Developmental profiles of phosphorylated and unphosphorylated CREBs in murine calvarial MC3T3-E1 cells.
The cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-E1 as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization 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 consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-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 the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.
Sakamoto
M K
MK
Department of Orthodontics Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0813, Japan. saka@den.hokudai.ac.jp
Suzuki
K
K
Takiya
S
S
Yoshimura
Y
Y
Imai
T
T
Matsumoto
A
A
Nakamura
S
S
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D016762:Genes, fos; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009841:Oligonucleotides / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012886:Skull / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D013997:Time Factors
D017362:Cyclic AMP Response Element-Binding Protein; D009841:Oligonucleotides; D015675:Osteocalcin; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP; D000469:Alkaline Phosphatase