Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines
2008; Elsevier BV; Volume: 284; Issue: 6 Linguagem: Inglês
10.1074/jbc.m804935200
ISSN1083-351X
AutoresZinaida A. Dubeykovskaya, Alexander Dubeykovskiy, Joel Solal-Cohen, Timothy C. Wang,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoThe secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca2+ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca2+ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo. The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca2+ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca2+ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo. Trefoil factor 2 (TFF2), 2The abbreviations used are: TFF, trefoil factor family; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; m, mouse; h, human; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SDF, stromal cell-derived factor; PKB/AKT, serine/threonin protein kinase B. previously known as spasmolytic polypeptide, is a unique member of the trefoil family that is expressed primarily in gastric mucous neck cells and is up-regulated in the setting of chronic inflammation. Experimental induction of ulceration in the rat stomach leads to rapid up-regulation of TFF2 expression with high levels observed 30 min after ulceration with persistence for up to 10 days (1Alison M.R. Chinery R. Poulsom R. Ashwood P. Longcroft J.M. Wright N.A. J. Pathol.. 1995; 175: 405-414Google Scholar). TFF2 is secreted into the mucus layer of the gastrointestinal tract of mammals where it stabilizes the mucin gel layer and stimulates migration of epithelial cells (2Hoffmann W. Jagla W. Int. Rev. Cytol.. 2002; 213: 147-181Google Scholar, 3Oertel M. Graness A. Thim L. Buhling F. Kalbacher H. Hoffmann W. Am. J. Respir. Cell Mol. Biol.. 2001; 25: 418-424Google Scholar, 4Graness A. Chwieralski C.E. Reinhold D. Thim L. Hoffmann W. J. Biol. Chem.. 2002; 277: 18440-18446Google Scholar), suggesting an important role in restitution and in maintenance of the integrity of the gut. Exogenous administration of recombinant TFF2, either orally or intravenously, provides mucosal protection in several rodent models of acute gastric or intestinal injury (5Babyatsky M.W. deBeaumont M. Thim L. Podolsky D.K. Gastroenterology.. 1996; 110: 489-497Google Scholar, 6Poulsen S.S. Kissow H. Hare K. Hartmann B. Thim L. Regul. Pept.. 2005; 126: 163-171Google Scholar). A TFF2-/- knock-out mouse model has confirmed the importance of TFF2 in the protection of gastrointestinal mucosa against chronic injury (7Farrell J.J. Taupin D. Koh T.J. Chen D. Zhao C.M. Podolsky D.K. Wang T.C. J. Clin. Investig.. 2002; 109: 193-204Google Scholar). It is widely accepted that trefoil factors exert their biological action through a cell surface receptor. This suggestion comes from studies on binding of 125I-labeled TFF2 that demonstrated specific binding sites in the gastric glands, intestine, and colon that could be displaced by non-radioactive TFF2 (6Poulsen S.S. Kissow H. Hare K. Hartmann B. Thim L. Regul. Pept.. 2005; 126: 163-171Google Scholar, 8Frandsen E.K. Regul. Pept.. 1988; 20: 45-52Google Scholar, 9Poulsen S.S. Thulesen J. Nexo E. Thim L. Gut.. 1998; 43: 240-247Google Scholar, 10Poulsen S.S. Thulesen J. Hartmann B. Kissow H.L. Nexo E. Thim L. Regul. Pept.. 2003; 115: 91-99Google Scholar). Structural studies have revealed potential binding sites for receptors for all members of the trefoil factor family (11Carr M.D. Bauer C.J. Gradwell M.J. Feeney J. Proc. Natl. Acad. Sci. U. S. A.. 1994; 91: 2206-2210Google Scholar, 12De A. Brown D.G. Gorman M.A. Carr M. Sanderson M.R. Freemont P.S. Proc. Natl. Acad. Sci. U. S. A.. 1994; 91: 1084-1088Google Scholar). In concordance with this hypothesis, several membrane proteins were found to interact with TFF2. First it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa peptide from membrane fractions of rat jejunum and two human adenocarcinoma cell lines, MCF-7 and Colony-29 (13Chinery R. Cox H.M. Peptides.. 