine-mediated SOCS1 and SOCS3 up-regulation modulates the signaling and function associated to a cytokine receptor, both in vitro and in vivo. The effect is mediated by SOCS binding to JAK2 and to the cytokine receptor, which blocks subsequent signaling events. The data reinforce the premise of cytokine-chemokine cross-talk, which helps contribute to modulating individual responses and in defining the functional plasticity of the immune system. Coordinated action between cytokines and chemokines is required for effective endocrine and immune responses. Proteins of both families promote receptor oligomerization, activation of the Janus kinase (JAK)/STAT pathway, and transcription of many genes, including the suppressor of cytokine signaling (SOCS) family. In this study, we show that chemokine-mediated SOCS1 and SOCS3 up-regulation modulates the signaling and function associated to a cytokine receptor, both in vitro and in vivo. The effect is mediated by SOCS binding to JAK2 and to the cytokine receptor, which blocks subsequent signaling events. The data reinforce the premise of cytokine-chemokine cross-talk, which helps contribute to modulating individual responses and in defining the functional plasticity of the immune system. Growth hormone (GH), 1The abbreviations used are: GH, growth hormone; GHR, GH receptor; hGH, human GH; hGHR, human GHR; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SOCS, suppressor of cytokine signaling; IGF-1, insulin-like growth factor-1; IGFBP, IGF-1-binding proteins; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; HSC, hematopoietic stem cells. secreted by the anterior pituitary gland into the circulation, has been long considered a pleiotropic molecule. It is the only hormone that stimulates somatic growth in a dose-dependent manner by promoting longitudinal bone growth (1Merimee T.J. Russell B. Quinn S. Riley W. J. Clin. Endocrinol. Metab. 1991; 73: 1031-1037Crossref PubMed Scopus (48) Google Scholar, 2Olney R.C. Med. Pediatr. Oncol. 2003; 41: 228-234Crossref PubMed Scopus (102) Google Scholar). GH is also involved in regulating metabolism, in transcription control and in cellular proliferation and differentiation (3Richter H.E. Albrektsen T. Billestrup N. J. Mol. Endocrinol. 2003; 30: 139-150Crossref PubMed Scopus (22) Google Scholar, 4Rudling M. Parini P. Angelin B. Growth Horm. IGF Res. 1999; 9: 1-7Crossref PubMed Scopus (21) Google Scholar). In addition to the somatotropic cells of the pituitary gland, many other cells produce GH, including hematopoietic and lymphoid cells (5Gagnerault M.C. Postel-Vinay M.C. Dardenne M. Endocrinology. 1996; 137: 1719-1726Crossref PubMed Scopus (67) Google Scholar). This suggests that, besides its endocrine function, GH may act in lymphoid tissues as an ancillary cytokine in an autocrine/paracrine manner (6Jeay S. Sonenshein G.E. Postel-Vinay M.C. Kelly P.A. Baixeras E. Mol. Cell. Endocrinol. 2002; 188: 1-7Crossref PubMed Scopus (107) Google Scholar). Indeed, bovine growth hormone-transgenic mice show reduced B cell lymphopoiesis, reduced myelopoiesis in fetal liver, and virtual absence of myelopoiesis in bone marrow (7Gonzalo J.A. Mazuchelli R. Mellado M. Frade J.M. Carrera A.C. von Kobbe C. Merida I. Martínez A.C. J. Immunol. 1996; 157: 3298-3304PubMed Google Scholar). Growth hormone effects are mediated directly or indirectly through induction and release of insulin-like growth factor-1 (IGF-1) (8Woelfle J. Chia D.J. Rotwein P. J. Biol. Chem. 2003; 278: 51261-51266Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). GH exerts its biological effects by binding to its receptor (GHR), a member of the cytokine receptor superfamily (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar); this results in receptor dimerization and tyrosine phosphorylation of an associated Janus kinase (JAK2). Activated JAK2 in turn phosphorylates the intracellular domain of the GHR, and the GHR-JAK2 complex provides docking sites for a variety of signaling molecules of the signal transducer and activator of transcription family (STAT) (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 10Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell Signal. 