INCREASED INTERLEUKIN-6 PRODUCTION IN MOUSE OSTEOBLASTIC MC3T3-E1 CELLS EXPRESSING ACTIVATING MUTANT OF THE STIMULATORY G-PROTEIN

The McCune-Albright syndrome (MAS) is characterized by polyostotic fib rous dysplasia, cafe-au-lait spots, and multiple endocrine hyperfuncti on, An activating missense mutation of the alpha subunit of the Gs pro tein (Gs alpha) was found in several affected tissues, resulting in pr olonged stimulation of adenylate cyclase. Our recent study has indicat ed that the cells derived from the fibrous bone dysplasia tissues in M AS patients produced increased levels of interleukin-6 (IL-6), which m ay be responsible for the increased bone resorption in this disease. I n 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 sho wed 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)-IL6, and NF-kappa B binding elements are important for the activating mutant Gs alpha-in duced gene expression. Electrophoretic mobility-shift assays using nuc lear extracts of the mutant Gs alpha-expressing cells showed that phos pho(Ser133)-cAMP-responsive element binding protein (CREB), AP-1, NF-I L6, and NF-kappa B were increased, compared with the control cells or the wild-type Gs alpha-expressing cells, These results indicate that a ctivating mutant Gs alpha increases the transcriptional factors bindin g to CRE, AP-1, NF-IL6, and NF-kappa B elements to induce IL-6 gene ex pression in the osteoblastic cells.