1995; 16: 749-755Google Scholar). Later it was found that recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein from the membrane of rat small intestinal cells (14Tan X.D. Hsueh W. Chang H. Wei K.R. Gonzalez-Crussi F. Biochem. Biophys. Res. Commun.. 1997; 237: 673-677Google Scholar). However, these 28- and 50-kDa proteins were characterized only by their molecular size without further identification. Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the β subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15Thim L. Mortz E. Regul. Pept.. 2000; 90: 61-68Google Scholar). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1(16Otto W.R. Patel K. McKinnell I. Evans M.D. Lee C.Y. Frith D. Hanrahan S. Blight K. Blin N. Kayademir T. Poulsom R. Jeffery R. Hunt T. Wright N.A. McGregor F. Oien K.A. Proteomics.. 2006; 6: 4235-4245Google Scholar). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown. Despite the absence of an identified cell surface receptor for TFF2, there is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal transduction pathways (17Emami S. Rodrigues S. Rodrigue C.M. Le Floch N. Rivat C. Attoub S. Bruyneel E. Gespach C. Peptides.. 2004; 25: 885-898Google Scholar, 18Hoffmann W. CMLS Cell. Mol. Life Sci.. 2005; 62: 2932-2938Google Scholar). TFF3 prevents cell death via activation of the serine/threonine kinase AKT in colon cancer cell lines (19Taupin D.R. Kinoshita K. Podolsky D.K. Proc. Natl. Acad. Sci. U. S. A.. 2000; 97: 799-804Google Scholar). The TFF3 protein also activates STAT3 signaling in human colorectal cancer cells, thus providing cells with invasion potential (20Rivat C. Rodrigues S. Bruyneel E. Pietu G. Robert A. Redeuilh G. Bracke M. Gespach C. Attoub S. Cancer Res.. 2005; 65: 195-202Google Scholar). TFF3 treatment leads to EGF receptor activation and β-catenin phosphorylation in HT-29 cells (21Liu D. el-Hariry I. Karayiannakis A.J. Wilding J. Chinery R. Kmiot W. McCrea P.D. Gullick W.J. Pignatelli M. Lab. Investig.. 1997; 77: 557-563Google Scholar) and to transient phosphorylation of ERK1/2 in oral keratinocytes (22Storesund T. Hayashi K. Kolltveit K.M. Bryne M. Schenck K. Eur. J. Oral Sci.. 2008; 116: 135-140Google Scholar). With respect to TFF2, recombinant peptide enhances the migration of human bronchial epithelial cell line BEAS-2B (4Graness A. Chwieralski C.E. Reinhold D. Thim L. Hoffmann W. J. Biol. Chem.. 2002; 277: 18440-18446Google Scholar). TFF2 has been shown to induce phosphorylation of c-Jun NH2-terminal kinase (JNK) and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is significantly inhibited by antagonists of ERK kinases and protein kinase C but not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is believed that the motogenic effect of trefoil factors and of TFF2 in particular, could contribute to in vivo restitution of gastric epithelium by enhancing cell migration. Although previous studies have suggested that TFF2 functions primarily in cytoprotection, accumulating evidence now suggests that TFF2 may also play a role in the regulation of host immunity. For example, recombinant TFF2 reduces inflammation in rat and mouse models of colitis (23Tran C.P. Cook G.A. Yeomans N.D. Thim L. Giraud A.S. Gut.. 1999; 44: 636-642Google Scholar, 24Soriano-Izquierdo A. Gironella M. Massaguer A. May F.E. Salas A. Sans M. Poulsom R. Thim L. Pique J.M. Panes J. J. Leukoc. Biol.. 2004; 75: 214-223Google Scholar). In addition, TFF2 was detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow) (25Cook G.A. Familari M. Thim L. Giraud A.S. FEBS Lett.. 1999; 456: 155-159Google Scholar). Recently we and others found TFF2 mRNA expression in primary and secondary lymphopoietic organs (26Baus-Loncar M. Kayademir T. Takaishi S. Wang T. CMLS Cell. Mol. Life Sci.. 2005; 62: 2947-2955Google Scholar, 27Kurt-Jones E.A. Cao L. Sandor F. Rogers A.B. Whary M.T. Nambiar P.R. Cerny A. Bowen G. Yan J. Takaishi S. Chi A.L. Reed G. Houghton J. Fox J.G. Wang T.C. Infect. Immun.. 