Functional Inactivation of CXC Chemokine Receptor 4–mediated Responses through SOCS3 Up-regulation Silvia F. Soriano, Patricia Hernanz-Falcón, José Miguel Rodríguez-Frade, Ana Martín de Ana, Ruth Garzón, Carla Carvalho-Pinto, Antonio J. Vila-Coro, Angel Zaballos, Dimitrios Balomenos, Carlos Martínez-A., and Mario Mellado Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientifícas, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Abstract Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated ac- tion between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cy- tokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger re- ceptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2ϩ flux and chemotaxis in vitro and in vivo. This effect is mediated ␣ by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and G i pathways, without interfering with cell surface chemokine receptor expression. The data pro- vide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system. Key words: SOCS3 • JAK-STAT activation • chemokine signaling • cytokine signaling Introduction The chemokines comprise a large family of low molecular only stromal cell–derived factor (SDF)-1␣ (CXCL12) (1, weight (8–10 kD) cytokines, with chemotactic and pro-acti- 2), a chemokine isolated from stromal cell culture superna- vatory effects on different leukocyte lineages (1–3). Studies tants (9). Its chemotactic properties have been described in have established the central role of chemokines in a number PBLs (10), CD34ϩ progenitor cells (11), and pre- and pro- of physiological situations, including T helper responses, B cell lines (12). Knockout mice lacking the CXCL12 pro- hematopoiesis, angiogenesis, and homeostasis, as well as in tein (10) and mice lacking the CXCR4 receptor (13, 14) pathological conditions such as asthma, tumor rejection, display similar phenotypes: animals die before birth, dis- HIV-1 infection, and arteriosclerosis (4–8). playing abnormalities in B cell lymphopoiesis and bone Chemokines mediate their biological effects after bind- marrow myelopoiesis, lack of blood vessel formation in the ing to specific receptors, members of the seven-transmem- gut, severe ventricular septal defects, and altered cerebellar brane domain G protein–coupled receptor family (1, 2). neuron migration (13–15). Chemokine receptors are promiscuous, as each can bind After binding to their receptors, chemokines trigger re- more than one chemokine; expression is heterogeneous ceptor association of guanine nucleotide-binding proteins among different cells of the leukocyte lineage and is tran- (G proteins; reference 16); this activates signaling molecules scriptionally regulated (3, 5). An exception is the CXC that mediate changes in the cytoskeletal apparatus and tran- chemokine receptor 4 (CXCR4)* receptor, which binds scription factors that regulate cell growth (17–19). We have Address correspondence to Carlos Martínez-A., Department of Immu- nology and Oncology, Centro Nacional de Biotecnología/CSIC, Uni- versidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 EMSA, electrophoretic mobility shift assay; GH, growth hormone; JAK, Madrid, Spain. Phone: 34-91-585-4660; Fax: 34-91-372-0493; E-mail: Janus kinase; RT, room temperature; SIE, sis-inducible element; siRNA, [email protected] silent RNA; SOCS, suppressors of cytokine signaling; STAT, signal *Abbreviations used in this paper: CXCR, CXC chemokine receptor; transducers and transactivators of transcription; Tg, transgenic. 311 J. Exp. Med. The Rockefeller University Press • 0022-1007/2002/08/311/11 $5.00 Volume 196, Number 3, August 5, 2002 311–321 http://www.jem.org/cgi/doi/10.1084/jem.20012041 shown that CXCL12 (16), as well as other chemokines hGHR-05 mAb (1 ␮g/50 ␮l/well, 60 min, 4ЊC). Cells were such as monocyte chemotactic protein (MCP)-1 (CCL2) washed twice in PBS with 2% BSA and 2% FCS and centrifuged and regulated upon activation, normal T cell–expressed and (250 g, 5 min, 4ЊC). Fluorescein isothiocyanate-labeled strepta- vidin (Southern Biotechnology Associates, Inc.) was added, in- secreted (RANTES; CCL5) (20, 21), exert their effects via Њ dimerization of their receptors and activation of Janus ki- cubated (30 min, 4 C), plates washed twice, and cell-bound fluorescence determined in a Profile XL flow cytometer at 525 nm nase (JAK) kinases, a protein family originally implicated in (Beckman Coulter). cytokine signaling (22). This in turn phosphorylates the Immunocytochemical Analysis. IM-9 cells were cultured in chemokine receptors in tyrosine residues and activates sig- RPMI with 1% BSA for 2 h, alone or CXCL12-stimulated (50 nal transducers and transactivators of transcription (STAT) nM), washed with PBS at room temperature (RT), fixed with transcription factors (23). 