Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation

doi:10.1038/nature24283

. 2017 Nov 2;551(7678):105-109.

doi: 10.1038/nature24283. Epub 2017 Oct 25.

Reversing SKI-SMAD4-mediated suppression is essential for T_H17 cell differentiation

Song Zhang^{1

2}, Motoki Takaku³, Liyun Zou^{1

2}, Ai-di Gu^{1

2}, Wei-Chun Chou^{1

2

4}, Ge Zhang^{1

2

5}, Bing Wu^{1

2}, Qing Kong^{1

2

6}, Seddon Y Thomas⁷, Jonathan S Serody^{1

2}, Xian Chen^{1

6}, Xiaojiang Xu⁸, Paul A Wade³, Donald N Cook⁷, Jenny P Y Ting^{1

2

4}, Yisong Y Wan^{1

2}

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
² Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
³ Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
⁴ Department of Genetics, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
⁵ Department of Immunology, Dalian Medical University, Dalian 116044, China.
⁶ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
⁷ Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
⁸ Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

PMID: 29072299
PMCID: PMC5743442
DOI: 10.1038/nature24283

Reversing SKI-SMAD4-mediated suppression is essential for T_H17 cell differentiation

Song Zhang et al. Nature. 2017.

. 2017 Nov 2;551(7678):105-109.

doi: 10.1038/nature24283. Epub 2017 Oct 25.

Authors

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
² Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
³ Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
⁴ Department of Genetics, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
⁵ Department of Immunology, Dalian Medical University, Dalian 116044, China.
⁶ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
⁷ Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
⁸ Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.

PMID: 29072299
PMCID: PMC5743442
DOI: 10.1038/nature24283

Abstract

T helper 17 (T_H17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T_H17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T_H17 cell differentiation remains elusive. Here we reveal that TGFβ enables T_H17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T_H17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T_H17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T_H17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T_H17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T_H17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T_H17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T_H17-related diseases.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

**Extended Data Figure 1. Th17 cell differentiation in the absence of Smad4**
1a, Naïve CD4⁺ T cells isolated from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were activated in the presence of TGFβR inhibitor (i), IL-6+TGFβR inhibitor (IL-6+i), or IL-6+TGF-β (IL6+TGF-β). IL-17A⁺ cells were assessed by flow-cytometry 1 and 2 days later. Representative results (left) and statistical analysis (right) of 5 experiments are shown. 1b, The percentage of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ cells in the peripheral lymph nodes (pLN) and spleens from WT, *Cd4cre;Smad4*^fl/fl (S4 KO), *Cd4cre;Tgfbr2*^fl/fl (RII KO) and *Cd4cre;Smad4*^fl/fl;*Tgfbr2*^fl/fl (S4-RII DKO) mice under steady state were assessed by flow-cytometry. Representative result (left) and statistics from 8 mice (right) are shown. (***p<0.001 per two sided t-test; NS, not significant per two-sided t-test; centers indicate mean values)

**Extended Data Figure 2. Smad4 suppresses RORγt expression**
2a, CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. The mRNA expression of Th17 related genes was analyzed at indicated time points post activation by qRT-PCR. Means ± s.d. of three experiments are shown. 2b, Naïve CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor for 3 and 12 hours. Total RNA was then collected for RNA-seq analysis. All genes were analyzed and presented as volcano plots based on the fold change (log₂) of S4 KO versus WT and p-value (-log₁₀). Differentially expressed genes (p<0.05) are highlighted in red. 2c, Naïve CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor. The mRNA expression of Th17 related genes was analyzed at indicated time points post activation by qRT-PCR. Means ± s.d. of three experiments are shown. 2d, Naïve CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl;*Tgfbr2*^fl/fl (S4-RII DKO) mice were sorted and activated in the presence of IL-6. The mRNA expression of Th17 related genes was analyzed at indicated time points post activation by qRT-PCR. Means ± s.d. of three experiments are shown. 2e, Naïve CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6 and TGFβR inhibitor. The RORγt protein expression was assessed by immuno-blotting 1 and 4 days post activation. Results are representative of three experiments with similar results. 2f, CD4⁺ T cells from WT and *Cd4cre;Tgfbr2*^fl/fl;Smad4^fl/fl (S4-RII DKO) mice were activated in the presence of IL-6. The RORγt protein expression was assessed by immuno-blotting 1 day post activation. Results are representative of three experiments with similar results. 2g, CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells were harvested after 12 hours. ChIP assay was performed with Ctrl IgG antibody and Smad4 antibody. The enrichment of Smad4 binding to *Rorc* promoter was determined. Means ± s.d. of 3 samples in one experiment of three are shown. 2h, CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells were harvested after 12 hours. ChIP-seq assay was performed with Smad4 antibody. The enrichment of Smad4 binding to *Il17a and Il17f* loci were determined by the mapped read coverage of Smad4 ChIP-seq data. The results of two independent experiments were show as #1 and #2. (*p<0.05, **p<0.01, ***p<0.001 per two sided t-test; NS, not significant per two-sided t-test)