2007; 75: 471-480Google Scholar). These data suggest that TFF2 may play some function in the immune system. In concordance with these findings, we detected an exacerbated inflammatory response to acute injury in TFF2 knock-out animals (27Kurt-Jones E.A. Cao L. Sandor F. Rogers A.B. Whary M.T. Nambiar P.R. Cerny A. Bowen G. Yan J. Takaishi S. Chi A.L. Reed G. Houghton J. Fox J.G. Wang T.C. Infect. Immun.. 2007; 75: 471-480Google Scholar, 28Fox J.G. Rogers A.B. Whary M.T. Ge Z. Ohtani M. Jones E.K. Wang T.C. Am. J. Pathol.. 2007; 171: 1520-1528Google Scholar). These observations prompted us to look at the possible function of TFF2 in immune cells. Unexpectedly we found that TFF2 modulates Ca2+ and AKT signaling in lymphoblastic Jurkat cells and that these effects appear to be mediated through the CXCR4 receptor. Chemicals, Chemokines, Peptides, and Antibodies—The CXCR4 receptor antagonist AMD3100, was purchased from Sigma. The Ca2+-sensing dye Indo-1 and the transfection reagent Lipofectamine 2000 were obtained from Molecular Probes/Invitrogen. The recombinant human chemokine stromal cell-derived factor (SDF)-1α was purchased from R&D Systems (Minneapolis, MN). Unlabeled (azide-free) and phycoerythrin-conjugated anti-human CXCR4 (12G5) and anti-mouse CXCR4 (clone 2B11) antibodies were purchased from BD Pharmingen. 12G5 antibodies recognize conformation-dependent epitope including amino acid 28 in the NH2 terminus, amino acids 179, 181, 182, and 190 in the second extracellular loop (ECL2); and amino acid 274 in the ECL3 (29Kalinkovich A. Tavor S. Avigdor A. Kahn J. Brill A. Petit I. Goichberg P. Tesio M. Netzer N. Naparstek E. Hardan I. Nagler A. Resnick I. Tsimanis A. Lapidot T. Cancer Res.. 2006; 66: 11013-11020Google Scholar). Antibody 2B11 was raised to 63 amino acids of the NH2 terminus of CXCR4 receptor (30Forster R. Kremmer E. Schubel A. Breitfeld D. Kleinschmidt A. Nerl C. Bernhardt G. Lipp M. J. Immunol.. 1998; 160: 1522-1531Google Scholar). Isotypic mouse IgG2a antibodies were from eBioscience (San Diego, CA). Antibodies to total human AKT, Ser-473 (or Thr-308)-phosphorylated AKT, and total and phosphorylated ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to α-tubulin were purchased from Oncogene Science, Inc. (Cambridge, MA). Rabbit anti-TFF2 antibodies to a carboxyl-terminal peptide (16 amino acids) of hTFF2 (which also recognize mTFF2) were developed previously and characterized in our laboratory (31Tu S. Chi A.L. Lim S. Cui G. Dubeykovskaya Z. Ai W. Fleming J.V. Takaishi S. Wang T.C. Am. J. Physiol.. 2007; 292: G1726-G1737Google Scholar). Cell Lines—KATO III, AsPC-1, AGS, NIH3T3, HEK293T/17, and Jurkat E6-1 cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA). AGS, NIH3T3, HEK293T/17, and AsPC-1 cells were cultured in DMEM supplemented with heat-inactivated 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 units/ml streptomycin. KATO III and Jurkat cells were cultivated in RPMI 1640 medium with 10% fetal bovine serum. All cell cultures were incubated at 37 °C in a humidified atmosphere (5% CO2). Where indicated, cells were starved in the respective medium with bovine serum albumin (0.5%) but lacking serum overnight. Cloning and Expression of mTFF2 in Eukaryotic Cells—To express recombinant mouse TFF2 in CHO-K1 and Jurkat cells, we used a retroviral vector, pMIG, which was kindly provided by Dr. David Baltimore (Caltech, Pasadena, CA). This vector contains a multiple cloning site followed by an internal ribosome binding site and