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Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Yoon JH, Sudo K, Kuroda M, Kato M, Lee IK, Han JS, Nakae S, Imamura T, Kim J, Ju JH, Kim DK, Matsuzaki K, Weinstein M, Matsumoto I, Sumida T, Mamura M - Nat Commun (2015)

Bottom Line: Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively.Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes.Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan [2] Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan [3] Department of Internal Medicine, Kyungpook National University School of Medicine, 50 SAMDUK-2GA, Jungu, Daegu 700-721, Republic of Korea.

ABSTRACT
Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

No MeSH data available.


Related in: MedlinePlus

Opposing roles of Smad2 and Smad3 in STAT3-induced TH17 differentiation.Purified CD4+ T cells were activated under TH17-polarizing condition for 3 days. (a) Flow cytometry analyses of IL-17A and RORγt in Smad2+/+,+/−, −/− and Smad3+/+,+/−, −/− CD4+ T cells. (b) Quantitative RT–PCR analysis of the Il17a and Rorc mRNA in Smad2+/+, −/− and Smad3+/+, −/− TH17 cells (n=7). (c) Effects of Smads on STAT3-induced activation of the Rorc promoter and the Il17a promoter constructs transfected in TH17 cells were analysed using luciferase assay. (d) Binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene in TH17 cells was determined using ChIP. (e) Requirement of STAT3 for the binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using STAT3 knockdown TH17 cells. Requirement of Smad2 and Smad3 for the binding of STAT3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using (f) Smad2−/− or (g) Smad3−/− TH17 cells. ChIP data are shown as differential occupancy fold changes. Data are from one experiment representative of seven (a,d), three (c), two (e) or five (f,g) independent experiments or pooled from seven experiments (b). Each experiment (a–g) was performed in triplicate (n=3). Data are mean+s.d. or mean+s.d. with P values (b, unpaired Student's t-test).
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f2: Opposing roles of Smad2 and Smad3 in STAT3-induced TH17 differentiation.Purified CD4+ T cells were activated under TH17-polarizing condition for 3 days. (a) Flow cytometry analyses of IL-17A and RORγt in Smad2+/+,+/−, −/− and Smad3+/+,+/−, −/− CD4+ T cells. (b) Quantitative RT–PCR analysis of the Il17a and Rorc mRNA in Smad2+/+, −/− and Smad3+/+, −/− TH17 cells (n=7). (c) Effects of Smads on STAT3-induced activation of the Rorc promoter and the Il17a promoter constructs transfected in TH17 cells were analysed using luciferase assay. (d) Binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene in TH17 cells was determined using ChIP. (e) Requirement of STAT3 for the binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using STAT3 knockdown TH17 cells. Requirement of Smad2 and Smad3 for the binding of STAT3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using (f) Smad2−/− or (g) Smad3−/− TH17 cells. ChIP data are shown as differential occupancy fold changes. Data are from one experiment representative of seven (a,d), three (c), two (e) or five (f,g) independent experiments or pooled from seven experiments (b). Each experiment (a–g) was performed in triplicate (n=3). Data are mean+s.d. or mean+s.d. with P values (b, unpaired Student's t-test).