2001; 13: 599-616Crossref PubMed Scopus (221) Google Scholar). GH activates mainly STAT5, and to a lesser degree, STAT3 and STAT1. After phosphorylation, the STATs form dimers, translocate to the nucleus, and bind to DNA, triggering gene transcription (11Herrington J. Smit L.S. Schwartz J. Carter-Su C. Oncogene. 2000; 19: 2585-2597Crossref PubMed Scopus (226) Google Scholar). Some cytokine actions differ, depending on signal persistence. JAK/STAT pathway activation is generally transient and cell type-dependent. Negative regulation is exerted by tyrosine phosphatases such as SHP-2 (12Yin T. Shen R. Feng G.S. Yang Y.C. J. Biol. Chem. 1997; 272: 1032-1037Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and by SOCS proteins, either by direct interaction with activated JAK or with cytokine receptors (13Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1135) Google Scholar, 14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (602) Google Scholar, 15Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1805) Google Scholar). JAK/STAT pathway activation is not limited to the cytokine receptor superfamily. SOCSs also have a regulatory role in other receptor systems, including the IGF-1 (16Dey B.R. Furlanetto R.W. Nissley P. Biochem. Biophys. Res. Commun. 2000; 278: 38-43Crossref PubMed Scopus (81) Google Scholar) and chemokine receptors (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar), suggesting that SOCS proteins may have wide-ranging effects on signaling cascades (18Chen X.P. Losman J.A. Rothman P. Immunity. 2000; 13: 287-290Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Chemokines comprise a large family of low molecular mass (8–10 kDa) cytokines, with chemotactic and proactivatory effects on different leukocyte lineages (19Mackay C.R. Nat. Immunol. 2001; 2: 95-101Crossref PubMed Scopus (715) Google Scholar). Several studies established the central role of chemokines in a number of physiological situations, including T helper cell responses, hematopoiesis, angiogenesis, and homeostasis, as well as in processes such as asthma, tumor rejection, human immunodeficiency virus-1 (HIV-1) infection, and arteriosclerosis (20Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1211) Google Scholar, 21Moser B. Loetscher P. Nat. Immunol. 2001; 2: 123-128Crossref PubMed Scopus (1035) Google Scholar, 22Luther S.A. Cyster J.G. Nat. Immunol. 2001; 2: 102-107Crossref PubMed Scopus (603) Google Scholar). CXCL12 was isolated from stromal cell culture supernatants (23Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1285) Google Scholar). Its chemotactic properties have been described in peripheral blood lymphocytes (24Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2016) Google Scholar), CD34+ progenitor cells (25Aiuti A. Webb I.J. Bleul C. Springer T.A. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 111-120Crossref PubMed Scopus (1205) Google Scholar), and pre- and pro-B cell lines (26D'Apuzzo M. Rolink A. Loetscher M. Hoxie J.A. Clark-Lewis I. Melchers F. Baggiolini M. Moser B. Eur. J. Immunol. 1997; 27: 1788-1793Crossref PubMed Scopus (284) Google Scholar). In contrast to proinflammatory chemokines, CXCL12 is constitutively produced in many organs, including human bone marrow, suggesting a major role for CXCL12/CXCR4 interactions in steady-state homeostatic processes such as controlling leukocyte trafficking and retaining undifferentiating and maturing hematopoietic cells within the bone marrow. Embryos of mice lacking either the CXCL12 protein (24Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (2016) Google Scholar) or its receptor CXCR4 (27Zou Y-R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2127) Google Scholar, 28Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Nishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-594Crossref PubMed Scopus (1323) Google Scholar) display multiple lethal defects, including abnormalities in B cell lymphopoiesis and bone marrow myelopoiesis, lack of blood vessel formation in the gut, severe ventricular septal defects, and altered cerebellar neuron migration (27Zou Y-R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2127) Google Scholar, 28Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Nishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-594Crossref PubMed Scopus (1323) Google Scholar, 29Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9448-9453Crossref PubMed Scopus (1428) Google Scholar). Chemokines mediate their biological effects by binding to specific receptors, members of the seven-transmembrane domain G protein-coupled receptor family. After binding, chemokines trigger receptor dimerization, association to, and activation of JAK in a process that is central to chemokine function. JAK in turn phosphorylates the chemokine receptors on tyrosine residues and activates STAT transcription factors (30Mellado M. Rodriguez-Frade J.M. Manes S. Martínez A.