Figure 4. 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.