Mentions: IL-6 is the main arthritogenic cytokine and TGF-β is produced and activated in the inflammatory lesions1225. Because IL-6 and TGF-β are the pivotal cytokines to induce TH17 differentiation, we cultured Smad2−/− or Smad3−/− CD4+ T cells under TH17-polarizing condition with IL-6 and TGF-β (ref. 3) to examine the mechanisms whereby R-Smads regulate TH17 differentiation. Expression levels of protein and mRNA of RORγt and IL-17A decreased in Smad2−/− CD4+ T cells, whereas those increased in Smad3−/− CD4+ T cells (Fig. 2a,b). The mRNA levels of TH17-inducing genes (Batf, Il23r, Il6, Il6ra, Il21 and Il21r) and TH17-suppressing genes (Il2, Il2ra, Tbet and Eomesodermin) were unaffected in both Smad2−/− and Smad3−/− CD4+ T cells (Supplementary Fig. 7), suggesting that R-Smads regulate TH17 differentiation by specifically targeting the Rorc and Il17a genes. Because IL-6 or TGF-β alone has little effect on TH17 differentiation3 and STAT3-mediated IL-6 signalling is crucial for TH17 differentiation16, we examined whether R-Smads regulate STAT3-induced transcription of RORγt and IL-17A in CD4+ T cells cultured under TH17-polarizing condition by promoter assays with the luciferase reporters spanning 2 kb upstream of the first exons of the Rorc and Il17a genes (Fig. 2c). STAT3 or Smad2 alone induced their promoter activities, whereas Smad3 alone had no effect. Smad2 further enhanced, whereas Smad3 suppressed STAT3-induced reporter activation. Co-transfection of Smad4 with R-Smads and STAT3 did not show the additive effects. We next determined the binding of R-Smads to the proximal promoter regions of the Rorc and Il17a genes in TH17 cells by chromatin immunoprecipitation (ChIP) using the primers to detect the DNA-binding sequences of Smads and STAT3 (refs 10, 26, 27). Smad2 and Smad3 were bound to the same sites in the Rorc promoter, whereas they were bound to the distinct sites in the Il17a promoter (Fig. 2d). Active promoters are characterized by histone acetylation and trimethylation of H3K4, whereas repressed inactive chromatin is marked by methylation of H3K27 and H3K9 (ref. 28). Smad2-binding sites in the Il17a promoter showed higher acetylation of histone H3 and trimethylation of histone H3K4, which correlate with transcriptionally active chromatin (Supplementary Fig. 8a). By contrast, Smad3-binding sites in the Il17a promoter showed higher trimethylation of histone H3K27, which correlate with transcriptionally inactive chromatin (Supplementary Fig. 8b). These data suggest that Smad2 and Smad3 have the opposing roles in STAT3-induced transcription of the Rorc and Il17a genes.


Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Yoon JH, Sudo K, Kuroda M, Kato M, Lee IK, Han JS, Nakae S, Imamura T, Kim J, Ju JH, Kim DK, Matsuzaki K, Weinstein M, Matsumoto I, Sumida T, Mamura M - Nat Commun (2015)

