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• W2012015660 abstract "Activation of T cell antigen receptor (TCR) signaling inhibits glucocorticoid (GC)-induced apoptosis of T cells. However, the detailed mechanism regarding how activated T cells are protected from GC-induced apoptosis is unclear. Previously, we have shown that the expression level of SRG3, a murine homolog of BAF155 in humans, correlated well with the GC sensitivity of T cells either in vitro or in vivo. Intriguingly, the expression of SRG3 decreased upon positive selection in the thymus. Here we have shown that TCR signaling inhibits the SRG3 expression via Ras activation and thereby renders primary thymocytes and some thymoma cells resistant to GC-mediated apoptosis. By using pharmacological inhibitors, we have shown that Ras-mediated down-regulation of the SRG3 gene expression is mediated by MEK/ERK and phosphatidylinositol 3-kinase pathways. Moreover, TCR signals repressed the SRG3 transcription through the putative binding sites for E proteins and Ets family transcription factors in the proximal region of the SRG3 promoter. Introduction of mutations in these elements rendered the SRG3 promoter immune to the Ras or TCR signals. Taken together, these observations suggest that TCR signals result in GC desensitization in immature T cells by repressing SRG3 gene expression via Ras activation. Activation of T cell antigen receptor (TCR) signaling inhibits glucocorticoid (GC)-induced apoptosis of T cells. However, the detailed mechanism regarding how activated T cells are protected from GC-induced apoptosis is unclear. Previously, we have shown that the expression level of SRG3, a murine homolog of BAF155 in humans, correlated well with the GC sensitivity of T cells either in vitro or in vivo. Intriguingly, the expression of SRG3 decreased upon positive selection in the thymus. Here we have shown that TCR signaling inhibits the SRG3 expression via Ras activation and thereby renders primary thymocytes and some thymoma cells resistant to GC-mediated apoptosis. By using pharmacological inhibitors, we have shown that Ras-mediated down-regulation of the SRG3 gene expression is mediated by MEK/ERK and phosphatidylinositol 3-kinase pathways. Moreover, TCR signals repressed the SRG3 transcription through the putative binding sites for E proteins and Ets family transcription factors in the proximal region of the SRG3 promoter. Introduction of mutations in these elements rendered the SRG3 promoter immune to the Ras or TCR signals. Taken together, these observations suggest that TCR signals result in GC desensitization in immature T cells by repressing SRG3 gene expression via Ras activation. Signals triggered by the activation of glucocorticoid (GC) 1The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; GRE, GC response element; TCR, T cell receptor; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; SP, single-positive; DP, double-positive; PMA, phorbol 12-myristate 13-acetate; Dex, dexamethasone; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FBS, fetal bovine serum; 2-ME, 2-mercaptoethanol; HEB, HeLa E box-binding; EMSA, electrophoretic mobility shift assay. 1The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; GRE, GC response element; TCR, T cell receptor; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; SP, single-positive; DP, double-positive; PMA, phorbol 12-myristate 13-acetate; Dex, dexamethasone; FITC, fluorescein isothiocyanate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FBS, fetal bovine serum; 2-ME, 2-mercaptoethanol; HEB, HeLa E box-binding; EMSA, electrophoretic mobility shift assay. receptor (GR) or T cell antigen receptor (TCR) alone potently induce death of T cells (1Liu Z.G. Smith S.W. McLaughlin K.A. Schwartz L.M. Osborne B.A. Nature. 1994; 367: 281-284Crossref PubMed Scopus (494) Google Scholar, 2Smith C.A. Williams G.T. Kingston R. Jenkinson E.J. Owen J.J. Nature. 