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 grow th by stimulating bone cell replication and differentiation. We previo usly showed that prostaglandin E-2 (PGE(2)) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteo blasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hor monal regulation of IGF-I gene expression, We now demonstrate that CCA AT/enhancer-binding protein (C/EBP) delta is a major component of a PG E(2)-stimulated DNA-protein complex involving HS3D and find that C/EBP delta 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 nucl ear proteins. Southwestern blotting add UV-cross-linking studies showe d that the HS3D probe recognized a similar to 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBP delta comprised mo st of the PGE(2)-activated gel-shifted complex, C/EBP delta was detect ed by Western immunoblotting in osteoblast nuclear extracts after trea tment of cells with PGE(2). An HS3D oligonucleotide competed effective ly with a high affinity C/EBP site from the rat albumin gene for bindi ng to osteoblast nuclear proteins, Cotransfection of osteoblast cell c ultures with a C/EBP delta expression plasmid enhanced basal and PGE(2 )-activated IGE-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site, By contrast, an expression plas mid for the related protein, C/EBP beta, did not alter basal IGF-I gen e activity but did increase the response to PGE,. In osteoblasts and i n COS-7 cells, C/EBP delta, but not C/EBP beta, transactivated a repor ter gene containing four tandem copies of HS3D fused to a minimal prom oter; 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/EBP delta as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D elem ent within IGF-I promoter 1 is a high affinity binding site for this p rotein.