the green fluorescent protein (GFP) gene. A strong viral long terminal repeat promoter controls transcription of both the cloned and GFP genes. The coding region of immature mouse TFF2 along with the noncoding 5′ flanking region and EcoRI/SalI flanking restriction sites was amplified from MGC clone (BC050086; Open Biosystems, Baltimore, MD) by PCR and introduced into corresponding restriction sites of the pMIG polylinker. The integrity of the resultant construct, pMIG-mTFF2 (GFP), was verified by sequencing. The stable CHO-K1 cell line secreting recombinant mouse TFF2 was generated by infection with the pMIG-mTFF2 retrovirus in the presence of Polybrene (5 μg/ml). The GFP-positive pool of cells was collected by flow cytometric sorting. Stable pools of Jurkat cells bearing empty pMIG or pMIG-mTFF2 were selected in identical fashion. Recombinant TFF2—Recombinant human TFF2 purified from Escherichia coli (>98% pure by SDS-PAGE and high pressure liquid chromatography analysis) was obtained from Peprotech (Rocky Hills, NJ). Human glycosylated TFF2 purified from yeast that have been used in the majority of the published studies on the biological function of TFF2 peptide was kindly provided by Dr. Lars Thim ("Novo Nordisk," Maaloev, Denmark). For murine TFF2 production, CHO/pMIG-mTFF2 cells were expanded, and the TFF2 was purified from the supernatant of adherent cells by a combination of gel filtration on Sephadex G-50 and ion-exchange chromatography. Homogeneity of the final product was validated through electrophoresis in an 18% polyacrylamide gel under both reducing and nonreducing conditions. The presence of the monomeric form of secreted recombinant TFF2 was confirmed in Western blot analysis by using antibodies developed to the carboxyl end of the human counterpart. Chemotaxis Assay—For the migration assay we used Jurkat T cells transfected with retroviral vector pMIG (control) or pMIG-TFF2. The latter expresses both TFF2 and GFP proteins from a single bicistronic transcription unit. For the migration assay, control or TFF2-expressing cells were washed, and 3 × 106 cells/ml were suspended in medium containing RPMI 1640 medium with 1% bovine serum albumin with low endotoxin content (Sigma, CAS 9048-46-8). 100-μl aliquots of cell suspensions were applied to the upper chambers of 24-well Transwell plates (Boyden chamber, Costar 3422, 5-μm-diameter pore size). 600 μl of RPMI 1640 medium/BSA supplemented with the indicated concentrations of SDF-1α (R&D Systems) were loaded into the lower chambers. Cells were allowed to migrate for 3 h at 37 °C. After incubation, the porous inserts were removed carefully, and the viable cells were counted using a hemocytometer. The results are expressed as the percentage of cells that migrated to the bottom chamber. Each experiment was performed seven to eight times in triplicate. In experiments with recombinant TFF2, Jurkat cells were applied to the upper chambers with different concentrations of recombinant protein (75–1000 nm) in the upper and lower chamber. 600 μl of RPMI 1640 medium/BSA supplemented with 100 ng/ml SDF-1 were loaded into the lower chambers. Generation of Stably Transfected AGS Cells Bearing the CXCR4-GFP Chimeric Receptor—The retroviral construct LZRS-CXCR4-GFP-IRES-Zeocin encoding simian CXCR4 was kindly provided by Drs. Eloise Anthony and Peter L. Hordijk (32van Buul J.D. Voermans C. van Gelderen J. Anthony E.C. van der Schoot C.E. Hordijk P.L. J. Biol. Chem.. 2003; 278: 30302-30310Google Scholar). Amphotrophic retrovirus was obtained, and virgin AGS cells (5 × 106) were infected with this retrovirus (at a multiplicity of 1:10) as described above. Infected GFP-positive cells (∼3%) were collected by flow cytometric sorting (twice), expanded, and stored in working aliquots in vapors of liquid nitrogen. AGS cells