1989; 337: 181-184Crossref PubMed Scopus (1106) Google Scholar, 3Thompson E.B. Trends Endocrinol. Metab. 1999; 10: 353-358Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 4Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar). However, simultaneous activation of both receptors fails to induce apoptosis by antagonizing the other signaling pathway (5Ashwell J.D. King L.B. Vacchio M.S. Stem Cells. 1996; 14: 490-500Crossref PubMed Scopus (61) Google Scholar, 6Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (201) Google Scholar, 7Iwata M. Ohoka Y. Kuwata T. Asada A. Stem Cells. 1996; 14: 632-641Crossref PubMed Scopus (44) Google Scholar, 8Philips A. Maira M. Mullick A. Chamberland M. Lesage S. Hugo P. Drouin J. Mol. Cell. Biol. 1997; 17: 5952-5959Crossref PubMed Scopus (174) Google Scholar, 9Vacchio M.S. Papadopoulos V. Ashwell J.D. J. Exp. Med. 1994; 179: 1835-1846Crossref PubMed Scopus (313) Google Scholar, 10Vacchio M.S. Ashwell J.D. Semin. Immunol. 2000; 12: 475-485Crossref PubMed Scopus (51) Google Scholar, 11Zacharchuk C.M. Mercep M. Chakraborti P.K. Simons Jr., S.S. Ashwell J.D. J. Immunol. 1990; 145: 4037-4045PubMed Google Scholar, 12Stephens G.L. Ashwell J.D. Ignatowicz L. Int. Immunol. 2003; 15: 623-632Crossref PubMed Scopus (12) Google Scholar). There has been some evidence that GCs inhibit TCR-induced apoptosis by repressing FasL expression (13Yang Y. Mercep M. Ware C.F. Ashwell J.D. J. Exp. Med. 1995; 181: 1673-1682Crossref PubMed Scopus (206) Google Scholar). In addition, by observing thymocyte development in mice with altered levels of GCs or GR, it has been shown that in interplay with TCR, GCs affect thymic development (14King L.B. Vacchio M.S. Dixon K. Hunziker R. Margulies D.H. Ashwell J.D. Immunity. 1995; 3: 647-656Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 15Tolosa E. King L.B. Ashwell J.D. Immunity. 1998; 8: 67-76Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Vacchio M.S. Ashwell J.D. J. Exp. Med. 1997; 185: 2033-2038Crossref PubMed Scopus (126) Google Scholar, 17Vacchio M.S. Lee J.Y. Ashwell J.D. J. Immunol. 1999; 163: 1327-1333PubMed Google Scholar). Reciprocally, proper stimulation through TCR/CD3 antagonized GR activity and might contribute to GC resistance in T cells largely through the Ras activation of MEK/ERK cascade (18Jamieson C.A. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7319-7324Crossref PubMed Scopus (83) Google Scholar). Activation of Ras alone, instead of TCR/CD3 engagement, was sufficient to inhibit GC-mediated apoptosis by activating Raf and Ral.GDS as mediators of the survival signals. Clonal deletion of immature T cells, which are nonfunctional or self-reactive, is crucial for maintenance of the normal immune system. In particular, glucocorticoids (GCs) have been suggested to trigger programmed cell death of T cells and may help to eliminate developing thymocytes that fail to differentiate properly (17Vacchio M.S. Lee J.Y. Ashwell J.D. J. Immunol. 1999; 163: 1327-1333PubMed Google Scholar, 19Ashwell J.D. Lu F.W. Vacchio M.S. Annu. Rev. Immunol. 2000; 18: 309-345Crossref PubMed Scopus (648) Google Scholar, 20Pazirandeh A. Xue Y. Prestegaard T. Jondal M. Okret S. FASEB J. 2002; 16: 727-729Crossref PubMed Scopus (83) Google Scholar). They are either produced by thymic epithelium and possibly thymocytes themselves or are transferred from the adrenal gland in an endocrine manner (9Vacchio M.S. Papadopoulos V. Ashwell J.D. J. Exp. Med. 1994; 179: 1835-1846Crossref PubMed Scopus (313) Google Scholar). Due to their lipophilic properties, GCs can diffuse into the cytoplasm of target cells including thymocytes, bind to and activate their receptor, GR, which upon ligand binding translocates into the nucleus and associates with specific GC-response elements (GREs) to evoke various effects by modulating the expression of specific genes either positively or negatively depending on cell and gene context. GC sensitivity of thymocytes is developmentally regulated during maturation in the thymus. Immature CD4+CD8+ double-positive (DP) thymocytes are known to be exquisitely susceptible to GC-induced apoptosis, whereas mature CD4+ or CD8+ single-positive (SP) T cells are relatively resistant (4Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 21Gruber J. Sgonc R. Hu Y.H. Beug H. Wick G. Eur. J. Immunol. 