The transcription factor Sox4 is a downstream target of signaling by the cytokine TGF-β and suppresses TH2 differentiation

Journal name:
Nature Immunology
Volume:
13,
Pages:
778–786
Year published:
DOI:
doi:10.1038/ni.2362
Received
Accepted
Published online

Abstract

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-β and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (TH2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a TH2 cytokine, and prevented binding of GATA-3 to this promoter. TH2 cell–driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-β to inhibit GATA-3 function, TH2 differentiation and TH2 cell–mediated inflammation.

At a glance

Figures

  1. TGF-[beta]-induced Sox4 expression in CD4+ T cells.
    Figure 1: TGF-β-induced Sox4 expression in CD4+ T cells.

    (a) Quantitative RT-PCR analysis of Sox4 mRNA in naive CD4+ T cells stimulated for 0–72 h with immobilized mAb to TCRβ in the presence of IL-2; results are presented relative to the expression of Hprt mRNA (encoding hypoxanthine guanine phosphoribosyl transferase). (b) Quantitative RT-PCR analysis of Sox4 mRNA in naive CD4+ T cells left inactivated (TCR−) or activated for 24 h with mAb to TCR (TCR+) in the presence (+) or absence (−) of TGF-β. (c) Immunoblot analysis of Sox4 in the nuclear fraction of naive CD4+ T cells cultured as in b; analysis with antibody to α-tubulin (Anti-α-tubulin) serves as a loading control for the cytosolic fraction (bottom). (d) ChIP assay of the binding of Smad2-Smad3 (Smad2,3) or immunoglobulin (Ig; control) to a 5′ region upstream of the Sox4 locus (Sox4u) or the promoter of the gene encoding the invariant signaling protein CD3ε (Cd3e) in naive CD4+ T cells cultured for 12 h in the presence of medium (Med) or TGF-β; results are presented relative to those of input DNA. (e) Quantitative RT-PCR analysis of Sox4 and Smad3 mRNA in naive CD4+ T cells mock transduced (Mock) or transduced to express Smad3, then stimulated with mAb to TCR, followed by stimulation for 24 h with TGF-β (presented as in a). (f) Quantitative RT-PCR analysis of Sox4 and Smad7 mRNA in naive CD4+ T cells mock transduced or transduced to express Smad7, then stimulated with mAb to TCR, followed by no stimulation (−) or stimulation with TGF-β (+; presented as in a). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of four (a; mean and s.d. of three samples), three (b,d) or two (c,e,f) independent experiments with similar results (error bars (b,df, s.d.).

  2. Involvement of Sox4 in the TGF-[beta]-mediated inhibition of TH2 differentiation.
    Figure 2: Involvement of Sox4 in the TGF-β-mediated inhibition of TH2 differentiation.

    (a) Intracellular staining of IL-4 and IFN-γ in cells cultured under TH1- or TH2-polarizing conditions with medium alone or TGF-β. Numbers in quadrants indicate percent cells in each throughout. (b) Quantitative RT-PCR analysis of cytokine-encoding mRNA in TH1 and TH2 cells left untreated (−) or treated with TGF-β (+), then restimulated for 24 h with immobilized anti-TCR (presented as in Fig. 1a). (c) ELISA of cytokines in supernatants of the cells in b. (d) Quantitative RT-PCR analysis of cytokine-encoding mRNA in cells treated with control shRNA (Mock) or Sox4-specific shRNA (shSox4) and left untreated or treated with TGF-β (presented as in Fig. 1a). (e) Quantitative RT-PCR analysis of Il5 and Sox4 mRNA in cells treated with control siRNA (Ctrl) or Sox4-specific siRNA (siSox4 (1) and siSox4 (2)) and left untreated or treated with TGF-β (presented as in Fig. 1a). (f) ChIP assay of the binding of GATA-3, Sox4 and immunoglobulin to the Il5 promoter (Il5p), the CGRE and the VA site in the Il4 enhancer (VA enh) in developing TH2 cells cultured with medium alone or TGF-β (presented as in Fig. 1d). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three (a,b,d,e) or two (c,f) independent experiments with similar results (error bars (bf), s.d.).

  3. Enforced expression of Sox4 inhibits TH2 differentiation.
    Figure 3: Enforced expression of Sox4 inhibits TH2 differentiation.