-C. Annu. Rev. Immunol. 2001; 19: 397-421Crossref PubMed Scopus (299) Google Scholar). We recently showed that CXCL12-induced STAT activation leads to SOCS3 up-regulation and interference with chemokine signaling and function (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). Here we show that GH-mediated JAK/STAT pathway activation is blocked in cells pretreated with chemokines. CXCL12-mediated up-regulation of SOCS1 and SOCS3 is linked to inhibition of GH-mediated responses both in vitro and in vivo. These data and previous observations are discussed in the context of cytokine-chemokine cross-talk, a two-way mechanism that modulates the biological functions of these protein families that coincide in many physiopathological settings. Biological Materials and Antibodies—IM9 cells were from the American Type Culture Collection (CCL159). Antibodies included anti-CXCR4 (CXCR4-01) (31Vila-Coro A.J. Rodríguez-Frade J.M. Martín De Ana A. Moreno-Ortíz M.C. Martínez A.-C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (442) Google Scholar) and anti-hGH receptor (hGHR-05) mAb generated in our laboratory (32Mellado M. Rodriguez-Frade J.M. Kremer L. Martínez A.-C. J. Clin. Endocrinol. Metab. 1996; 81: 1613-1618PubMed Google Scholar), rabbit anti-JAK2 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-PTyr (Promega, Madison, WI), and rabbit anti-STAT5b, goat anti-SOCS1, and rabbit anti-SOCS3 (Santa Cruz Biotechnology, Santa Cruz, CA). Recombinant hGH (Genotonorm) was from Amersham Biosciences. Flow Cytometry Analysis—Cells were centrifuged (250 × g, 10 min, room temperature), plated in V-bottom 96-well plates (2.5 × 105 cells/well), and incubated with biotin-labeled GH (1 μg/100 μl/well) followed by fluorescein isothiocyanate (FITC)-labeled streptavidin (Southern Biotechnology) (30 min, 4 °C). When mAbs were used, cells were incubated as above with biotin-hGHR-05 (1 μg/50 μl/well, 60 min, 4 °C) or biotin-CXCR4-01 (1 μg/50 μl/well). Cells were washed twice in PBS with 2% bovine serum albumin and 2% fetal calf serum and then centrifuged (250 × g, 5 min, 4 °C). FITC-labeled streptavidin was added (30 min, 4 °C), and cell-bound fluorescence was determined in a Profile XL flow cytometer at 525 nm (Coulter, Miami, FL). When necessary, cell cycle status was measured by cellular DNA staining with propidium iodide followed by cytometric determination as above. Immunoprecipitation, SDS-PAGE, and Western Blot Analysis—After serum depletion (60 min, 37 °C in RPMI containing 0.1% bovine serum albumin), IM9 cells were CXCL12-treated (50 nm, 60 min, 37 °C) prior to stimulation with GH (10 μg/ml). Cells were lysed in detergent buffer (10 mm triethanolamine, pH 8.0, 150 mm NaCl, 1 nm EDTA, 10% glycerol, 2% digitonin, with 10 μm sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin; 30 min, 4 °C, continuous rocking) and then centrifuged (15,000 × g, 15 min). Immunoprecipitation was as described (31Vila-Coro A.J. Rodríguez-Frade J.M. Martín De Ana A. Moreno-Ortíz M.C. Martínez A.-C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (442) Google Scholar). Precipitates or protein extracts were separated in SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was as described (31Vila-Coro A.J. Rodríguez-Frade J.M. Martín De Ana A. Moreno-Ortíz M.C. Martínez A.