1994; 24: 1115-1121Crossref PubMed Scopus (121) Google Scholar). Thus, it is conceivable that the immature thymocytes to be positively selected should be protected from apoptotic actions of GCs. Thus far, many studies have addressed the cross-talk pathway for the inhibition of GC-induced apoptosis by TCR- or Notch-mediated signaling (6Iwata M. Hanaoka S. Sato K. Eur. J. Immunol. 1991; 21: 643-648Crossref PubMed Scopus (201) Google Scholar, 22Deftos M.L. He Y.W. Ojala E.W. Bevan M.J. Immunity. 1998; 9: 777-786Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 23Wagner Jr., D.H. Hagman J. Linsley P.S. Hodsdon W. Freed J.H. Newell M.K. J. Exp. Med. 1996; 184: 1631-1638Crossref PubMed Scopus (58) Google Scholar). Only a small population of DP thymocytes that acquire GC resistance by signaling through TCR/CD3 or NotchI appear to survive GC-triggered cell death and differentiate into mature SP thymocytes. Relatively little is known, however, regarding the specific nuclear target that couples these surface signals with GC resistance and thereby contributes to distinguishing thymocytes fated to die by GCs from those destined to survive. SRG3 (for SWI3-related gene), a murine homolog of yeast SWI3 or human BAF155, was originally isolated as a gene expressed at higher levels in the thymus than in the periphery (24Jeon S.H. Kang M.G. Kim Y.H. Jin Y.H. Lee C. Chung H.Y. Kwon H. Park S.D. Seong R.H. J. Exp. Med. 1997; 185: 1827-1836Crossref PubMed Scopus (42) Google Scholar). It is presumed to play crucial roles as a core component of the SWI/SNF complex by remodeling chromatin structure (25Kim J.K. Huh S.O. Choi H. Lee K.S. Shin D. Lee C. Nam J.S. Kim H. Chung H. Lee H.W. Park S.D. Seong R.H. Mol. Cell. Biol. 2001; 21: 7787-7795Crossref PubMed Scopus (167) Google Scholar, 26Phelan M.L. Sif S. Narlikar G.J. Kingston R.E. Mol. Cell. 1999; 3: 247-253Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Recently, we found that the level of SRG3 expression is critical in determining GC sensitivity of developing thymocytes (24Jeon S.H. Kang M.G. Kim Y.H. Jin Y.H. Lee C. Chung H.Y. Kwon H. Park S.D. Seong R.H. J. Exp. Med. 1997; 185: 1827-1836Crossref PubMed Scopus (42) Google Scholar, 27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar, 28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). The SRG3 protein was required for GC-induced apoptosis of the S49.1 thymoma cells (24Jeon S.H. Kang M.G. Kim Y.H. Jin Y.H. Lee C. Chung H.Y. Kwon H. Park S.D. Seong R.H. J. Exp. Med. 1997; 185: 1827-1836Crossref PubMed Scopus (42) Google Scholar). SRG3 associated with the GR and enhanced GC-induced apoptosis by potentiating its transcriptional activity (28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). In wild-type mice, the SRG3-GR complex was readily detected in GC-sensitive immature thymocytes but barely detectable in GC-resistant mature T lymphocytes. Intriguingly, the sensitivity to GCs in T cells could be modified by altering the expression level of SRG3 in transgenic mice, demonstrating clear correlation between SRG3 protein levels and GC sensitivity. Peripheral T lymphocytes in transgenic mice overexpressing SRG3 became more susceptible to GCs than in wild-type mice because of the increase in the SRG3-GR complex (28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). Conversely, immature thymocytes in transgenic mice overexpressing SRG3 mRNA in an antisense orientation were more GC-resistant than wild-type