    (a) Staining of IL-4 and IFN-γ in CD4+ T cells left uninfected (hNGFR) or infected (hNGFR+), and transduced with empty vector (mock transduced; Mock) or with a Sox4-expressing retroviral vector (Sox4) and then cultured for 3 d under TH1- or TH2-polarizing conditions. (b) ELISA of cytokines in TH2 cells mock transduced (Mock) or transduced with a Sox4-expressing retroviral vector (Sox4) and restimulated with immobilized mAb to TCRβ. (c) Quantitative RT-PCR analysis of mRNA encoding various transcription factors (vertical axes) in TH2 cells transduced as in b (presented as in Fig. 1a). (d) Staining of IL-4 and IFN-γ in wild-type (WT) and Sox4-transgenic (TG) naive CD4+ T cells cultured under TH2-polarizing conditions with various concentrations (above plots) of anti-TCRβ (α-TCRβ) or under TH1-polarizing conditions (far right). (e) ELISA of cytokines in supernatants of wild-type and Sox4-transgenic TH2 cells generated in vitro and stimulated for 16 h with immobilized mAb to TCRβ at a concentration of 0.3 or 3 μg/ml (key). (f) Quantitative RT-PCR analysis of mRNA encoding various transcription factors (vertical axes) in wild-type and Sox4-transgenic CD4+ T cells (presented as in Fig. 1a). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three (a,b,df) or four (c) independent experiments with similar results (error bars (b,c,e,f), s.d.).

  4. Physical association of Sox4 with GATA-3.
    Figure 4: Physical association of Sox4 with GATA-3.

    (a) Immunoprecipitation (IP) and immunoblot analysis (IB) of the association of Sox4 and GATA-3 in 293T cells left untransfected (−) or transfected (+) to express Myc-tagged Sox4 (Myc-Sox4) and/or Flag-tagged GATA-3 (Flag–GATA-3); below (Input), parallel analysis of total cell lysates (without immunoprecipitation). (b) Immunoprecipitation and immunoblot analysis of the association of endogenous GATA-3 with endogenous Sox4 in TG40 cells (Input (middle right), as in a). NS, nonspecific band. (c) Precipitation assay (Ppt) of the association of Sox4 and GATA-3 in cells transfected to express Myc-tagged recombinant Sox4 (Myc-Sox4), glutathione S-transferase–tagged recombinant GATA-3 (GST–GATA-3) and/or glutathione S-transferase alone (GST), probed with anti-Myc; below (Input), immunoblot analysis of an aliquot of input samples (1/15 volume) with anti-Myc or anti-GST. (d) Immunoprecipitation and immunoblot analysis of the association of Sox4 and GATA-3 in 293T cells mock transfected (–) or transfected to express Flag-tagged GATA-3 and Myc-tagged wild-type Sox4 (WT) or mutant Sox4 lacking amino acids 60–71 (Δ60–71) or amino acids 203–214 (Δ203–214) or with substitution of alanine residues for arginine and proline (RP(61–62)AA), phenylalanine and methionine (FM(66–67)AA) or methionine and glutamic acid (ME(78–79)AA); Input (below), as in a. (e) Staining of IL-4, IL-5 and IFN-γ in TH2 cells left untransduced (hNGFR) or transduced (hNGFR+) and mock transfected or transfected to express wild-type or mutant Sox4 (as in d), assessed 3 d after retroviral infection. Data are representative of at least three independent experiments with similar results.

  5. Sox4 interferes with the binding of GATA-3 to DNA.
    Figure 5: Sox4 interferes with the binding of GATA-3 to DNA.

    (a) Precipitation assay of the binding of GATA-3 to a GATA-binding consensus motif in 293T cells transfected to express Myc-tagged Sox4 and/or Flag-tagged GATA-3 (Input (below), immunoblot analysis of whole-cell lysates without precipitation (control). Wedges indicate threefold 'titration' of input lysates. (b) ChIP analysis of the binding of GATA-3 and immunoglobulin at the CGRE, Il4 VA site and Il5 promoter region in Sox4-transgenic TH2 cells (presented as in Fig. 1d). (c) Quantitative RT-PCR analysis of mRNA encoding TH2 cytokines in TH2 cells mock infected (Mock; far left) or infected with retroviral vector encoding Sox4 or GATA-3 alone (middle) or together (GATA-3 + Sox4; far right) and stimulated with immobilized mAb to TCRβ (presented as in Fig. 1a). ND, not detected. (d) Probes used in e, containing the wild-type Il5 promoter (Il5p WT) or Il5 promoter with mutation (red) of GATA-binding site 1 (GATA-BS1 (blue outline)) alone (Il5p Mut1) or in combination with mutation of the Sox-binding site (Sox-BS (green); Il5p Mut2), which overlaps GATA-binding site 2 (GATA-BS2 (blue outline)). (e) Precipitation assay of mixtures of lysates of 293T cells mock transfected (−) or transfected (+) to express a Myc-tagged Sox4 mutant lacking the carboxy-terminal region (Myc–Sox4-ΔC) or various concentrations (wedges) of Flag-tagged GATA-3, assessed with the mutated probes in d (Input (below), as in a). (f) Luciferase activity in M12 B cells left untransfected (−) or transfected (+) to express wild-type Sox4 or the Sox4 mutant in e, plus GATA-3, as well as a firefly luciferase reporter for the Il5 promoter, then left unstimulated (Med) or stimulated (Stim) with the phorbol ester PMA (30 ng/ml) and dibuteryl cAMP (100 μM); results are presented relative to renilla luciferase activity. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of at least three independent experiments with similar results (mean and s.d. of three samples in c; mean and s.d. in b and f).