-C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (442) Google Scholar), using 3% bovine serum albumin in Trissaline buffer as a blocking agent for anti-phosphotyrosine analyses. For stripping, membranes were incubated (60 min, 60 °C) with 62.5 mm Tris-HCl, pH 7.8, containing 2% SDS and 0.5% β-mercaptoethanol. Protein loading was controlled using a protein detection kit (Pierce) and by reprobing the membrane with the immunoprecipitating antibody. When necessary, splenocytes from untreated and CXCL12-treated mice were extracted and activated in vitro with GH (10 μg/ml, 37 °C) for different periods of time before processing. In Vivo Assays to Determine the GH Biological Activity—One-month-old BALB/c mice received an intrasplenic injection of CXCL12 (1 μg in 75 μl of sterile PBS) or PBS. Mice were sacrificed 60 min after injection, and splenocytes were extracted and activated in vitro with GH (10 μg/ml, 37 °C). In other experiments, mice received an intravenous injection of CXCL12 (1 μg in 200 μl of sterile PBS) or PBS. After 60 min, mice received an intravenous injection of GH (0.1 μgin100 μl of sterile PBS). Blood, spleen, and liver were extracted at different times for later analysis. Quantification of IGF-1 Serum Levels—Blood was collected at different times after GH injection from the retro-orbital sinus of mice stimulated as above. After acid-ethanol extraction of IGF-1-binding proteins (IGFBP) (33Daughaday W.H. Mariz I.K. Blethen S.L. J. Clin. Endocrinol. Metab. 1980; 51: 781-788Crossref PubMed Scopus (702) Google Scholar), serum IGF-1 levels were measured in a radioimmunoassay using polyclonal anti-IGF-1 antibody (Gropep, Adelaide, Australia) and rhIGF-1 (Amersham Biosciences and Kabi Peptide Hormones, Stockholm, Sweden) as a standard. To avoid interference from the remaining IGFBP-coupled IGF after extraction, a truncated 125I-des(1–3) IGF-1 (Amersham Biosciences and Kabi Peptide Hormones) was used as tracer (34Bang P. Eriksson U. Sara V. Wivall I.L. Hall K. Acta Endocrinol. (Copenh.). 1991; 124: 620-629Crossref PubMed Scopus (303) Google Scholar). Northern Blot Analysis—Total spleen and liver RNA from 4-week-old BALB/c mice, treated with CXCL12 (1 μg/ml, 60 min) or PBS, were extracted using Tri reagent (Sigma). RNA samples were resolved on denaturing formaldehyde-agarose gels and transferred to nylon membrane (Hybond N+; Amersham Biosciences). Membranes were hybridized with 32P-labeled cDNA from pEF-FLAG-I/mSOCS1, /mSOCS3, and β-actin (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). In some cases, mice received intravenous injections of CXCL12 (1 μg in 200 μl of sterile PBS) or PBS followed by an intravenous injection of GH (0.1 μg in 100 μl of sterile PBS); spleen and liver were extracted, and total RNA was processed as above. Membranes were hybridized with 32P-labeled cDNA from pGEM-mIGF-1 exon 4, pGEM-rIGFBP3, and β-actin. Data were quantified using ImageJ software and expressed as a percentage of the optical density (OD) of the specific band as compared with that of controls. IGFBP3 Analysis by Ligand Blot—Mice received CXCL12 or PBS injections as above followed 60 min later by an intravenous injection of GH (0.1 μg in 100 μl of sterile PBS); 1 h later, they were bled from the retro-orbital sinus, and serum IGFBP-3 was analyzed in Western ligand blot as described (35Hossenlopp P. Seutin D. Segovia-Quinson B. Hardouin S. Binoux M. Anal. Biochem. 1986; 154: 138-143Crossref PubMed Scopus (1199) Google Scholar). In brief, 1.5 μl of serum was treated with 2× SDS loading buffer containing Tris (0.5 m, pH 6.8), glycerol (68% v/v), and SDS (2% w/v) and then electrophoresed on 15% SDS-polyacrylamide gels under non-reducing conditions. Proteins were transferred to nitrocellulose and treated sequentially with Tris (0.1 m) pH 7.4 buffer containing NaCl (0.15 m), 3% (v/v) Nonidet P-40, and 1% (w/v) bovine serum albumin and then probed overnight with 125I-labeled IGF-1 and IGF-II (106 cpm each/sheet). After extensive washing in buffer containing 0.1% (v/v) Tween 20, the nitrocellulose was autoradiographed with Kodak X-omat AR film (4–8 days, –70 °C). Human serum (1.5 μl) was included as a positive control. When necessary, data were quantified using ImageJ software and expressed as the area of the spot (arbitrary units). CXCL12 Modulates GH-mediated Function and Affects Circulating IGF-1 Levels—GH binding to its target cell triggers production of IGF-1 (8Woelfle J. Chia D.J. Rotwein P. J. Biol. Chem. 2003; 278: 51261-51266Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), the main mediator of GH function. We observed that an intravenous injection of GH (1 μg/ml) into 4-week-old BALB/c mice triggered an increase in serum IGF-1 levels, as determined by radioimmunoassay (maximum effect at 60 min after GH injection) (Fig. 1A). The data concur with up-regulation of IGF-1 mRNA levels in the liver of GH-treated animals (Fig. 1B). Nonetheless, animals that received an intravenous injection of the chemokine CXCL12 (50 nm) 60 min prior to GH treatment showed neither IGF-1 mRNA up-regulation nor modification of circulating IGF-1 levels (Fig. 1, A and B). GH is reported to increase circulating IGFBP-3 levels (36Beauloye V. Muaku S.M. Lause P. Portetelle D. Renaville R. Robert A.R. Ketelslegers J.M. Maiter D. Am. J. Physiol. 