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Prostaglandin E₂ promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase.

Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S - Nat Commun (2013)

Bottom Line: Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28.Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo.These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606 8501, Japan.

ABSTRACT
T helper 1 (Th1) cells have critical roles in various autoimmune and proinflammatory diseases. cAMP has long been believed to act as a suppressor of IFN-γ production and Th1 cell-mediated immune inflammation. Here we show that cAMP actively promotes Th1 differentiation by inducing gene expression of cytokine receptors involved in this process. PGE2 signalling through EP2/EP4 receptors mobilizes the cAMP-PKA pathway, which induces CREB- and its co-activator CRTC2-mediated transcription of IL-12Rβ2 and IFN-γR1. Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28. Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo. These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

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Recruitment of p-CREB and CRTC2 to Il12rb2 and Ifngr1 gene loci upon cAMP stimulation.(a) Schematic diagram of the mouse Il12rb2 (left) and Ifngr1 (right) genes and their promoter/enhancer regions illustrating potential CRE sites and the positions of the primers. (b,c) Enrichment of p-CREB(S133) (upper) and CRTC2 (lower) at the Il12rb2 (b) and Ifngr1 (c) gene loci. T cells were rested for 2 days followed by stimulation with db-cAMP or vehicle for 1 h. Cells were fixed, and chromatin immunoprecipitation was performed using anti-p-CREB (S133) and anti-CRTC2. Data are normalized to the input DNA and represent eight individual chromatin immunoprecipitations from four independent experiments (mean±s.e.m.). (d–g) Luciferase activity of EL4 cells stably expressing the Il12rb2 luciferase reporter, stimulated for 24 h with indicated concentrations of db-cAMP, forskolin or vehicle (d), or stimulated for 24 h with db-cAMP after 40 h treatment with scrambled or Creb1 siRNA (e), after 24 h transfection with WT CREB or the CREB(S133A) mutant (f) or after 40 h treatment with scrambled or Crtc2 siRNA (g). Relative light unit (RLU) is normalized to vehicle control of each transfection condition (e–g). Data shown as mean±s.e.m. are representative of two (d–g) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01.
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f6: Recruitment of p-CREB and CRTC2 to Il12rb2 and Ifngr1 gene loci upon cAMP stimulation.(a) Schematic diagram of the mouse Il12rb2 (left) and Ifngr1 (right) genes and their promoter/enhancer regions illustrating potential CRE sites and the positions of the primers. (b,c) Enrichment of p-CREB(S133) (upper) and CRTC2 (lower) at the Il12rb2 (b) and Ifngr1 (c) gene loci. T cells were rested for 2 days followed by stimulation with db-cAMP or vehicle for 1 h. Cells were fixed, and chromatin immunoprecipitation was performed using anti-p-CREB (S133) and anti-CRTC2. Data are normalized to the input DNA and represent eight individual chromatin immunoprecipitations from four independent experiments (mean±s.e.m.). (d–g) Luciferase activity of EL4 cells stably expressing the Il12rb2 luciferase reporter, stimulated for 24 h with indicated concentrations of db-cAMP, forskolin or vehicle (d), or stimulated for 24 h with db-cAMP after 40 h treatment with scrambled or Creb1 siRNA (e), after 24 h transfection with WT CREB or the CREB(S133A) mutant (f) or after 40 h treatment with scrambled or Crtc2 siRNA (g). Relative light unit (RLU) is normalized to vehicle control of each transfection condition (e–g). Data shown as mean±s.e.m. are representative of two (d–g) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01.

Mentions: Computational analysis identified several putative half or full CRE sites in the promoter/enhancer regions of Il12rb2 and Ifngr1 gene loci (Fig. 6a). We performed chromatin immunoprecipitation analysis and observed that upon cAMP stimulation, p-CREB(S133) and CRTC2 were recruited to several of these sites located from −3.6 to +0.8 kb in Il12rb2 gene locus (Fig. 6b, sites E–L). Similarly, cAMP increased binding of p-CREB(S133) and CRTC2 to two sites around the transcription start site and one site at position +4.6 kb in the first intron of Ifngr1 gene (Fig. 6c, sites M, N and P). These results suggest that CREB and CRTC2 activated by cAMP are recruited to the sites in the promoter/enhancer regions of Il12rb2 or Ifngr1 gene loci.