Opposing roles of Smad2 and Smad3 in STAT3-induced TH17 differentiation.Purified CD4+ T cells were activated under TH17-polarizing condition for 3 days. (a) Flow cytometry analyses of IL-17A and RORγt in Smad2+/+,+/−, −/− and Smad3+/+,+/−, −/− CD4+ T cells. (b) Quantitative RT–PCR analysis of the Il17a and Rorc mRNA in Smad2+/+, −/− and Smad3+/+, −/− TH17 cells (n=7). (c) Effects of Smads on STAT3-induced activation of the Rorc promoter and the Il17a promoter constructs transfected in TH17 cells were analysed using luciferase assay. (d) Binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene in TH17 cells was determined using ChIP. (e) Requirement of STAT3 for the binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using STAT3 knockdown TH17 cells. Requirement of Smad2 and Smad3 for the binding of STAT3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using (f) Smad2−/− or (g) Smad3−/− TH17 cells. ChIP data are shown as differential occupancy fold changes. Data are from one experiment representative of seven (a,d), three (c), two (e) or five (f,g) independent experiments or pooled from seven experiments (b). Each experiment (a–g) was performed in triplicate (n=3). Data are mean+s.d. or mean+s.d. with P values (b, unpaired Student's t-test).
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f2: Opposing roles of Smad2 and Smad3 in STAT3-induced TH17 differentiation.Purified CD4+ T cells were activated under TH17-polarizing condition for 3 days. (a) Flow cytometry analyses of IL-17A and RORγt in Smad2+/+,+/−, −/− and Smad3+/+,+/−, −/− CD4+ T cells. (b) Quantitative RT–PCR analysis of the Il17a and Rorc mRNA in Smad2+/+, −/− and Smad3+/+, −/− TH17 cells (n=7). (c) Effects of Smads on STAT3-induced activation of the Rorc promoter and the Il17a promoter constructs transfected in TH17 cells were analysed using luciferase assay. (d) Binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene in TH17 cells was determined using ChIP. (e) Requirement of STAT3 for the binding of Smad2 and Smad3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using STAT3 knockdown TH17 cells. Requirement of Smad2 and Smad3 for the binding of STAT3 to the proximal promoter regions of the Rorc gene and the Il17a gene was determined with ChIP using (f) Smad2−/− or (g) Smad3−/− TH17 cells. ChIP data are shown as differential occupancy fold changes. Data are from one experiment representative of seven (a,d), three (c), two (e) or five (f,g) independent experiments or pooled from seven experiments (b). Each experiment (a–g) was performed in triplicate (n=3). Data are mean+s.d. or mean+s.d. with P values (b, unpaired Student's t-test).
Mentions: IL-6 is the main arthritogenic cytokine and TGF-β is produced and activated in the inflammatory lesions1225. Because IL-6 and TGF-β are the pivotal cytokines to induce TH17 differentiation, we cultured Smad2−/− or Smad3−/− CD4+ T cells under TH17-polarizing condition with IL-6 and TGF-β (ref. 3) to examine the mechanisms whereby R-Smads regulate TH17 differentiation. Expression levels of protein and mRNA of RORγt and IL-17A decreased in Smad2−/− CD4+ T cells, whereas those increased in Smad3−/− CD4+ T cells (Fig. 2a,b). The mRNA levels of TH17-inducing genes (Batf, Il23r, Il6, Il6ra, Il21 and Il21r) and TH17-suppressing genes (Il2, Il2ra, Tbet and Eomesodermin) were unaffected in both Smad2−/− and Smad3−/− CD4+ T cells (Supplementary Fig. 7), suggesting that R-Smads regulate TH17 differentiation by specifically targeting the Rorc and Il17a genes. Because IL-6 or TGF-β alone has little effect on TH17 differentiation3 and STAT3-mediated IL-6 signalling is crucial for TH17 differentiation16, we examined whether R-Smads regulate STAT3-induced transcription of RORγt and IL-17A in CD4+ T cells cultured under TH17-polarizing condition by promoter assays with the luciferase reporters spanning 2 kb upstream of the first exons of the Rorc and Il17a genes (Fig. 2c). STAT3 or Smad2 alone induced their promoter activities, whereas Smad3 alone had no effect. Smad2 further enhanced, whereas Smad3 suppressed STAT3-induced reporter activation. Co-transfection of Smad4 with R-Smads and STAT3 did not show the additive effects. We next determined the binding of R-Smads to the proximal promoter regions of the Rorc and Il17a genes in TH17 cells by chromatin immunoprecipitation (ChIP) using the primers to detect the DNA-binding sequences of Smads and STAT3 (refs 10, 26, 27). Smad2 and Smad3 were bound to the same sites in the Rorc promoter, whereas they were bound to the distinct sites in the Il17a promoter (Fig. 2d). Active promoters are characterized by histone acetylation and trimethylation of H3K4, whereas repressed inactive chromatin is marked by methylation of H3K27 and H3K9 (ref. 28). Smad2-binding sites in the Il17a promoter showed higher acetylation of histone H3 and trimethylation of histone H3K4, which correlate with transcriptionally active chromatin (Supplementary Fig. 8a). By contrast, Smad3-binding sites in the Il17a promoter showed higher trimethylation of histone H3K27, which correlate with transcriptionally inactive chromatin (Supplementary Fig. 8b). These data suggest that Smad2 and Smad3 have the opposing roles in STAT3-induced transcription of the Rorc and Il17a genes.

Bottom Line: Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively.Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes.Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan [2] Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan [3] Department of Internal Medicine, Kyungpook National University School of Medicine, 50 SAMDUK-2GA, Jungu, Daegu 700-721, Republic of Korea.

ABSTRACT
Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

No MeSH data available.


Related in: MedlinePlus