1999; 277: E308-E315PubMed Google Scholar); CXCL12 pretreatment also abrogated GH-induced up-regulation of IGFBP-3, as shown by the determination of IGFBP-3 mRNA levels in the liver of treated mice (Fig. 1C). This was confirmed by ligand blot analysis of sera from untreated and CXCL12-treated mice (Fig. 1D). We previously showed GH-mediated modulation of chemokine responses based on SOCS3 up-regulation (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). SOCS3 binding to CXCR4 blocks activation of the CXCR4-triggered signaling cascade and its function. To evaluate whether a similar mechanism could explain the effect of CXCL12 on GH function, we used the human B cell line IM9, which endogenously co-expresses CXCR4 and GHR as analyzed by flow cytometry with biotin-anti-CXCR4 or -GHR mAb followed by FITC-streptavidin (Fig. 2A).Fig. 2CXCL12 treatment alters GH-mediated early signaling events in IM9 cells. A, CXCR4 and GHR levels were measured by flow cytometry in IM9 cells incubated with biotin-labeled hGHR-05 or CXCR4 mAb followed by FITC-streptavidin. mAb binding is compared with that of negative controls. B, untreated or CXCL12-treated IM9 cells were GH-stimulated for the time indicated and then lysed. Lysates were immunoprecipitated with anti-PTyr, and the Western blot was developed with anti-JAK2 antibody. Mr, molecular mass; i.s., intrasplenic. C, IM9 cell lysates as in B were immunoprecipitated with anti-PTyr and developed with anti-STAT5b antibody.View Large Image Figure ViewerDownload (PPT) CXCL12 Treatment Alters GH-mediated Early Signaling Events—GH-induced SOCS3 up-regulation is mediated by activation of the JAK2/STAT5 pathway (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). We evaluated the effect of CXCL12 treatment on GH-mediated JAK2/STAT5 activation in IM9 cells. Untreated or CXCL12-treated (50 nm, 60 min, 37 °C) IM9 cells were activated with GH (10 μg/ml, 37 °C). Cells were lysed, immunoprecipitated with anti-PTyr polyclonal antibody, and analyzed in Western blot with anti-JAK2 antibody (Fig. 2B) or precipitated with anti-JAK2 and developed with anti-PTyr polyclonal antibody (not shown). GH induced a rapid tyrosine phosphorylation of JAK2, which was completely abrogated by CXCL12 pretreatment (Fig. 2B). To confirm that CXCL12 affects the entire GH-induced JAK/STAT pathway, cells as above were immunoprecipitated with anti-pTyr antibody and analyzed in Western blot with anti-STAT5 antibodies. As predicted, GH-induced STAT5 phosphorylation was abrogated in CXCL12-pretreated cells (Fig. 2C). Our data thus indicate that CXCL12 treatment blocks GH-mediated signaling events; together with previous observations (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar), this suggests that chemokine-cytokine interference is a two-way phenomenon. CXCL12 treatment modifies neither GHR levels nor GH binding, as shown by flow cytometry analysis using biotin-GH followed by FITC-streptavidin (Fig. 3A). Cytokine activation of the JAK/STAT pathway leads to up-regulation of SOCS, a protein family implicated in the negative feedback of cytokine and chemokine signaling (13Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1135) Google Scholar, 14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (602) Google Scholar, 15Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1805) Google Scholar, 17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). We thus measured SOCS up-regulation in CXCL12-treated IM9 cells. Northern blot analysis of CXCL12-stimulated IM9 cells showed SOCS1 and SOCS3 up-regulation after chemokine treatment (Fig. 3B) (maximum at 60 min); β-actin hybridization was used as a loading control. We studied the mechanism by which CXCL12-mediated SOCS up-regulation interferes in GH signaling