Regulated Nuclear-Cytoplasmic Localization of CCAAT/ Enhancer-binding Protein δ in Osteoblasts

Abstract

Insulin-like growth factor I (IGF-I) plays a central role in skeletal growth by promoting bone cell replication and differentiation. Prostaglandin E 2 (PGE 2 ) 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 PGE 2 activated the transcription factor CCAAT/enhancer-binding protein ␦ (C/EBP␦) 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/EBP␦. Under basal conditions, C/EBP␦ was cytoplasmic but rapidly accumulated in the nucleus after PGE 2 treatment (t1 ⁄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/EBP␦ was not a PKA substrate. Upon removal of hormonal stimulus, C/EBP␦ quickly exited the nucleus (t1 ⁄2 < 12 min) through a pathway blocked by leptomycin B. Mutagenesis studies indicated that the basic domain of C/EBP␦ 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/EBP␦ through its regulated nuclear import.

3-Ketosteroid Reductase Activity and Expression by Fetal Rat Osteoblasts

“…This finding is analogous to those from analyses of insulin-like growth factor I and transforming growth factor ␤ receptor type III gene promoters, which also respond rapidly to PKA-dependent activation and translocation of pre-existing C/EBP␦ in rat and human osteoblasts (31,39,50). Original inspection of the AKR1C9 gene promoter identified multiple C/EBP response elements.…”

“…CREB and Runx2 are constitutively expressed at high levels in differentiated osteoblasts, but within several contexts their abilities to bind DNA do not appear to be PGE2-sensitive (32,47). However, Runx2 drives the expression of C/EBP␦ (32), which then accumulates in the nucleus in osteoblasts and induces gene expression after exposure to PKA activating hormones like PGE2 (31,50). The possibility for involvement by C/EBP␦ was therefore assessed within the context of the 2.0 kb AKR1C9 promoter region.…”

C/EBPδ expression in a BCR-ABL-positive cell line induces growth arrest and myeloid differentiation

“…Earlier studies demonstrated that expression of C/EBPd in mammary cells results in G0/ G1 growth arrest and apoptosis along with upregulation of p27 expression (O'Rourke et al, 1999;Billiard et al, 2001). These results suggest that C/EBPd regulates growth in different cell types by influencing p27 levels.…”

“…In different cell types, C/EBPd can be found either in the cytoplasm in an inactive form, or in the nucleus where it can be functionally active (Billiard et al, 2001). DAPI staining and immunohistochemistry with a C/ EBPd antibody were used to determine the localization of C/EBPd in the KCL22 cells.…”

Functional Cooperation between CCAAT/Enhancer-Binding Proteins and the Vitamin D Receptor in Regulation of 25-Hydroxyvitamin D3 24-Hydroxylase

“…8D). A PKA-dependent accumulation of C/EBP␦ and C/EBP␤ in the nucleus has been suggested as an additional mechanism involved in the second messenger-mediated enhancement of gene expression via activation of C/EBPs (9,13,38). C/EBP␤ has also been reported to be a substrate for PKA, and its phosphorylation has been shown to be required for its nuclear translocation (13).…”