controls (27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). In the present study, we found that TCR/CD3 signaling inhibits GC-induced apoptosis of primary thymocytes and immature DP cells through SRG3 down-modulation. Signals from TCR/CD3 repressed the SRG3 transcription via Ras activation. As a result, the physical interaction between SRG3 and GR was inhibited, resulting in GC desensitization. However, thymocytes in transgenic mice overexpressing SRG3 were more sensitive to GC-triggered apoptosis compared with wild-type controls, and TCR signals did not suppress the cell death as potently as in wild-type mice. TCR repression of SRG3 expression was largely mediated through activation of the Ras/MEK/ERK or PI3K pathway, which was previously suggested to be critical for TCR inhibition of GC-induced apoptosis. In addition, we found that TCR signals suppress the SRG3 transcription through the putative binding sites for E proteins and Ets family transcription factors in the SRG3 promoter. Collectively, these observations suggest a model for the role of SRG3 in the cross-talk pathway for inhibition of GC-mediated apoptosis by TCR signals. Mice and Cells—Transgenic mice overexpressing SRG3 in FVB background were previously described (28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). The S49.1 murine thymoma cell was purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). The murine T cell hybridoma, KCIT1-8.5, was obtained from Dr. Y. Choi (University of Pennsylvania School of Medicine, Philadelphia) and maintained in RPMI 1640 containing 10% FBS supplemented with 50 μm 2-mercaptoethanol (2-ME). The murine double-positive (DP) thymoma, 16610D9, was provided by Dr. C. Murre (University of California, San Diego, La Jolla, CA) and cultured in Opti-MEM (Invitrogen) containing 10% FBS supplemented with 50 μm 2-ME. All media were supplemented with penicillin and streptomycin. Reagents and Antibodies—We purchased PD98059 and SB203580 from Calbiochem; wortmannin, phorbol 12-myristate 13-acetate (PMA), A23187 (ionomycin), and dexamethasone (Dex) from Sigma; anti-actin (sc-1615), anti-GR (M-20), anti-HEB (A-20X), and anti-hamster IgG (H-4772) antibodies from Santa Cruz Biotechnology; fluorescein isothiocyanate (FITC)-conjugated anti-CD69 (01504D), FITC-conjugated annexin V (556419), anti-Lck (554281), anti-E47 (554077), and anti-E12 (556509) antibodies from Pharmingen; and anti-Ras (R02120) antibody from Transduction Laboratories. The anti-TCRβ, anti-CD3ϵ, and anti-CD4 antibodies were purified from hybridoma supernatants from the H57.597, YCD3, and GK1.5 lines, respectively. Antiserum against SRG3 was raised from rabbits in our laboratory as described previously (28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). Cell Stimulation, Immunoprecipitation, Immunoblotting, and Flow Cytometry—Single cell suspensions of thymocytes (1–2 × 106) from 4- to 5-week-old mice were suspended in 1 ml of RPMI 1640 medium containing 10% FBS and 50 μm 2-ME supplemented with glutamine, penicillin, and streptomycin and cultured in medium alone or treated with PMA + ionomycin for 3 h. After further incubation for 12 h in the absence or presence of Dex treatment, the cells were stained with FITC-conjugated annexin V and propidium iodide (PI) according to the manufacturer's protocol. PI-negative (live) cells were electronically gated and analyzed by flow cytometry (20,000–30,000 events) with the CellQuest™ software using FACStar (BD Biosciences). Whole-cell extracts from the cells unstimulated or stimulated with PMA + ionomycin for 12 h were immunoprecipitated with anti-GR antibodies (M-20x; Santa Cruz Biotechnology) as described previously (29Fryer C.J. Archer T.K. Nature. 