and function. The SOCS block cytokine signaling via interactions with JAK2 (SOCS1) and/or the cytoplasmic tail of GHR (cytokine-inducible SH2 protein and SOCS3) (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Untreated and CXCL12-treated (60 min, 37 °C) IM9 cells were activated with GH (10 μg/ml, 37 °C) as indicated and then lysed. Cell extracts were immunoprecipitated with anti-JAK2 and blotted with anti-SOCS1 antibodies. Results showed that CXCL12 treatment up-regulated SOCS1 expression (Fig. 3B) and promoted its association to JAK2 (Fig. 3C). Protein loading equivalence was controlled with a protein detection kit (Pierce). Notably, JAK2/SOCS1 association did not increase after GH activation, suggesting that this complex may not require further JAK2 activation (Fig. 3C). Further, untreated and CXCL12-treated (60 min, 37 °C) IM9 cells were activated with GH as above before lysis. Cell extracts were immunoprecipitated with anti-GHR and blotted with anti-SOCS3 antibody. SOCS3, which is up-regulated by CXCL12 treatment (Fig. 3B), associates with GHR (Fig. 3D). This association increases after GH binding, indicating that the hormone promotes a conformational change in the GHR that exposes or stabilizes a SOCS3 binding site. These data suggest that CXCL12 interference in GH signaling includes up-regulation of some SOCS proteins, SOCS1 and SOCS3, that associate in turn to JAK2 and GHR, blocking later signaling events. CXCL12 Treatment Modifies GH-mediated Responses in Primary Cells—We tested whether the mechanism identified in vitro is also implicated in modulation of in vivo responses. CXCL12 (50 nm) was injected intrasplenically into 4-week-old BALB/c mice. After 60 min, mice were killed, and splenocytes were used to measure SOCS1 and SOCS3 levels by Northern blot. The analysis showed that both SOCS proteins were up-regulated in vivo after CXCL12 treatment (Fig. 4A, upper); as a control, the membrane was rehybridized with the β-actin probe (Fig. 4A, lower). These splenocytes, which express CXCR4 and GHR as confirmed by flow cytometry analysis (not shown), were stimulated in vitro with GH (10 μg/ml, 37 °C) for the times indicated and then lysed. Cell extracts were immunoprecipitated with anti-pTyr and blotted using anti-JAK2 (Fig. 4B) and anti-STAT5 (Fig. 4C) antibodies. CXCL12 treatment completely abrogated GH-mediated JAK2 and STAT5 phosphorylation (Fig. 4, B and C). The potential toxic effects of intrasplenic injection were discarded by evaluating splenocyte cell cycle status after propidium iodide incorporation and flow cytometry analysis (Fig. 4D). To analyze the mechanism behind this effect, splenocytes from untreated or CXCL12-pretreated (60 min) mice were stimulated in vitro with GH (10 μg/ml, 37 °C) for the times indicated. Cells were lysed, and cell extracts were immunoprecipitated with anti-SOCS1 antibody and analyzed in Western blot using anti-JAK2 antibody. As for IM9 cells, SOCS1 associated with JAK2 in an in vivo model (Fig. 4E). GH activation did not modify JAK2/SOCS1 association, supporting the observation that association between these molecules does not require JAK2 kinase activity. In accordance, SOCS1 is described to inhibit JAK catalytic activity by an interaction between the SOCS1 kinase inhibitory region and the JAK activation loop (37Alexander W. Nature Rev. 2002; 2: 1-7Google Scholar). Interaction among different cell systems is essential for the survival of all species; coordination of various stimuli is thus a critical step in achieving optimal cell responses. Interactions between the endocrine and the immune systems have been described (38Chappel S. Murphy W. Oppenheim J. Feldman M. Durum S. Hirano T. Vilcek J. Nicola N. Cytokine Reference. First Ed. Academic Press, London2002: 251-265Google Scholar); some immune mediators have a direct effect on endocrine function (39Aller M.A. Arias J.L. Nava M.P. Arias J. Exp. Biol. Med. 2004; 229: 170-181Crossref PubMed Scopus (59) Google Scholar), and endocrine system proteins affect immune cell activity (40Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2080) Google Scholar, 41Akdis M. Verhagen J. Taylor A. Karamloo F. Karagiannidis C. Crameri R. Thunberg S. Deniz G. Valenta R. Fiebig H. Kegel C. Disch R. Schmidt-Weber C.B. Blaser K. Akdis C.A. J. Exp. Med. 