DEVELOPMENTAL PROFILES OF PHOSPHORYLATED AND UNPHOSPHORYLATED CREBS IN MURINE CALVARIAL MC3T3-E1 CELLS

The cAMP-responsive element (CRE) binding protein/activating transcrip tion factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development, We analyzed t he changes of the amount, degree of phosphorylation and binding activi ty of the CREB/ATF family in the course of development of the murine c alvarial osteoblastic cell line MC3T3-E1 as an in, vitro model system of bone formation, The amount of CREB in the whole-cell extract detect able by Western blot analysis was high through all stages of developme nt and maximal in the proliferation stage, The degree of phosphorylati on estimated with anti-phosphorylated CREB antibody changed greatly an d reached high levels in the proliferation stage and early mineralizat ion stage, The ratio of phosphorylated CREB to total CREB in the CREB- CRE complex was also examined by gel shift assay, Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages, In the early mineralization stage, most CREB bound to con sensus/CRE was phosphorylated, while both phosphorylated and unphospho rylated CREB were bound to consensus/CRE in the proliferation stage, A TF-1 was also detected as a minor component bound to the consensus/CRE probe, The alteration of the binding of CREB to consensus/CRE over th e course of osteoblast development supports the hypothesis that CREB m ay regulate the expression of genes defining the developmental sequenc e of MC3T3-E1 cells.