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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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SIK2-CRTC2 signalling mediates cAMP-induced expression of Il12rb2 and Ifngr1.(a) cAMP induces CREB phosphorylation and CRTC2 dephosphorylation in T cells. (b) cAMP and STS reduces cytoplasmic CRTC2 and increases nuclear CRTC2 in T cells that were rested for 2 days and then stimulated for 1 h. (c) cAMP does not increase nuclear CRTC2 in SIK2−/− T cells. (d) GAL4 reporter assay of CRTC2 activity of SIK2+/+ or SIK2−/− T cells that were transfected with GAL4-fusion pM-mCRTC2 and pTAL-5xGAL4-Luciferase reporter and stimulated with db-cAMP for the indicated times. The activity is shown as the ratio of firefly luciferase activity to renilla pRL-TK luciferase activity. (e,f) T cells were transfected with scrambled or Crtc2 siRNA for 36 h, followed by immunobloting of CRTC2 (e) or stimulating with db-cAMP for another 12 h for detection of mRNA expression (f). mRNA expression in db-cAMP-treated cells is normalized to vehicle-treated of each siRNA-transfected cells. (g) Expression of indicated mRNA in T cells stimulated with STS or db-cAMP for 12 h. (h) CRTC2(S171A) increases basal expression of Il12rb2 and Ifngr1, while SIK2(S587A) reduces cAMP-induced expression of these genes in TCR-activated T cells. (i) SIK2 WT and S587A mutant reduce basal expression of Il12rb2 and Ifngr1 in SIK2-deficient T cells. Data shown as mean±s.e.m. are representative of two (a–e,i) or three (f,g) independent experiments with triplicates or are from four independent experiments (h). Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant; a.u., arbitrary units.
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f5: SIK2-CRTC2 signalling mediates cAMP-induced expression of Il12rb2 and Ifngr1.(a) cAMP induces CREB phosphorylation and CRTC2 dephosphorylation in T cells. (b) cAMP and STS reduces cytoplasmic CRTC2 and increases nuclear CRTC2 in T cells that were rested for 2 days and then stimulated for 1 h. (c) cAMP does not increase nuclear CRTC2 in SIK2−/− T cells. (d) GAL4 reporter assay of CRTC2 activity of SIK2+/+ or SIK2−/− T cells that were transfected with GAL4-fusion pM-mCRTC2 and pTAL-5xGAL4-Luciferase reporter and stimulated with db-cAMP for the indicated times. The activity is shown as the ratio of firefly luciferase activity to renilla pRL-TK luciferase activity. (e,f) T cells were transfected with scrambled or Crtc2 siRNA for 36 h, followed by immunobloting of CRTC2 (e) or stimulating with db-cAMP for another 12 h for detection of mRNA expression (f). mRNA expression in db-cAMP-treated cells is normalized to vehicle-treated of each siRNA-transfected cells. (g) Expression of indicated mRNA in T cells stimulated with STS or db-cAMP for 12 h. (h) CRTC2(S171A) increases basal expression of Il12rb2 and Ifngr1, while SIK2(S587A) reduces cAMP-induced expression of these genes in TCR-activated T cells. (i) SIK2 WT and S587A mutant reduce basal expression of Il12rb2 and Ifngr1 in SIK2-deficient T cells. Data shown as mean±s.e.m. are representative of two (a–e,i) or three (f,g) independent experiments with triplicates or are from four independent experiments (h). Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant; a.u., arbitrary units.

Mentions: One candidate for such factors is CRTC2 that is regulated negatively by SIK2 and functions as a CREB coactivator in many types of cells19202122. Stimulation of T cells with db-cAMP induced not only phosphorylation of CREB but also dephosphorylation of CRTC2 (Fig. 5a), decreased the amount of CRTC2 in the cytoplasm and increased the amount of dephosphorylated CRTC2 in the nucleus (Fig. 5b). This distribution is SIK-dependent, because SIK2–/– T cells22 constitutively showed more nuclear CRTC2 than WT cells, which increased little by cAMP (Fig. 5c). As reported1819, protein kinase inhibitor staurosporine (STS) induced dephosphorylation and nuclear translocation of CRTC2 without enhancing CREB phosphorylation (Fig. 5b). To investigate whether cAMP affects transcriptional activity of CRTC2, we performed a reporter assay with GAL4-fusion CRTC2 in WT and SIK2–/– T cells. While db-cAMP increased GAL4-transcriptional activity in WT cells, the basal GAL4 activity was already higher in SIK2–/– T cells and no further increase was observed upon cAMP addition (Fig. 5d). These findings suggest that SIK2-CRTC2 pathway functions downstream of cAMP in T cells.