2004; 199: 1567-1575Crossref PubMed Scopus (902) Google Scholar). Immune processes involve many cell types and molecules, including cytokines and chemokines, indicating the need for coordination among these molecules for an effective response. Cytokines, chemokines, and growth factors share some signaling pathways in which ligand binding stabilizes receptor oligomerization and activates JAK; several proteins including the STAT transcription factors. are then phosphorylated by this tyrosine kinase. Activated STAT proteins dimerize and translocate to the nucleus, where they mediate transcription of a broad range of genes (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 30Mellado M. Rodriguez-Frade J.M. Manes S. Martínez A.-C. Annu. Rev. Immunol. 2001; 19: 397-421Crossref PubMed Scopus (299) Google Scholar), including those of the SOCS proteins. Most cytokines analyzed to date induce several SOCS in a tissue-specific manner (18Chen X.P. Losman J.A. Rothman P. Immunity. 2000; 13: 287-290Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar); SOCS could thus have a central role in coordinating individual responses from different immune system components. We recently described an active role for SOCS interfering with chemokine responses and determined that, via SOCS, stimulation of the GHR reduced the migratory capacity of different immune cell types (17Soriano S.F. Hernanz-Falcón P. Rodríguez-Frade J.M. Martín de Ana A. Garzón R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martínez A.-C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). Here we show a reciprocal interaction between these two families of mediators as activation of the CXCR4 chemokine receptor modulates GH cytokine responses through SOCS proteins, both in vivo and in vitro. GH exerts its effects by inducing the synthesis of its main mediator, IGF-1, which circulates in complex with high affinity IGFBP. These binding proteins act as carriers to transport IGF from the synthesis sites to target tissues; they also protect IGF from proteolytic degradation (42Martin J.L. Baxter R.C. Rosenfeld R. Roberts C. The IGF System: Molecular Biology, Physiology and Clinical Applications. Humana, Totowa, NJ1999: 227-255Crossref Google Scholar). Circulating IGF-1 is produced mainly by hepatic cells, although it presumably does not act directly on hepatocytes as they do not express detectable levels of IGF-1 receptor mRNA (43Le Roith D. Scavo L. Butler A. Trends Endocrinol. Metab. 2001; 12: 48-52Abstract Full Text Full Text PDF PubMed Google Scholar). In this model, GH triggers IGF-1 up-regulation and an increase in IGFBP3 levels, which may be an indirect effect as it is not seen in GH-stimulated, liver-specific IGF-1 gene-deleted mice (LID mice) (44Yakar S. Liu J.L. Stannard B. Butler A. Accili D. Sauer B. Le Roith D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7324-7329Crossref PubMed Scopus (1185) Google Scholar). Our observation that CXCL12 pretreatment reduces circulating IGF-1 levels and blunts the GH-induced IGF-1 increase in serum suggests an inhibitory effect on liver IGF-1 production. The co-expression of CXCR4 (45Wald O. Pappo O. Safadi R. Dagan-Berger M. Beider K. Wald H. Franitza S. Weiss I. Avniel S. Boaz P. Hanna J. Zamir G. Eid A. Mandelboim O. Spengler U. Galun E. Peled A. Eur. J. Immunol. 2004; 34: 1164-1174Crossref PubMed Scopus (93) Google Scholar) and GH receptors in hepatocytes (9Herrington J. Carter-Su C. Trends Endocrinol. Metab. 2001; 12: 252-257Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) hints at cross-talk between the signaling pathways activated through these receptors. CXCL2 could thus indirectly regulate IGF-1 expression in the liver; indeed, CXCL12 was shown to stimulate hematopoiesis in adult liver (46Kollet O. Shivtiel S. Chen Y.Q. Suriawinata J. Thung S.N. Dabeva M.D. Kahn J. Spiegel A. Dar A. Samira S. Goichberg P. Kalinkovich A. Arenzana-Seisdedos F. Nagler A. Hardan I. Revel M. Shafritz D.A. Lapidot T. J. Clin. Invest. 