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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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Activation of PKA-CREB pathway is required but insufficient for cAMP-induced IL-12Rβ2 and IFN-γR1 expression.(a) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with indicated concentrations of a PKA agonist, N6-Bnz-cAMP, for 12 h. (b) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with db-cAMP in the presence of PKA inhibitor, Rp-8-Br-cAMPS and/or Rp-8-CPT-cAMPS for 12 h. (c,d) T cells treated with scrambled or Creb1 siRNA for 48 h, followed by immunoblot for CREB (c) or stimulated with db-cAMP for another 12 h for detection of mRNA expression (d). mRNA expression is normalized to vehicle-treated of each siRNA-transfected cells. Icer and Nr4a2 were used as positive controls for CREB-dependent genes. (e) Immunoblot for p-CREB(S133) in IFN-γR1−/− T cells stimulated without (unactivated) or with either αCD3/CD28, PMA (20 ng ml−1) plus ionomycin (500 ng ml−1) (PMA+Iono), or db-cAMP for 30 min. (f) Expression of Il12rb2 and Ifngr1 mRNA in IFN-γR1−/− T cells stimulated as in e in the presence of anti-IL-2 for 12 h. Data shown as mean±s.e.m. are representative of two (a–c,e,f) or four (d) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
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f4: Activation of PKA-CREB pathway is required but insufficient for cAMP-induced IL-12Rβ2 and IFN-γR1 expression.(a) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with indicated concentrations of a PKA agonist, N6-Bnz-cAMP, for 12 h. (b) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with db-cAMP in the presence of PKA inhibitor, Rp-8-Br-cAMPS and/or Rp-8-CPT-cAMPS for 12 h. (c,d) T cells treated with scrambled or Creb1 siRNA for 48 h, followed by immunoblot for CREB (c) or stimulated with db-cAMP for another 12 h for detection of mRNA expression (d). mRNA expression is normalized to vehicle-treated of each siRNA-transfected cells. Icer and Nr4a2 were used as positive controls for CREB-dependent genes. (e) Immunoblot for p-CREB(S133) in IFN-γR1−/− T cells stimulated without (unactivated) or with either αCD3/CD28, PMA (20 ng ml−1) plus ionomycin (500 ng ml−1) (PMA+Iono), or db-cAMP for 30 min. (f) Expression of Il12rb2 and Ifngr1 mRNA in IFN-γR1−/− T cells stimulated as in e in the presence of anti-IL-2 for 12 h. Data shown as mean±s.e.m. are representative of two (a–c,e,f) or four (d) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.

Mentions: We next investigated the molecular mechanisms of how cAMP directly induces IL-12Rβ2 and IFN-γR1 expression. The db-cAMP-induced Il12rb2 and Ifngr1 expression was mimicked by a PKA-specific agonist N6-Bnz-cAMP (ref. 34) (Fig. 4a) and attenuated by PKA inhibitors (Fig. 4b) in unactivated T cells, which produced neither IL-2 nor IFN-γ. We transfected unactivated T cells with a small interfering RNA (siRNA) for Creb1 or a scrambled siRNA and then stimulated T cells with db-cAMP. Creb1-specific siRNA reduced CREB expression in transfected cells (Fig. 4c) and suppressed cAMP-induced Il12rb2 and Ifngr1 expression compared with those in T cells transfected with scrambled siRNA (Fig. 4d). CREB is usually activated by phosphorylation at Ser133, p-CREB(S133), which promotes transcription by recruitment of the coactivator CREB-binding protein to gene loci17. However, although TCR or phorbol-12-myristate-13-acetate (PMA) plus ionomycin could induce CREB activation (Fig. 4e), they did not induce, but rather reduced Il12rb2 or Ifngr1 expression under the condition where cAMP induced both mRNAs (Fig. 4f). These results suggest requirement of other factors downstream of PKA in addition to p-CREB(S133).


