Fig. 3. ISO induces IL-18BP expression via CREB.
A, Time-dependent effects of ISO on CREB activation. Quiescent ACM were treated with the cAMP antagonist Rp-8-Br-cAMPS (100 µM in water for 1 h) or infected with Ad.KCREB (right hand panel) prior to ISO addition (10−7M for 30 min). Total CREB, phospho-CREB (Ser133) and αTubulin were quantified by immunoblotting (n=3). B, ISO induced CREB DNA-binding activity. ACM treated with ISO (10−7 M) for up to 1 h were analyzed for CREB DNA-binding activity by EMSA using gene-specific primers and nuclear protein extracts. Specificity of DNA binding, indicated by an arrow, was verified in competition experiments as described in ‘Materials and methods’. Unbound labeled probe at the bottom of gel is not shown. Lamin A/C (nuclear) and GAPDH (cytoplasmic) served as loading and purity controls, and are shown in the bottom panel. C, ISO-mediated CREB activation was confirmed by a reporter assay. *P < 0.001 versus Ad.MCS-Luc + IL-18. D, ISO-stimulated CREB binding to Il18bp promoter in vivo was confirmed by the ChIP assay (n = 3). E, ISO induced Akt activation. ACM treated with ISO were analyzed by immunoblotting using phospho-Akt (Thr308) and Akt antibodies (n=3). F, ISO-induced Akt activation is attenuated by PI3K and Akt inhibitors and adenoviral transduction of dnPI3Kp85 and dnAkt1 (n=3). G, ISO induces ERK1/2 activation via PI3K, Akt, and MEK1/2. ACM were pre-treated with specific inhibitors of PI3K, Akt, MEK1/2 and ERK1/2, or transduced with adenoviral mutant vectors prior to ISO addition. Total and phospho-ERK1/2 were analyzed by immunoblotting (n=3). H, ISO activates CREB via PI3K, Akt, and MEK1/2. ACM treated as in A and G were analyzed for phospho-CREB levels (n=3). I, ISO induces IL-18BP mRNA expression via PI3K, Akt, and MEK1/2. ACM treated as in A and G were analyzed for IL-18BP mRNA expression by Northern blotting (n = 3).