1998; 393: 88-91Crossref PubMed Scopus (407) Google Scholar) and subjected to immunoblotting (28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). For stimulating thymoma cells, 6-well plates were coated with appropriate antibodies at 10 μg/ml at 4 °C overnight followed by washing with phosphate-buffered saline. Five million cells at 106 cells/ml were added to each well and incubated at 37 °C for 24 h. For stimulation of T cell lines by PMA + ionomycin, cells were treated with 7.4 ng/ml PMA and 0.26 μg/ml ionomycin or 0.2 ng/ml PMA and 0.25 μg/ml ionomycin. The cells (106 cells) were then stained with FITC-conjugated anti-CD69 antibody and propidium iodide (PI). After washing with phosphate-buffered saline, the surface expression of CD69 was analyzed by flow cytometry. The remaining cells were used for immunoblotting. Northern Blot Analysis—Total RNA was prepared from cells by resuspension in TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (7.5–10 μg) was resolved on a 1.2% formaldehyde gel and then blotted to Hybond™-N (Amersham Biosciences). Northern blots were probed with Id3 and SRG3 probes. The 405-bp Id3 probe was generated by random priming using the EcoRI fragment of Id3/S-003 vector, and the specific probe for SRG3 was generated as described previously (27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). Plasmids, Mutagenesis, Transient Transfection, and Reporter Assay—The pSRG3-Luc reporter construct was described previously (27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). K-RasV12, K-RasV12S35, K-RasV12G37, and K-RasV12C40 were from Dr. S. Yonehara (Kyoto University, Japan). H-RasV12 was from Dr. Han W. Lee (Sung Kyun Kwan University, Suwon, Korea). Mutagenesis of the E box sequences (CATCTG into CTGCAG) or Ets-binding site (CCGGAAGA to CCGAGAGA) in the SRG3 promoter, transient transfection, and luciferase assay were performed as described previously (27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). The PCR primers for the mutagenesis are as follows: E84 element, 5′-AGGAGGTGGCTGCAGCGCGCGCGG-3′ and 5′-CCGCGCGCGCTGCAGCCACCTCCT-3′; EBS130 element, 5′-CCGCGCCTCGAGCCGAGAGAGGGTTGGCTG-3′ and 5′-CAGCCAACCCTCTCTCGGCTCGAGGCGCGG-3′. Electrophoretic Mobility Shift Assay (EMSA)—Preparation of nuclear extracts was performed as described (30Kim D. Xu M. Nie L. Peng X.C. Jimi E. Voll R.E. Nguyen T. Ghosh S. Sun X.H. Immunity. 2002; 16: 9-21Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). EMSA was performed as follows. Double-stranded oligonucleotides were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase and purified over a MicroSpin™ G-25 column (Amersham Biosciences). Nuclear extracts (2–5 μg) were incubated with an appropriate probe and 2 μg of poly(dI-dC) for 30 min at room temperature in 20 μl of binding buffer containing 20 mm HEPES (pH 7.9), 40 mm KCl, 2.5 mm MgCl2, 1 mm dithiothreitol, and 5% glycerol. For antibody-mediated supershifts or competition experiments, the extracts were pre-incubated with each antibody for 30 min or DNA competitor for 30 min at room temperature, respectively, before addition of the radiolabeled oligonucleotide probe. The resulting protein-DNA complexes were resolved in a nondenaturating 5% acrylamide gel in 0.5× TBE buffer. Gels were dried and visualized by autoradiography. The sequence of each oligonucleotide used for EMSA is as follows: Sp152, 5′-GGTCCAGAAGGGGCGTGGCCGCGCCTCGAG-3′; Sp114, 5′-AGGGTTGGCTGGGCGGGGCTAGGAGGAGGA-3′; EBS130, 5′-CCGCGCCTCGAGCCGGAAGAGGGTTGGCTG-3′; E84, 5′-AGGAGGTGGCATCTGCGCGCGCGG-3′; μE5, 5′-TCGAAGAACACCTGCAGCAGCT-3′; Sp1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′; and Oct-1, 5′-TGTCGAATGCAAATCACTAGAA-3′. Other oligonucleotides used for competition studies are as follows: 1) mb-1, 5′-TCGAGTGAACAGGAAGTGAGGCGGAGTCGA-3′; 2) PEG-3, 5′-TGGAGAGAGGAAAACAACAC-3′; 3) major histocompatibility complex class II promoter, 5′-TCGAGAGTGAGGAACCAATCAG-3′; 4) Fas, 5′-TGGCCAGGAAATAATGAGTAACGAAGGACAGGAAGTAATTGT-3′; 5) IgH enhancer π, 5′-TCGACTGGCAGGAAGCAGGTCATGC-3′; 6) glycoprotein IX (GPIX), 5′-ATTTTCATCATCACTTCCTTCCGC-3′; 7) polyomavirus