The McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the alpha subunit of the Gs protein (Gs alpha) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin-6 (IL-6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL-6 production by activating mutant Gs alpha in bone cells, we established mouse osteoblastic MC3T3-E1 cells stably transfected with the activating mutant Gs alpha expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL-6 than the cells transfected with control vector or wild-type Gs alpha expression vector. Analysis of the IL-6 promoter revealed that any of the AP-1, nuclear factor (NF)-IL-6, and NF-kappa B binding elements are important for the activating mutant Gs alpha-induced gene expression. Electrophoretic mobility-shift assays using nuclear extracts of the mutant Gs alpha-expressing cells showed that phospho(Ser133)-cAMP-responsive element binding protein (CREB), AP-1, NF-IL6, and NF-kappa B were increased, compared with the control cells or the wild-type Gs alpha-expressing cells. These results indicate that activating mutant Gs alpha increases the transcriptional factors binding to CRE, AP-1, NF-IL6, and NF-kappa B elements to induce IL-6 gene expression in the osteoblastic cells.