**Extended Data Figure 3. Ski identification and its degradation upon low dose of TGF-β**
3a, Schematic of quantitative IP-MS (immuno-precipitation and mass spectrometry) proteomic strategy to identify Smad4 binding proteins under different conditions. In one scheme, to identify Smad4 binding protein in the absence of TGF-β signaling, CD4⁺ T cell from *Cd4cre;Smad4*^fl/fl (S4 KO) mice were sorted and activated in the presence of IL-6+i in the SILAC/AACT medium provided either with amino acids containing light (L) isotopes or with amino acids containing heavy (H) isotopes. Cells were then transduced with retroviruses expressing either Flag tag (RV Flag) or Flag tag and Smad4 fusion protein (RV Flag-S4). Cells were harvested and mixed 4 days post activation. Immuno-precipitation was performed using anti-Flag. IP’ed proteins were processed and subjected to quantitative MS analysis. Proteins with an H/L ratio of more than 2 were identified. In the other scheme, to identify the proteins whose Smad4 interaction was perturbed upon TGF-β stimulation, CD4⁺ T cell from WT mice were sorted and activated either in the presence of IL-6+i in the SILAC/AACT medium provided with amino acids containing light (L) isotopes or in the presence of IL-6+TGF-β in the SILAC/AACT medium provided with amino acids containing heavy (H) isotopes. Cells were harvested and mixed on 4 days post activation. Immuno-precipitation was performed using anti-Smad4 antibody. IP’ed proteins were processed and subjected to quantitative MS analysis. Proteins with an H/L ratio of less than 1 were identified. The commonly identified protein Ski in the two experiments was subjected to further investigation. 3b, CD4⁺ T cell from WT mice were activated in the presence of IL-6 and indicated dose of TGF-β. Cells were harvested after 24 hours. Ski protein expression was detected by immuno-blotting. Results are representative of three experiments with similar results. 3c, CD4⁺ T cell from WT mice were activated in the presence of IL-6 and indicated dose of TGF-β. IL-17A⁺ and Foxp3⁺ cells were assessed by flow-cytometry on day 4. Representative results (upper) and statistical analysis (lower) of 5 experiments are shown (Centers indicate mean values). 3d, CD4⁺ T cells from WT or *Cd4cre;Smad2*^fl/fl (S2 KO) mice were activated in the presence of IL-6 for 24 hours. Cells were then stimulated with indicated conditions for additional 1 hour with or without 10μM SIS3 (specific inhibitor of Smad3 phosphorylation). Ski protein expression and Smad3 phosphorylation were assessed by immuno-blotting. Results are representative of three experiments with similar results.

**Extended Data Figure 4. Ski and Smad4 cooperatively suppress Th17 differentiation**
4a, Bone marrow cells were isolated from the femur bones of sex- and age-matched *Cd4cre;CdC* (Cas9, CD45.2⁺) mice and wild-type (WT, CD45.1⁺) mice. Cells were mixed, and transduced with two different gRNA expressing virus (as indicated) and transferred into sub-lethally irradiated (400 cGy) *Rag1*^−/− recipient mice. CD4⁺ T cells isolated from lymph nodes and spleen of generated bone marrow chimeric mice were activated in the presence of IL-6 and TGFβR inhibitor. Cells transduced with gRNA in wild-type donor indicated as WT. Cells transduced with gRNA in *CD4Cre;CdC* donor indicated as Ski KO. IL-17A⁺ cells were assessed by flow-cytometry on day 4. Representative results (left) and statistical analysis (right) of 5 experiments are shown. 4b, CD4⁺ T cells from WT mice were activated in the presence of IL-6 and TGF-β, and then transduced with MSCV-IRES-GFP (RV), MSCV-Ski-IRES-GFP (RV Ski) or co-transduced with MSCV-Ski-IRES-GFP and MSCV-RORγt-IRES-Thy1.1 (RV Ski+RORγt) retroviruses. IL-17A expression of transduced (GFP⁺) or co-transduced (GFP⁺Thy1.1⁺) T cells was assessed by flow-cytometry. Representative results (left) and statistical analysis (right) of 5 experiments are shown. 4c, CD4⁺ T cells from WT and *Cd4cre;Smad4*^fl/fl (S4 KO) mice were activated in the presence of IL-6+TGFβR inhibitor (i) or IL-6+TGF-β. Cells were harvested 3 days later. ChIP assay was performed with Control IgG antibody or Ski antibody. The relative enrichment of Ski binding to *Rorc* locus was determined. Means ± s.d. of 3 samples in one experiment of three are shown. (**P<0.01, ***P<0.001 per two sided t-test; NS, not significant per two sided t-test; centers indicate mean values)

**Extended Data Figure 5. TGF-β superfamily signaling overcomes Ski-Smad4 complex mediated suppression of RORγt expression in activated T cells to license Th17 cell differentiation**
5a, RORγt expression is potentiated by IL-6-STAT3 signaling but restrained by the HDAC-activity-containing Ski-Smad4 complex that associates with and de-acetylates *Rorc* locus. 5b, Additional TGF-β or Activin signaling triggers Ski degradation. The disruption of Ski-Smad4 complex dissociates HDAC activity from *Rorc* locus and to license RORγt expression and Th17 cell differentiation.