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)

SIK2-CRTC2 signalling mediates cAMP-induced expression of Il12rb2 and Ifngr1.(a) cAMP induces CREB phosphorylation and CRTC2 dephosphorylation in T cells. (b) cAMP and STS reduces cytoplasmic CRTC2 and increases nuclear CRTC2 in T cells that were rested for 2 days and then stimulated for 1 h. (c) cAMP does not increase nuclear CRTC2 in SIK2−/− T cells. (d) GAL4 reporter assay of CRTC2 activity of SIK2+/+ or SIK2−/− T cells that were transfected with GAL4-fusion pM-mCRTC2 and pTAL-5xGAL4-Luciferase reporter and stimulated with db-cAMP for the indicated times. The activity is shown as the ratio of firefly luciferase activity to renilla pRL-TK luciferase activity. (e,f) T cells were transfected with scrambled or Crtc2 siRNA for 36 h, followed by immunobloting of CRTC2 (e) or stimulating with db-cAMP for another 12 h for detection of mRNA expression (f). mRNA expression in db-cAMP-treated cells is normalized to vehicle-treated of each siRNA-transfected cells. (g) Expression of indicated mRNA in T cells stimulated with STS or db-cAMP for 12 h. (h) CRTC2(S171A) increases basal expression of Il12rb2 and Ifngr1, while SIK2(S587A) reduces cAMP-induced expression of these genes in TCR-activated T cells. (i) SIK2 WT and S587A mutant reduce basal expression of Il12rb2 and Ifngr1 in SIK2-deficient T cells. Data shown as mean±s.e.m. are representative of two (a–e,i) or three (f,g) independent experiments with triplicates or are from four independent experiments (h). Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant; a.u., arbitrary units.
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f5: SIK2-CRTC2 signalling mediates cAMP-induced expression of Il12rb2 and Ifngr1.(a) cAMP induces CREB phosphorylation and CRTC2 dephosphorylation in T cells. (b) cAMP and STS reduces cytoplasmic CRTC2 and increases nuclear CRTC2 in T cells that were rested for 2 days and then stimulated for 1 h. (c) cAMP does not increase nuclear CRTC2 in SIK2−/− T cells. (d) GAL4 reporter assay of CRTC2 activity of SIK2+/+ or SIK2−/− T cells that were transfected with GAL4-fusion pM-mCRTC2 and pTAL-5xGAL4-Luciferase reporter and stimulated with db-cAMP for the indicated times. The activity is shown as the ratio of firefly luciferase activity to renilla pRL-TK luciferase activity. (e,f) T cells were transfected with scrambled or Crtc2 siRNA for 36 h, followed by immunobloting of CRTC2 (e) or stimulating with db-cAMP for another 12 h for detection of mRNA expression (f). mRNA expression in db-cAMP-treated cells is normalized to vehicle-treated of each siRNA-transfected cells. (g) Expression of indicated mRNA in T cells stimulated with STS or db-cAMP for 12 h. (h) CRTC2(S171A) increases basal expression of Il12rb2 and Ifngr1, while SIK2(S587A) reduces cAMP-induced expression of these genes in TCR-activated T cells. (i) SIK2 WT and S587A mutant reduce basal expression of Il12rb2 and Ifngr1 in SIK2-deficient T cells. Data shown as mean±s.e.m. are representative of two (a–e,i) or three (f,g) independent experiments with triplicates or are from four independent experiments (h). Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant; a.u., arbitrary units.
Mentions: One candidate for such factors is CRTC2 that is regulated negatively by SIK2 and functions as a CREB coactivator in many types of cells19202122. Stimulation of T cells with db-cAMP induced not only phosphorylation of CREB but also dephosphorylation of CRTC2 (Fig. 5a), decreased the amount of CRTC2 in the cytoplasm and increased the amount of dephosphorylated CRTC2 in the nucleus (Fig. 5b). This distribution is SIK-dependent, because SIK2–/– T cells22 constitutively showed more nuclear CRTC2 than WT cells, which increased little by cAMP (Fig. 5c). As reported1819, protein kinase inhibitor staurosporine (STS) induced dephosphorylation and nuclear translocation of CRTC2 without enhancing CREB phosphorylation (Fig. 5b). To investigate whether cAMP affects transcriptional activity of CRTC2, we performed a reporter assay with GAL4-fusion CRTC2 in WT and SIK2–/– T cells. While db-cAMP increased GAL4-transcriptional activity in WT cells, the basal GAL4 activity was already higher in SIK2–/– T cells and no further increase was observed upon cAMP addition (Fig. 5d). These findings suggest that SIK2-CRTC2 pathway functions downstream of cAMP in T cells.

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