  6. Attenuated OVA-induced allergic airway inflammation in Sox4-transgenic mice.
    Figure 6: Attenuated OVA-induced allergic airway inflammation in Sox4-transgenic mice.

    (a) Quantification of eosinophils (Eos), neutrophils (Neu), lymphocytes (Lym), macrophages (Mφ) and total cells in BAL fluid from wild-type and Sox4-transgenic mice (n = 5 per group) left unimmunized (ctrl) or immunized with OVA (imm). *P < 0.001 (analysis of variance (ANOVA) and Bonferroni test). (b) Quantitative RT-PCR analysis of Il4, Il5 and Il13 mRNA in cells from the BAL fluid of mice as in a. *P < 0.01 (Student's t-test). (c,d) Microscopy (c (left) and d) of lungs from mice as in a, fixed and stained with hematoxylin and eosin (c) or periodic acid–Schiff reagent (d). Original magnification, ×200 (scale bars, 10 μm). Right (c), quantification of leukocytes in the peribronchiolar region (cells per mm2). *P < 0.01 (Student's t-test). (e) Quantitative RT-PCR analysis of Clca3 and Muc2 mRNA in lungs from mice as in a (n = 3 per group). (f) Airway resistance of mice as in a (n = 6 per group) treated with various concentrations of methacholine (horizontal axis), presented as lung resistance (RL) relative to the lung resistance without methacholine treatment. *P < 0.01 and **P < 0.001 (ANOVA and Bonferroni test). Data are representative of three independent experiments with similar results (mean and s.d. in ac,e,f).

  7. Enhanced OVA-induced allergic airway inflammation in mice with CD4+ T cell-specific Sox4 deficiency.
    Figure 7: Enhanced OVA-induced allergic airway inflammation in mice with CD4+ T cell–specific Sox4 deficiency.

    (a) Staining of IL-4 and IFN-γ in wild-type naive CD4+ T cells (WT) and Sox4fl/flCD4-Cre naive CD4+ T cells (KO) cultured under TH2-polarizing conditions with various concentrations (above plots) of IL-4 or under TH1-polarizing conditions (far right). (b) Quantitative RT-PCR analysis of Il4, Il5, Il13 and Ifng mRNA in wild-type and Sox4fl/flCD4-Cre TH2 cells generated in vitro and treated with IL-4 at a concentration of 1 or 10 ng/ml (key) and stimulated with immobilized mAb to TCRβ (presented as in Fig. 1a). *P < 0.01 (Student's t-test). (c) Quantification of eosinophils, neutrophils, lymphocytes, macrophages and total cells of the BAL fluid from wild-type or Sox4fl/flCD4-Cre mice (n = 5 per group) left unimmunized or immunized with OVA. *P < 0.05 and **P < 0.01 (ANOVA and Bonferroni test). (d) Microscopy of lungs from the mice in c, fixed and stained with hematoxylin and eosin. Quantification of mononuclear cells (per mm2): wild-type, 552 ± 37.6 (unimmunized) or 2,654 ± 308.6 (immunized); Sox4fl/flCD4-Cre, 474.7 ± 80.5 (unimmunized) or 5,694 ± 574.7 (immunized). P < 0.01, wild-type immunized versus Sox4fl/flCD4-Cre immunized (Student's t-test). (e) Microscopy of lungs from the mice in c, fixed and stained with periodic acid–Schiff reagent. Original magnification (d,e), ×200 (scale bars, 10 μm). (f) Airway resistance of lungs from the mice in c (n = 6 per group; presented as in Fig. 6f). *P < 0.01 and **P < 0.001 (ANOVA and Bonferroni test). Data are representative of three (a,b) or two (cf) independent experiments with similar results (mean and s.d. in b,c,f).

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Affiliations

  1. Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

    • Makoto Kuwahara,
    • Masakatsu Yamashita,
    • Kenta Shinoda,
    • Soichi Tofukuji,
    • Atsushi Onodera,
    • Ryo Shinnakasu,
    • Shinichiro Motohashi,
    • Hiroyuki Hosokawa,
    • Damon Tumes,
    • Chiaki Iwamura &
    • Toshinori Nakayama
  2. Laboratory of Medical Genomics, Department of Human Genome Research, Kazusa DNA Research Institute, Chiba, Japan.

    • Makoto Kuwahara,
    • Masakatsu Yamashita &
    • Soichi Tofukuji
  3. Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Chiba, Japan.

    • Masakatsu Yamashita
  4. Department of Cell Biology and Orthopedic Research Center, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

    • Veronique Lefebvre
  5. Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Chiba, Japan.

    • Toshinori Nakayama

Contributions

M.K. and M.Y. designed and did experiments, analyzed data and wrote the manuscript; K.S., S.T., A.O., R.S., S.M., D.T., H.H. and C.I. designed and did experiments and edited the manuscript; V.L. established Sox4 deficient mice; and T.N. conceptualized the research, directed the study and wrote and edited the manuscript.

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The authors declare no competing financial interests.

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