Regulated nuclear-cytoplasmic localization of CCAAT/enhancer-binding protein delta in osteoblasts.
Insulin-like growth factor I (IGF-I) plays a central role in skeletal growth by promoting bone cell replication and differentiation. Prostaglandin E2 (PGE2) and parathyroid hormone enhance cAMP production in cultured rat osteoblasts and stimulate IGF-I expression through a transcriptional mechanism mediated by cAMP-dependent protein kinase (PKA). We previously showed that PGE2 activated the transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta) in osteoblasts and induced its binding to a DNA element within the IGF-I promoter. We report here that a PKA-dependent pathway stimulates nuclear translocation of C/EBPdelta. Under basal conditions, C/EBPdelta was cytoplasmic but rapidly accumulated in the nucleus after PGE2 treatment (t(1/2) < 30 min). Nuclear translocation occurred without concurrent protein synthesis and was maintained in the presence of hormone. Nuclear localization required PKA and was blocked by a dominant-interfering regulatory subunit of the enzyme, even though C/EBPdelta was not a PKA substrate. Upon removal of hormonal stimulus, C/EBPdelta quickly exited the nucleus (t(1/2) < 12 min) through a pathway blocked by leptomycin B. Mutagenesis studies indicated that the basic domain of C/EBPdelta was necessary for nuclear localization and that the leucine zipper region permitted full nuclear accumulation. We thus define a pathway for PKA-mediated activation of C/EBPdelta through its regulated nuclear import.
Oregon Health Sciences University, Molecular Medicine Division, Department of Medicine, Portland, Oregon 97201-3098, USA.
J
J
Billiard
Y
Y
Umayahara
K
K
Wiren
M
M
Centrella
T L
TL
McCarthy
P
P
Rotwein
D000818:Animals; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D015232:Dinoprostone / Q000494:pharmacology; D005260:Female; D007150:Immunohistochemistry; D016350:Leucine Zippers; D008297:Male; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011247:Pregnancy; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone