Abstract
Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.
Highlights
¶ To whom correspondence and reprint requests should be addressed: Oregon Health Sciences University, Dept. of Medicine, Molecular Medicine Division, 3181 S
Our previous studies defined HS3D as an atypical cAMP response element located in the 5Ј-untranslated region of rat Insulin-like growth factor-I (IGF-I) exon 1 that mediated hormonally activated IGF-I gene transcription in primary rat osteoblasts [13, 16]
The current experiments were designed to investigate the physical-chemical properties of the interactions between C/EBP␦ and the HS3D site and to determine whether C/EBP␦ was involved as a mediator of cAMP-activated transcription in IGF-I genes from species other than rats
Summary
Cell Culture—Primary osteoblast-enriched cell cultures were prepared from dissected and collagenase-digested parietal bones of 22-dayold Sprague-Dawley rat fetuses, as described previously [12, 29]. After sequencing the amplified portion, the entire C/EBP␦ coding region was inserted into BamHI- and SalI-digested pET29a(ϩ) (Novagen, Madison, WI) to make pET29a-C/EBP␦. A bacterial expression plasmid for C/EBP was constructed by directionally cloning rat C/EBP [22] into NcoI- and SalI-digested pET29a(ϩ) to generate pET29a-C/EBP. In this plasmid, the C/EBP coding sequence has been fused in frame to an NH2-terminal 27 amino acid S-tag. The medium was aspirated and cultures were rinsed with phosphate-buffered saline and lysed in cell lysis buffer (Promega Corp.), and luciferase activity was measured as described [13, 16].
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