enhancer, 5′-GATCTTTAAGCAGGAAGTGACTAACTGACCGCAGGTGGATC-3′; 8) E74, 5′-TCGAGTAACCGGAAGTAACTCAG-3′; 9) consensus Ets1/PEA3 (Santa Cruz Biotechnology, sc-2555), 5′-GATCTCGAGCAGGAAGTTCGA-3′; 10) consensus PU.1/GABPα (Santa Cruz Biotechnology, sc-2549), 5′-GGGCTGCTTGAGGAAGTATAAGAAT-3′; 11) consensus mutant Ets-1 (Santa Cruz Biotechnology, sc-2556), 5′-GATCTCGAGCAAGAAGTTCGA-3′; 12) consensus serum-response element (Santa Cruz Biotechnology, sc-2523), 5′-GGATGTCCATATTAGGACATCT-3′. Repression of SRG3 Expression upon TCR/CD3 Engagement Independently of TCR-induced Cell Death—We found previously that SRG3 acts as a coactivator for GR to up-regulate the transcriptional activity of this receptor and thereby render thymocytes sensitive to GC-mediated apoptosis (24Jeon S.H. Kang M.G. Kim Y.H. Jin Y.H. Lee C. Chung H.Y. Kwon H. Park S.D. Seong R.H. J. Exp. Med. 1997; 185: 1827-1836Crossref PubMed Scopus (42) Google Scholar, 28Han S. Choi H. Ko M.G. Choi Y.I. Sohn D.H. Kim J.K. Shin D. Chung H. Lee H.W. Kim J.B. Park S.D. Seong R.H. J. Immunol. 2001; 167: 805-810Crossref PubMed Scopus (33) Google Scholar). The relative sensitivity of developing thymocytes to the GCs was modulated depending on the expression level of SRG3 proteins. Intriguingly, the level of SRG3 mRNA in the positively selected thymocytes (CD3highCD69+) in the thymus was about three times lower than in the pre-selected thymocytes (CD3lowCD69–) (27Choi Y.I. Jeon S.H. Jang J. Han S. Kim J.K. Chung H. Lee H.W. Chung H.Y. Park S.D. Seong R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10267-10272Crossref PubMed Scopus (31) Google Scholar). Because selection events in the thymus are active processes that involve various signals emanating from TCR/CD3 complex and other accessory molecules, and TCR/CD3 signaling has been shown to antagonize GC-induced apoptosis of T cells (19Ashwell J.D. Lu F.W. Vacchio M.S. Annu. Rev. Immunol. 2000; 18: 309-345Crossref PubMed Scopus (648) Google Scholar), it is possible that TCR-mediated signals render thymocytes resistant to GCs by modulating the SRG3 expression level. To assess whether SRG3 expression is directly modulated by antibody-mediated TCR/CD3 engagement, the murine DP thymoma cell line, 16610D9, was cultured in plates coated with anti-CD3ϵ or anti-TCRβ antibody, alone or in combination with anti-CD4 antibody. The 16610D9 cell line is derived from a spontaneous thymoma in a p53-deficient mouse and exhibits characteristics typical of primary DP thymocytes (HSAhighTCRmedCD5lowCD44lowCD69low) (31Bain G. Quong M.W. Soloff R.S. Hedrick S.M. Murre C. J. Exp. Med. 1999; 190: 1605-1616Crossref PubMed Scopus (107) Google Scholar). The expression of CD69, an early T cell activation marker (32Swat W. Dessing M. von Boehmer H. Kisielow P. Eur. J. Immunol. 1993; 23: 739-746Crossref PubMed Scopus (255) Google Scholar), was specifically induced by antibody-mediated TCR/CD3 engagement, whereas it remained unchanged when treated with anti-hamster IgG control antibody (Fig. 1A). Subsequently, the level of SRG3 proteins in the whole-cell extracts from control and TCR-stimulated cells was examined by immunoblotting. As shown in Fig. 1C, the expression level of SRG3 protein decreased maximally by 55.3% upon T cell activation, whereas that of GR did not. The degree of SRG3 down-regulation correlated well with that of CD69 induction, suggesting that SRG3 repression may depend on the intensity of TCR/CD3 signals. When another thymoma cell line, S49.1, was activated with anti-CD3ϵ, the expression of SRG3 also decreased maximally by 64% (Fig. 1D). These findings suggest that the expression of SRG3 in immature thymoma cells is down-regulated in response to signals through TCR/CD3 ligation. In addition to antibody-mediated TCR/CD3 cross-linking, cotreatment of a defined combination of PMA, a protein kinase C activator and ionomycin, a calcium ionophore also resulted in a decrease in SRG3 protein expression (Fig. 1D). Intracellular signaling triggered by the combinations of these drugs was shown to mimic the anti-apoptotic effect of proper cross-linking of the TCR/CD3 complex and induce thymocyte survival and differentiation (6Iwata M. Hanaoka S. Sato K. Eur. J. 