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CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblastsInsulin-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.
Document ID
20040142016
Acquisition Source
Legacy CDMS
Document Type
Reprint (Version printed in journal)
Authors
Umayahara, Y.
(Oregon Health Sciences University, Department of Medicine Molecular Medicine Division, Portland, Oregon 97201-3098, United States)
Billiard, J.
Ji, C.
Centrella, M.
McCarthy, T. L.
Rotwein, P.
Date Acquired
August 22, 2013
Publication Date
April 9, 1999
Publication Information
Publication: The Journal of biological chemistry
Volume: 274
Issue: 15
ISSN: 0021-9258
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: 5-RO1-DK37449
CONTRACT_GRANT: 5-PO1-HD20805
Distribution Limits
Public
Copyright
Other
Keywords
NASA Discipline Musculoskeletal
Non-NASA Center

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