expressing GFP comprised 96% of the final purified cell population. Measurement of Ca2+ Level by Flow Cytometry—Jurkat cells (2.5 × 106 cells/ml) were resuspended in RPMI 1640 medium containing 0.5% BSA and incubated with the Ca2+-binding dye Indo-1 AM at a final concentration of 5 μm for 1 h at 37 °C in the dark with agitation. Loaded cells were washed, resuspended in Hanks' balanced salt solution medium containing 2 mm CaCl2 and 1 mm MgCl2, and left for 20 min at room temperature. Cells were aliquoted into fluorescence-activated cell sorter tubes that were immediately transferred into a 37 °C water bath for an additional 5 min prior to measurements. Equilibrated cells were then used for flow cytometric analysis of the Ca2+ level using an LSRII machine (BD Biosciences). The base-line intracellular Ca2+ level was recorded for an initial 25–30 s followed by a stimulation with the indicated concentrations of SDF-1α, human or murine TFF2, gastrin, ionomycin, or diluent (phosphate-buffered saline). Data collection was continued at the speed of 2000 events/s for an additional 4–10 min. An increase in binding of cytosolic Ca2+ to Indo-1 results in a change of the emission spectrum of Indo-1 from 510 nm (free form) to 420 nm (Ca2+-bound form). Thus, blue (4′,6-diamidino-2-phenylindole channel, 420 nm) and violet (Indo channel, 510 nm) cell fluorescence was measured, and data were plotted using FlowJo software (version 6.4; Tree Star, Inc.). Intracellular calcium mobilization in response to SDF-1α or recombinant mouse/human TFF2 in the presence of AMD3100 or anti-CXCR antibody was measured after Jurkat cells were preincubated for 40 min at 37 °C with AMD3100 at a concentration of 0.5 μg/ml or with 13 μg/ml 12G5 antibody (eBioscience) accordingly. Immunodetection of Phosphorylation Status of Proteins—Jurkat cells were washed twice in RPMI 1640 medium, suspended at 10 × 106 cells/ml in the same medium with 0.5% BSA, and starved for 20 h at 37 °C in 5% CO2. Cancer lines AGS, KATO III, or AsPC-1 or recombinant cells AGS/CXCR4 were seeded in 60-mm dishes (5 × 105 cells/dish), grown for 24 h in DMEM supplemented with 10% fetal bovine serum, and starved overnight in DMEM supplemented with 0.5% BSA. Cells were stimulated with of SDF-1 (100 nm) or TFF2 (500 nm) at different time points, washed with ice-cold phosphate-buffered saline, and resuspended in lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 2 mm sodium vanadate, 2 mm sodium fluoride, and 0.25 mm sodium pyrophosphate) for 20 min. After centrifugation (15 min at 13,000 rpm), the soluble fractions were kept at -80 °C until use. Protein concentration was detected with a kit (Bio-Rad). Equivalent amounts of protein from each sample were run on 10% Tricine-glycine SDS-PAGE (Invitrogen) and transferred to 0.45-μm nitrocellulose membranes (Bio-Rad) according to standard protocols. The blots were probed with anti-phospho-AKT (Thr-308) or -AKT (Ser-473) and then anti-total AKT or with anti-phospho-ERK1/2 (Thr-202/Tyr-204) mouse monoclonal antibodies followed by anti-total ERK1/2 rabbit polyclonal antibodies. Bound primary antibodies were visualized with appropriate peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) using Enhanced Luminol Chemiluminescence Reagent (Amersham Biosciences). Proliferation Assay—To assess a proliferative effect of cytokines on epithelial cells, AGS and AGS/CXCR4 cells were plated at density 12 × 103 cells/well in a 96-well plate in complete DMEM for 24 h. The next day this medium was replaced with DMEM supplemented with 0.1% BSA. The following day various concentrations of cytokines were added to quiescent cells in triplicates for an additional 72 h. At the end cell growth was analyzed using either Cell Counting Kit-8 (WST-8, Dojindo Laboratories, Rockville, MD) or the BrdU Cell Proliferation