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Because combination of PMA + ionomycin may bypass the early consequences of TCR cross-linking, including membrane proximal signals in thymocytes, these results suggest that TCR-proximal signals are not required for TCR-mediated repression of SRG3 gene expression. The viability of the activated cells was not significantly affected by antibody-mediated TCR/CD3 cross-linking, although cotreatment of PMA + ionomycin led to a slight increase in cell death (data not shown). However, to exclude the possibility that the TCR/CD3-mediated SRG3 down-regulation resulted from nonspecific protein degradation in cells undergoing TCR-induced cell death, a T cell hybridoma, KCIT1-8.5, was used. The KCIT1-8.5 cell line is resistant to TCR-induced apoptosis due to the defect in the T cell death-associated gene 51 (TDAG51), which couples TCR stimulation with CD95 (Fas) expression but is still competent to be activated by TCR triggering (39Park C.G. Lee S.Y. Kandala G. Choi Y. Immunity. 1996; 4: 583-591Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 40Wong B. Park C.G. Choi Y. Semin. Immunol. 1997; 9: 7-16Crossref PubMed Scopus (15) Google Scholar). Stimulation of KCIT1-8.5 with anti-CD3ϵ or anti-TCRβ antibody alone was sufficient for CD69 induction with no significant effect on the cell viability (Fig. 1B). As shown in Fig. 1E, activation of KCIT1-8.5 also resulted in a dramatic reduction in SRG3 proteins by 82%. These results indicate that SRG3 expression is reduced upon T cell activation independently of TCR-induced cell death. TCR-mediated Repression of SRG3 Transcription via Ras Activation of MEK/ERK and PI3K Pathways—To determine whether TCR signals modulate the SRG3 expression at the transcriptional level, 16610D9 DP cells were stimulated with PMA + ionomycin for varying amounts of time, and the mRNA levels of SRG3 and Id3 were quantified by Northern blot analysis. As reported previously (41Bain G. Cravatt C.B. Loomans C. Alberola-Ila J. Hedrick S.M. Murre C. Nat. Immun. 2001; 2: 165-171Crossref Scopus (224) Google Scholar), activation of these T cells rapidly induced Id3 transcripts (Fig. 2A). Id3 induction became much stronger as the period of stimulation lasted longer than 6 h. However, the level of SRG3 transcripts began to be down-regulated, albeit slowly, in the early phase but led to a dramatic decrease 6 h after T cell activation (Fig. 2A). The suppression of SRG3 transcription upon T cell stimulation by either antibody-mediated TCR/CD3 cross-linking or cotreatment of PMA + ionomycin was well reflected by a change in the SRG3 protein expression (Fig. 2C and data not shown). Because protein expression of the SRG3 gene seemed to depend on the strength of TCR signals (Fig. 1, A and C), we further investigated how SRG3 transcription is regulated when much weaker activation signals are transduced. At lower concentrations of PMA + ionomycin, Id3 transcripts were not significantly induced until 18 h (Fig. 2B). Concomitantly, SRG3 transcripts were not reduced until 18 h. Collectively, these results indicate that SRG3 transcription is repressed upon T cell activation. Engagement of TCR/CD3 complex was shown to activate intricate intracellular signaling networks, including Ras/MAPK pathway (42Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (683) Google Scholar, 43Downward J. Curr. Opin. Genet. Dev. 1998; 8: 49-54Crossref PubMed Scopus (503) Google Scholar, 44Dustin M.L. Chan A.C. Cell. 2000; 103: 283-294Abstract Full Text Full Text PDF PubMed Scopus (201) G" @default.
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