100% methanol (10 min, Ϫ20ЊC), and permeabilized with 0.2% The suppressors of cytokine signaling (SOCS) family Triton X-100 in TBS (15 min, RT). After washing with TBS proteins have been identified as feedback regulators of (3ϫ, 5 min, RT), nonspecific binding sites were blocked with 5% JAK/STAT activation, through their binding to JAK ki- goat serum in TBS (45 min, RT). Cells were washed with TBS, nases or cytokine receptors (24–28). The majority of cyto- then incubated with anti-STAT3 or anti-phospho-STAT3 mAb kines analyzed to date, such as LIF, IL-2, IL-3, IL-6, diluted in TBS, 2% BSA, 2% normal goat serum, followed by growth hormone (GH), IFN-␥, and leptin, induce several Cy3-labeled anti–rabbit IgG antibody (Jackson ImmunoResearch Laboratories). Vectashield mounting medium with 4Ј,6-diami- SOCS family members in a tissue-specific manner (24, 28). dino-2-phenylindole (DAPI; Vector Laboratories) was added and The regulatory role of SOCS molecules is not limited to cells analyzed in a Leica fluorescence microscope. the cytokine receptor superfamily, however, as interaction Cell Migration. Cells were placed (0.25 ϫ 106 cells in 0.1 ml) has been described between SOCS and other cellular tar- in the upper well of 24-well transmigration chambers (5 ␮m gets, such as the IGF-I receptor, suggesting that SOCS pro- pore; Transwell; Costar Corp.) whose membrane was precoated teins may have an extensive role in receptor signaling (29). with type VI collagen (20 ␮g/ml; Sigma-Aldrich); 50 nM Here we show that through JAK/STAT activation, CXCL12 or 10 nM CCL20 (in 0.6 ml RPMI with 0.25% BSA; CXCR4 occupancy by CXCL12 upregulates a member of PeproTech) was then added to the lower well. Plates were incu- Њ the SOCS protein family, SOCS3. Cytokine-stimulated bated (120 min, 37 C) and cells that migrated to the lower cham- overexpression or upregulation of SOCS3 interferes with ber were counted as described (23). When recombinant hGH (Genotropin; Pharmacia AB) treatment was used, cells (106/ml) signaling by certain chemokines, including CXCL12. By were incubated in RPMI 1640 with 0.1% BSA and 10 ␮g/ml binding to CXCR4, SOCS3 blocks JAK/STAT com- hGH for the time indicated. plex activation and association to CXCR4, inhibiting To evaluate primary cells, mouse spleen, lymph node, and CXCL12-mediated responses both in vivo and in vitro. bone marrow cells (0.25 ϫ 106 cells in 0.1 ml) were placed in the These data, discussed here in the context of cross-talk be- upper well of 24-well transmigration chambers (3 ␮m pore; tween cytokine and chemokine responses, will aid in un- Transwell) as described above. In spleen and bone marrow cell Њ derstanding the functional role of this chemokine/chemo- preparations, erythrocytes were lysed with NH4Cl (5 min, 37 C). kine receptor pair and add a new element to the complex In assays in which depletion of fresh isolated primary cells was function of the immune system. necessary, cells were plated (106 cells in 1 ml) in RPMI 1640 sup- plemented with 0.1% BSA and cultured (2 h, 37ЊC). After wash- ing, migration in response to CXCL12 was evaluated as above. HEK-293 cell migration was studied in a 96-well microcham- Materials and Methods ber (NeuroProbe Inc.; reference 31). Chemokines at several con- Biological Materials. IM-9 (ATCC CCL159) and HEK-293 centrations were loaded into lower wells (30 ␮l/well), and cells cells (ATCC TIB202) were from the American Type Culture (200 ␮l/well, 3 ϫ 106 cells/ml) in upper wells. Polyvinylpyrroli- Collection. Antibodies used include anti-CXCR4 (CXCR4–01) done-free filters (10 ␮m pore; NeuroProbe Inc.) were precoated and anti-hGH receptor (hGHR-05) mAb generated in our labo- (2 h, 37ЊC) with 20 ␮g/ml type VI collagen (Sigma-Aldrich). ratory (16, 30), horseradish peroxidase (PO)-labeled anti-PTyr The chamber was incubated (5 h, 37ЊC), after which filters were mAb (4G10; Upstate Biotechnology), anti-phosphothreonine and - removed and the cells on the upper part wiped off. Cells on the ␤ phosphoserine mAb (Calbiochem), anti- 2 microglobulin mAb filters were fixed and stained (0.5% crystal violet,