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

• Published in: The Journal of biological chemistry Vol. 272; no. 50; pp. 31793 - 31800
• Main Authors: Umayahara, Y., Ji, C., Centrella, M., Rotwein, P., McCarthy, T. L.
• Format: Journal Article
• Language: English
• Published: Legacy CDMS 12.12.1997
• Subjects: Animals; Base Sequence; CCAAT-Enhancer-Binding Proteins; COS Cells; Cyclic AMP - physiology; Dinoprostone - pharmacology; DNA-Binding Proteins - pharmacology; Female; Insulin-Like Growth Factor I - genetics; Life Sciences (General); Molecular Sequence Data; Nuclear Proteins - pharmacology; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - metabolism; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Signal Transduction; Transcription Factors - pharmacology; Transcription, Genetic - drug effects; Transcriptional Activation - drug effects; Non-Nasa Center; Nasa Discipline Musculoskeletal; NASA Discipline Musculoskeletal; Non-NASA Center
• ISSN: 0021-9258
• DOI: 10.1074/jbc.272.50.31793

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.