2003; 112: 160-169Crossref PubMed Scopus (557) Google Scholar). Our data confirm that CXCL12 pretreatment blocks GH in human IM9 lymphoblastoid cells. Chemokine treatment affected neither GH binding nor GH receptor levels, suggesting that interference is downstream of ligand/receptor interaction. As GH and CXCL12 both activate the JAK/STAT/SOCS pathway, we tested whether the effect was mediated through SOCS proteins. We found that CXCL12 treatment up-regulated SOCS1 and SOCS3 in vitro and in splenocytes from chemokine-treated mice. SOCS1 and SOCS3, by binding to JAK2 and to GHR, respectively, cooperate to abrogate GH-mediated activation. SOCS1/JAK2 association was unaffected by GH stimulation, indicating that JAK2 kinase activity is not involved in complex formation in this system. It has been established that hematopoietic cell growth, differentiation, and function are controlled by the coordinated action of the cytokine-chemokine network (47Youn B.S. Mantel C. Broxmeyer H.E. Immunol. Rev. 2000; 177: 150-174Crossref PubMed Scopus (109) Google Scholar). All leukocyte lineages are derived from pluripotent hematopoietic stem cells (HSC) that localize predominantly in the fetal liver and adult bone marrow. These cells can be mobilized to the periphery by cytokine treatment. Among other cytokine receptors, GHR is expressed in both hematopoietic and non-hematopoietic cells (48Kopchick J.J. Andry J.M. Mol. Genet. Metab. 2000; 71: 293-314Crossref PubMed Scopus (187) Google Scholar), and recombinant hGH significantly increases in vitro colony formation by human myeloid and erythroid progenitors (49Golde D.W. Bersch N. Li C.H. Science. 1997; 196: 1112-1113Crossref Scopus (143) Google Scholar, 50Reinerova M. Reiner P. Neoplasma (Bratisl.). 1991; 38: 175-183PubMed Google Scholar). Administration of recombinant hGH to mice after syngeneic bone marrow transplant significantly accelerates multilineage hematopoietic recovery (51Tian Z.G. Woody M.A. Sun R. Welniak L.A. Raziuddin A. Funakoshi S. Tsarfaty G. Longo D.L. Murphy W.J. Stem Cells (Mi-amisburg). 1998; 16: 193-199Crossref PubMed Scopus (57) Google Scholar). CXCL12 and its receptor CXCR4 play a key role in HSC trafficking to bone marrow (52Wright D.E. Bowman E.P. Wagers A.J. Butcher E.C. Weissman I.L. J. Exp. Med. 2002; 195: 1145-1154Crossref PubMed Scopus (418) Google Scholar). A reduction in CXCL12 and CXCR4 activity has also been implicated in HSC mobilization by G-CSF; this treatment leads to increased granulocyte accumulation in bone marrow and subsequent release of proteases that inactivate both CXCL12 and CXCR4 (53Levesque J.P. Hendy J. Takamatsu Y. Simmons P.J. Bendall L.J. J. Clin. Invest. 2003; 111: 187-196Crossref PubMed Scopus (641) Google Scholar), triggering HSC mobilization to the periphery (54Petit I. Szyper-Kravitz M. Nagler A. Lahav M. Peled A. Habler L. Ponomaryov T. Taichman R.S. Arenzana-Seisdedos F. Fujii N. Sandbank J. Zipori D. Lapidot T. Nat. Immunol. 2002; 3: 687-694Crossref PubMed Scopus (1154) Google Scholar). The mechanism we propose could also participate in this process since, by affecting cytokine or chemokine responses, SOCS could alter the equilibrium between HSC formation and mobilization. In aged mice with impaired bone marrow hematopoietic function and reduced stem cell mobilizing capacity, hGH is reported to restore bone marrow progenitor cell growth, as well as cytokine-elicited stem cell mobilization (55Carlo-Stella C. Di Nicola M. Milani R. Longoni P. Milanesi M. Bifulco C. Stucchi C. Guidetti A. Cleris L. Formelli F. Garotta G. Gianni A.M. Exp. Hematol. 2004; 32: 171-178Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In conclusion, the results show that individual cytokine responses are influenced by the presence of chemokines and vice versa. An overall view of these systems must thus consider the effect of cytokine/chemokine modulation of ligands and of receptors, as well as their influence on individual signaling pathways. We thank Drs T. Willson, R. Starr and D. Hilton (Walter and Eliza Hall Institute of Medical Research) for the gift of the SOCS expression vectors, Dr. D. Le Roith (National Institutes of Health) for the gift of pGEM-mIGF-1 exon 4 and pGEM-rIGFBP3 vectors, Dr. E. Montoya for critical reading of the manuscript, J. Pomada (Endocrine Care, Pfizer Spain) for recombinant hGH, M. C. Moreno-Ortíz for help with FACS analysis, and C. Bastos and C. Mark for secretarial and editorial assistance, respectively. The Department of Immunology and Oncology was founded and is supported by the CSIC and by Pfizer.
Referência(s)