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)

Activation of PKA-CREB pathway is required but insufficient for cAMP-induced IL-12Rβ2 and IFN-γR1 expression.(a) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with indicated concentrations of a PKA agonist, N6-Bnz-cAMP, for 12 h. (b) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with db-cAMP in the presence of PKA inhibitor, Rp-8-Br-cAMPS and/or Rp-8-CPT-cAMPS for 12 h. (c,d) T cells treated with scrambled or Creb1 siRNA for 48 h, followed by immunoblot for CREB (c) or stimulated with db-cAMP for another 12 h for detection of mRNA expression (d). mRNA expression is normalized to vehicle-treated of each siRNA-transfected cells. Icer and Nr4a2 were used as positive controls for CREB-dependent genes. (e) Immunoblot for p-CREB(S133) in IFN-γR1−/− T cells stimulated without (unactivated) or with either αCD3/CD28, PMA (20 ng ml−1) plus ionomycin (500 ng ml−1) (PMA+Iono), or db-cAMP for 30 min. (f) Expression of Il12rb2 and Ifngr1 mRNA in IFN-γR1−/− T cells stimulated as in e in the presence of anti-IL-2 for 12 h. Data shown as mean±s.e.m. are representative of two (a–c,e,f) or four (d) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3644078&req=5

f4: Activation of PKA-CREB pathway is required but insufficient for cAMP-induced IL-12Rβ2 and IFN-γR1 expression.(a) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with indicated concentrations of a PKA agonist, N6-Bnz-cAMP, for 12 h. (b) Expression of Il12rb2 and Ifngr1 mRNA in T cells stimulated with db-cAMP in the presence of PKA inhibitor, Rp-8-Br-cAMPS and/or Rp-8-CPT-cAMPS for 12 h. (c,d) T cells treated with scrambled or Creb1 siRNA for 48 h, followed by immunoblot for CREB (c) or stimulated with db-cAMP for another 12 h for detection of mRNA expression (d). mRNA expression is normalized to vehicle-treated of each siRNA-transfected cells. Icer and Nr4a2 were used as positive controls for CREB-dependent genes. (e) Immunoblot for p-CREB(S133) in IFN-γR1−/− T cells stimulated without (unactivated) or with either αCD3/CD28, PMA (20 ng ml−1) plus ionomycin (500 ng ml−1) (PMA+Iono), or db-cAMP for 30 min. (f) Expression of Il12rb2 and Ifngr1 mRNA in IFN-γR1−/− T cells stimulated as in e in the presence of anti-IL-2 for 12 h. Data shown as mean±s.e.m. are representative of two (a–c,e,f) or four (d) independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed student’s t-test, *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
Mentions: We next investigated the molecular mechanisms of how cAMP directly induces IL-12Rβ2 and IFN-γR1 expression. The db-cAMP-induced Il12rb2 and Ifngr1 expression was mimicked by a PKA-specific agonist N6-Bnz-cAMP (ref. 34) (Fig. 4a) and attenuated by PKA inhibitors (Fig. 4b) in unactivated T cells, which produced neither IL-2 nor IFN-γ. We transfected unactivated T cells with a small interfering RNA (siRNA) for Creb1 or a scrambled siRNA and then stimulated T cells with db-cAMP. Creb1-specific siRNA reduced CREB expression in transfected cells (Fig. 4c) and suppressed cAMP-induced Il12rb2 and Ifngr1 expression compared with those in T cells transfected with scrambled siRNA (Fig. 4d). CREB is usually activated by phosphorylation at Ser133, p-CREB(S133), which promotes transcription by recruitment of the coactivator CREB-binding protein to gene loci17. However, although TCR or phorbol-12-myristate-13-acetate (PMA) plus ionomycin could induce CREB activation (Fig. 4e), they did not induce, but rather reduced Il12rb2 or Ifngr1 expression under the condition where cAMP induced both mRNAs (Fig. 4f). These results suggest requirement of other factors downstream of PKA in addition to p-CREB(S133).

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