**Figure 1. Smad4 deletion leads to a Th17 differentiation in the absence of TGF-β signaling**
**a, c**, Flow-cytometry of cells cultured with or without TGFβR inhibitor (i) (n=6 experiments). **b,d**, qRT-PCR of cells cultured with IL-6+i (b) or IL-6 (d) for 4 days (n=3 experiments, mean ± s.d.). **e-g**, Flow-cytometry of spinal-cord-infiltrating cells (e), clinical scores (f) of EAE-inflicted mice (n=12 each group, mean ± s.e.m.), representative pathology (g, n=3 experiments, scale bar 500μm)**. h**, Flow-cytometry of spinal-cord-infiltrating, transferred cells in EAE mice (n=6 each group). (NS, not significant, *p<0.05, **p<0.01, ***p<0.001 per two-sided t-test; centers indicate mean values)

**Figure 2. Smad4 controls Th17 cell program by directly suppressing *Rorc* expression**
a, Differential expression of S4 KO/WT cells cultured with IL-6+TGFβR inhibitor by RNA-seq (scale bar is indicated). **b-d,f**, Flow-cytometry of cells without (b) or with (c, d, f) retrovirus (RV) transduction (n=5 experiments in b, d, f, n=6 experiments in c). e, qRT-PCR of S4 KO cells cultured with IL-6+TGFβR inhibitor, 18-hour post retrovirus transduction (n=3 experiments, mean ± s.d.). g, ChIP-seq analysis of Smad4 binding at *Rorc* locus in cells cultured with IL-6+TGFβR inhibitor for 24 hours (n=2 experiments). (**p<0.01, ***p<0.001 per two-sided t-test; centers indicate mean values)

**Figure 3. TGF-β signaling disrupts Ski-Smad4 complex to license Th17 differentiation**
**a, g** Flow-cytometry of cells transduced with retrovirus expressing wild-type or mutant Smad4 (n=7 experiments in a; n=5 experiments in g). b, ChIP of Smad4 binding at *Rorc* promoter (n=3 samples, mean ± s.d.). **c-f**, Immuno-precipitation and immune-blotting of S4 KO (c, f) or WT (d, e) cells treated as indicated (n=3 experiments). In e, cells were pre-cultured with IL-6 for 24 hours. (NS, not significant, ***p<0.001 per two-sided t-test; centers indicate mean values)

**Figure 4. Ski suppresses Th17 differentiation in a Smad4-dependent manner**
**a, d, h-j**, Flow-cytometry of cultured cells (n=7 in a, n=8 in d and n=6 in h-j experiments). b, qRT-PCR of WT cells cultured with IL-6+TGF-β, 18-hour post retrovirus transduction (n=3 experiments, mean ± s.d.). c, Flow-cytometry of spinal-cord-infiltrating, transferred cells in EAE mice (n=5). e, f, ChIP of H3K9Ac at *Rorc* promoter (n=3 samples in e; n=6 samples in f, mean ± s.d.). g, Immune-blotting of WT cells cultured for 24 hours (n=3 experiments). (NS, not significant, **p<0.01, ***p<0.001 per two-sided t-test; centers indicate mean values)

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New traffic light on Th17 Avenue.
Xu H, Littman DR. Xu H, et al. Cell Res. 2018 Feb;28(2):139-140. doi: 10.1038/cr.2017.156. Epub 2017 Dec 8. Cell Res. 2018. PMID: 29219146 Free PMC article.

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References

1. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. - PubMed
1. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27:485–517. - PubMed
1. Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. The New England journal of medicine. 2009;361:888–898. - PubMed
1. Singh RP, et al. Th17 cells in inflammation and autoimmunity. Autoimmunity reviews. 2014 - PubMed
1. Patel DD, Kuchroo VK. Th17 Cell Pathway in Human Immunity: Lessons from Genetics and Therapeutic Interventions. Immunity. 2015;43:1040–1051. - PubMed

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[1] Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. - PubMed

[2] Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. - PubMed

[3] Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27:485–517. - PubMed

[4] Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27:485–517. - PubMed

[5] Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. The New England journal of medicine. 2009;361:888–898. - PubMed

[6] Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. The New England journal of medicine. 2009;361:888–898. - PubMed

[7] Singh RP, et al. Th17 cells in inflammation and autoimmunity. Autoimmunity reviews. 2014 - PubMed

[8] Singh RP, et al. Th17 cells in inflammation and autoimmunity. Autoimmunity reviews. 2014 - PubMed

[9] Patel DD, Kuchroo VK. Th17 Cell Pathway in Human Immunity: Lessons from Genetics and Therapeutic Interventions. Immunity. 2015;43:1040–1051. - PubMed

[10] Patel DD, Kuchroo VK. Th17 Cell Pathway in Human Immunity: Lessons from Genetics and Therapeutic Interventions. Immunity. 2015;43:1040–1051. - PubMed

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Reversing SKI-SMAD4-mediated suppression is essential for T_H17 cell differentiation

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Reversing SKI-SMAD4-mediated suppression is essential for T_H17 cell differentiation

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