Assay (Chemicon, Temecula, CA) according to the manufacturer's instructions. Studies of Binding of Recombinant TFF2 to Jurkat Cells—Binding experiments were performed with 3 × 105 Jurkat cells resuspended in 50 μl of phosphate-buffered saline, 3% BSA containing anti-CXCR4-phycoerythrin monoclonal antibody 2B11 (10 μg/ml) or 12G5 (10 μg/ml) in the presence of SDF-1 or TFF2 (in the concentrations indicated in the figure legends) at 4 °C for 1 h. The cells were washed twice in phosphate-buffered saline, 3% BSA buffer. As a negative control, cells were stained with phycoerythrin-labeled isotype-matched antibodies. Finally the cells were analyzed on an LSRII flow cytometer. Statistical Analysis—If not specially stated, each experiment was repeated three to eight times. Statistical significance of the differences observed between experimental groups was determined by a two-tailed t test. p values less than 0.05 were considered to be significant. TFF2 Attenuates SDF-1α/CXCR4-mediated Chemotaxis and Ca2+ Signaling in Jurkat Cells—We and others have previously demonstrated TFF2 mRNA expression in the secondary mouse lymphoid organs, namely the spleen and thymus (25Cook G.A. Familari M. Thim L. Giraud A.S. FEBS Lett.. 1999; 456: 155-159Google Scholar, 26Baus-Loncar M. Kayademir T. Takaishi S. Wang T. CMLS Cell. Mol. Life Sci.. 2005; 62: 2947-2955Google Scholar, 27Kurt-Jones E.A. Cao L. Sandor F. Rogers A.B. Whary M.T. Nambiar P.R. Cerny A. Bowen G. Yan J. Takaishi S. Chi A.L. Reed G. Houghton J. Fox J.G. Wang T.C. Infect. Immun.. 2007; 75: 471-480Google Scholar). To evaluate possible TFF2 regulation in splenocytes by mitogens, we stimulated mouse splenocytes with T cell and B cell mitogens, concanavalin A and lipopolysaccharide, respectively. Total mRNA was isolated from activated splenocytes, and the level of TFF2 mRNA was analyzed by quantitative real time reverse transcription PCR. This analysis revealed a distinct increase in TFF2 mRNA abundance in response to concanavalin A (40-fold up-regulation), whereas lipopolysaccharide induced a more modest 2.5-fold increase compared with unstimulated cells (data not shown). To study the possibility of TFF2 function in T cells, we used as a model the well characterized Jurkat clone E6-1 T cell line, which does not express endogenous TFF2 mRNA (data not shown). TFF2 gene expression has been shown in a number of malignancies (33May F.E. Semple J.I. Prest S.J. Westley B.R. Peptides.. 2004; 25: 865-872Google Scholar), but the absence of TFF2 expression in Jurkat cells provided us with an opportunity to study TFF2 function through stable overexpression. We cloned the TFF2 cDNA (including its own secretory signal) into a retroviral vector (pMIG) that contained an additional marker, GFP, for easier selection of the infected Jurkat cells. Jurkat cells were infected with recombinant retrovirus pMIG-TFF2 (GFP) or empty retrovirus pMIG (GFP). Respective pools of GFP-positive stable clones (>99%; not shown) were collected after sorting by flow cytometry. Western blot analysis revealed secretion of TFF2 protein into the culture medium by the pooled Jurkat clones containing the pMIG-TFF2 retrovirus but not the empty pMIG retrovirus (supplemental Fig. 1A). Although GFP expression was strictly localized to the cytoplasm, the majority of TFF2 protein was found in the culture medium. Under reducing conditions, the secreted recombinant mouse TFF2 migrated in concordance with the expected calculated molecular mass of around 12 kDa for the secreted monomer form. Nonreduced recombinant murine TFF2 migrates slightly slower than the reduced form, and both migrated as a single band (supplemental Fig. 1B). Because no oligomeric forms of recombinant murine TFF2 were detected we suggested that the arrangement of disulfide bonds at the positions of Cys residues and that folding for the recombinant protein were likely to be identical to that of the native protein. A common and important property of hematopoietic cells is chemotaxis (34Johnston B. Butcher E.C. Semin. Immunol.. 