We have recently reported that interleukin-1α (IL-1α) can induce human macrophage colony-stimulating factor (M-CSF) expression through nuclear factor κB (NF-κB) activation, and treatment of human pancreatic MIA PaCa-2 cancer cells with forskolin or cAMP attenuated the NF-κB activation as well as M-CSF expression. In this study, we have further investigated the mechanism of cAMP attenuation. MIA PaCa-2 cells were incubated with forskolin or dibutyryl-cAMP and then stimulated with IL-1 for 1h. Cell lysates were immunoprecipitated by anti-inhibitory κB (IκB) kinase-β (IKKβ) antibody and the immune complex assayed for kinase activity using recombinant inhibitor of NF-κB (IκBα) as substrate. The levels of IKKβ in the respective cellular proteins were measured by subsequent Western blot. The results show that the level of IKK protein remains constant in the presence of cAMP, forskolin and/or IL-1, whereas IKK activity was robustly stimulated by IL-1. Nonetheless, dibutyryl-cAMP or forskolin did not affect the IKK activation induced by IL-1. This experiment suggests that elevated cAMP has no effect on IKK activity. IκBα protein level decreased markedly in IL-1-treated cells compared with the untreated. By contrast, cells treated with cAMP or forskolin possessed discernibly higher IκBα levels. In addition, we observed that forskolin potentiated and prolonged the IL-1-induced IκBα mRNA levels, whereas it did not stabilize the IκBα mRNA message. Wholly, these studies indicate that elevated cAMP antagonizes IL-1-induced M-CSF transcription by up-regulating IκBα gene induction and its consequent attenuation of NF-κB activation.

Abbreviations used: M-CSF, macrophage colony-stimulating factor; IL, interleukin; TNF, tumour necrosis factor; DB-cAMP, dibutyryl-cAMP; EMSA, electrophoretic mobility-shift assay; NF-κB, nuclear factor κB; IκB, inhibitory κB; NIK, NF-κB-inducing kinase; IKK, IκB kinase; LPS, lipopolysaccharide; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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