Accelerated cell cycle progression in osteoblasts overexpressing the c-fos proto-oncogene: induction of cyclin A and enhanced CDK2 activity.
Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cell cycle genes. Here we have generated osteoblast cell lines expressing c-fos under the control of a tetracycline-regulatable promoter to investigate the role of c-Fos in osteoblast cell cycle control in vitro. Three stable subclones, AT9.2, AT9.3, and AT9.7, derived from MC3T3-E1 mouse osteoblasts, expressed high levels of exogenous c-fos mRNA and protein in the absence of tetracycline. Functional contribution of ectopic c-Fos to AP-1 complexes was confirmed by electromobility shift assays and transactivation of AP-1 reporter constructs. Induction of exogenous c-Fos in quiescent AT9.2 cells caused accelerated S-phase entry following serum stimulation, resulting in enhanced growth rate. Ectopic c-Fos resulted in increased expression of cyclins A and E protein levels, and premature activation of cyclin A-, cyclin E-, and cyclin-dependent kinase (CDK) 2-associated kinase activities, although cyclin D levels and CDK4 activity were not affected significantly in these cell lines. The enhanced CDK2 kinase activity was associated with a rapid, concomitant dissociation of p27 from CDK2-containing complexes. Deregulated cyclin A expression and CDK2 activity was also observed in primary mouse osteoblasts overexpressing c-Fos, but not in fibroblasts, and c-Fos transgenic tumor-derived osteosarcoma cells constitutively expressed high levels of cyclin A protein. These data suggest that overexpression of c-Fos in osteoblasts results in accelerated S phase entry as a result of deregulated cyclin A/E-CDK2 activity. This represents a novel role for c-Fos in osteoblast growth control and may provide c-Fos-overexpressing osteoblasts with a growth advantage during tumorigenesis.
Sunters
Andrew
A
Department of Craniofacial Development, King's College London, Guy's Hospital, Guy's Tower, United Kingdom.
Thomas
David P
DP
Yeudall
W Andrew
WA
Grigoriadis
Agamemnon E
AE
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005434:Flow Cytometry; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016196:S Phase; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection
D019925:Cyclin A; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases