CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone.
Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast nuclear proteins. These results identify C/EBPdelta as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a high affinity binding site for this protein.
Umayahara
Y
Y
Oregon Health Sciences University, Department of Medicine, Molecular Medicine Division, Portland, Oregon 97201-3098, USA.
Ji
C
C
Centrella
M
M
Rotwein
P
P
McCarthy
T L
TL
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D019556:COS Cells; D000242:Cyclic AMP / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D015232:Dinoprostone / Q000494:pharmacology; D005260:Female; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014157:Transcription Factors / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP; D015232:Dinoprostone