2002; 14: 83-92Google Scholar). The directional movement of many hematopoietic cells, including T cells, is regulated in part by the chemokine SDF-1α/CXCL12 through the CXCR4 receptor. Therefore, we tested whether TFF2 expression affects SDF-1α/CXCR4-mediated chemotaxis of Jurkat cells. We quantified the migration of Jurkat pMIG and pMIG-mTFF2 cells in response to various concentrations of SDF-1α. As shown in Fig. 1 (top), the pool of TFF2-expressing Jurkat cells migrated ∼30–40% less efficiently than Jurkat cells containing the empty pMIG retrovirus. This inhibitory effect of TFF2 on SDF-1α-mediated migration was statistically significant for all ligand concentrations tested (p < 0.003). To support the notion that TFF2 modulates chemokine-dependent cellular migration through a cell surface mechanism, we tested the effect of exogenous recombinant TFF2 on SDF-1α-dependent chemotaxis of non-transfected, parental Jurkat cells. Purified murine TFF2 was added to Jurkat cells in the upper chamber of a Boyden chamber assay, and Jurkat cells were allowed to migrate toward SDF-1α. A significant (up to 30%) inhibition of SDF-1α-dependent Jurkat cell migration was observed when recombinant murine TFF2 was applied at a concentration 500–600 nm (Fig. 1, bottom). TFF2 did not affect growth or proliferation of Jurkat cell (not shown), reducing the likelihood of a nonspecific toxic effect. However, we could not further discriminate whether TFF2 affects chemotaxis and/or chemokinesis of Jurkat cells because other chemotactic compounds (like IGF-1, serum, etc.) did not work in this assay. Recombinant TFF2 on its own was unable to stimulate chemotaxis at concentrations ranging from 10 to 1000 nm (data not shown). The inhibitory effect of TFF2 on SDF-1α-dependent chemotaxis suggested the existence of some sort of interaction between TFF2 signaling and CXCR4 signaling in Jurkat cells. One explanation for the interaction might include down-regulation of CXCR4 receptor expression on the surface of Jurkat cells in response to TFF2 stimulation. However, flow cytometry did not reveal any variations in the surface expression of the CXCR4 receptor in Jurkat pMIG-mTFF2 compared with Jurkat pMIG cells (data not shown). The SDF-1α-induced migration of Jurkat cells is accompanied by the intracellular mobilization of Ca2+ ions (35Majka M. Ratajczak J. Kowalska M.A. Ratajczak M.Z. Eur. J. Haematol.. 2000; 64: 164-172Google Scholar). Therefore, we tested for a Ca2+ spike in Jurkat cells with or without TFF2 expression in response to SDF-1α stimulation (Fig. 2). TFF2 expression (in pMIG-mTFF2 Jurkat cells) resulted in a marked decrease in the magnitude of calcium flux after stimulation with 5 nm SDF-1α (Fig. 2, top panel). However, this inhibitory effect on calcium signaling was mostly abrogated when a much higher dose of SDF-1α (50 nm) was applied (Fig. 2, bottom panel). The latter observation raises the possibility of competition between two ligands for a common receptor, although heterologous desensitization of CXCR4 signaling by TFF2 through a distinct receptor could not be excluded. TFF2 Stimulates Calcium Mobilization and Protein Kinase B/AKT Activation in Jurkat Cells—We next tested recombinant TFF2 protein for the ability to directly stimulate signaling pathways in Jurkat cells. To this end, we measured Ca2+ flux in Jurkat cells after exposure to different concentrations of highly purified recombinant mouse TFF2. As shown in Fig. 3 (top), murine TFF2 was able to induce a prominent calcium rise in Jurkat cells with a
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