Prostaglandin E₂ promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase.

Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S - Nat Commun (2013)

Recruitment of p-CREB and CRTC2 to Il12rb2 and Ifngr1 gene loci upon cAMP stimulation.(a) Schematic diagram of the mouse Il12rb2 (left) and Ifngr1 (right) genes and their promoter/enhancer regions illustrating potential CRE sites and the positions of the primers. (b,c) Enrichment of p-CREB(S133) (upper) and CRTC2 (lower) at the Il12rb2 (b) and Ifngr1 (c) gene loci. T cells were rested for 2 days followed by stimulation with db-cAMP or vehicle for 1 h. Cells were fixed, and chromatin immunoprecipitation was performed using anti-p-CREB (S133) and anti-CRTC2. Data are normalized to the input DNA and represent eight individual chromatin immunoprecipitations from four independent experiments (mean±s.e.m.). (d–g) Luciferase activity of EL4 cells stably expressing the Il12rb2 luciferase reporter, stimulated for 24 h with indicated concentrations of db-cAMP, forskolin or vehicle (d), or stimulated for 24 h with db-cAMP after 40 h treatment with scrambled or Creb1 siRNA (e), after 24 h transfection with WT CREB or the CREB(S133A) mutant (f) or after 40 h treatment with scrambled or Crtc2 siRNA (g). Relative light unit (RLU) is normalized to vehicle control of each transfection condition (e–g). Data shown as mean±s.e.m. are representative of two (d–g) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01.
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f6: Recruitment of p-CREB and CRTC2 to Il12rb2 and Ifngr1 gene loci upon cAMP stimulation.(a) Schematic diagram of the mouse Il12rb2 (left) and Ifngr1 (right) genes and their promoter/enhancer regions illustrating potential CRE sites and the positions of the primers. (b,c) Enrichment of p-CREB(S133) (upper) and CRTC2 (lower) at the Il12rb2 (b) and Ifngr1 (c) gene loci. T cells were rested for 2 days followed by stimulation with db-cAMP or vehicle for 1 h. Cells were fixed, and chromatin immunoprecipitation was performed using anti-p-CREB (S133) and anti-CRTC2. Data are normalized to the input DNA and represent eight individual chromatin immunoprecipitations from four independent experiments (mean±s.e.m.). (d–g) Luciferase activity of EL4 cells stably expressing the Il12rb2 luciferase reporter, stimulated for 24 h with indicated concentrations of db-cAMP, forskolin or vehicle (d), or stimulated for 24 h with db-cAMP after 40 h treatment with scrambled or Creb1 siRNA (e), after 24 h transfection with WT CREB or the CREB(S133A) mutant (f) or after 40 h treatment with scrambled or Crtc2 siRNA (g). Relative light unit (RLU) is normalized to vehicle control of each transfection condition (e–g). Data shown as mean±s.e.m. are representative of two (d–g) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01.
Mentions: Computational analysis identified several putative half or full CRE sites in the promoter/enhancer regions of Il12rb2 and Ifngr1 gene loci (Fig. 6a). We performed chromatin immunoprecipitation analysis and observed that upon cAMP stimulation, p-CREB(S133) and CRTC2 were recruited to several of these sites located from −3.6 to +0.8 kb in Il12rb2 gene locus (Fig. 6b, sites E–L). Similarly, cAMP increased binding of p-CREB(S133) and CRTC2 to two sites around the transcription start site and one site at position +4.6 kb in the first intron of Ifngr1 gene (Fig. 6c, sites M, N and P). These results suggest that CREB and CRTC2 activated by cAMP are recruited to the sites in the promoter/enhancer regions of Il12rb2 or Ifngr1 gene loci.

Bottom Line: Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28.Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo.These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606 8501, Japan.

ABSTRACT
T helper 1 (Th1) cells have critical roles in various autoimmune and proinflammatory diseases. cAMP has long been believed to act as a suppressor of IFN-γ production and Th1 cell-mediated immune inflammation. Here we show that cAMP actively promotes Th1 differentiation by inducing gene expression of cytokine receptors involved in this process. PGE2 signalling through EP2/EP4 receptors mobilizes the cAMP-PKA pathway, which induces CREB- and its co-activator CRTC2-mediated transcription of IL-12Rβ2 and IFN-γR1. Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28. Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo. These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

Show MeSH
Related in: MedlinePlus