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

Julia BilliardDagger , Yutaka UmayaharaDagger , Kristine Wiren§, Michael Centrella, Thomas L. McCarthy, and Peter RotweinDagger ||

From the Dagger  Oregon Health Sciences University, Molecular Medicine Division, Department of Medicine, Portland, Oregon 97201-3098, the § Oregon Health Sciences University, Departments of Cell and Developmental Biology and Medicine, Portland, Oregon 97201-3098, and the  Yale University School of Medicine, Section of Plastic Surgery, New Haven, Connecticut 06520-8041

Received for publication, November 1, 2000, and in revised form, January 22, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (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/EBPdelta was not a PKA substrate. Upon removal of hormonal stimulus, C/EBPdelta quickly exited the nucleus (t1/2 < 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factor-I (IGF-I),1 a 70-amino acid secreted protein, plays a central role in regulating growth and development in mammals and other vertebrates (1, 2). IGF-I promotes the survival, proliferation, and differentiation of many cell types and tissues, including bone, where it enhances osteoblast replication and type I collagen synthesis, among other actions (3, 4). IGF-I is produced by various cells within the skeleton, including osteoblasts (5), and its synthesis is enhanced by systemic and locally produced hormones that regulate skeletal function, such as parathyroid hormone and prostaglandin E2 (PGE2) (5, 6). The increase in IGF-I induced by these hormones may explain their anabolic actions within the skeleton, and IGF-I may serve as a coupling factor to balance the remodeling sequence of resorption and new bone formation (5, 7, 8).

In cultured bone cells, both PGE2 and parathyroid hormone stimulate IGF-I gene and protein expression by a transcriptional mechanism (9-11). These effects on IGF-I gene transcription are mediated by hormone-induced increases in cAMP and subsequent activation of cAMP-dependent protein kinase (PKA) (5, 6, 12). As evidence for this pathway, the major IGF-I promoter can be induced in transient transfection experiments in osteoblasts by a co-transfected catalytic subunit of PKA to the level seen with PGE2 treatment (10). Furthermore, a dominant-interfering mutant regulatory subunit of PKA that does not bind cAMP blocks hormone-activated gene expression (10). In past studies, we mapped a functional cAMP response element to the 5'-untranslated region of IGF-I exon 1 within a previously footprinted site termed HS3D (10, 13) and showed that this sequence was required for full hormonal responsiveness of the IGF-I promoter in osteoblasts (13). More recently, we identified CCAAT/enhancer-binding protein delta  (C/EBPdelta ) as the critical hormone-regulated transcription factor responsible for PKA-stimulated IGF-I gene transcription through the HS3D sequence (14, 15) and showed that hormones that activate PKA induce binding of C/EBPdelta to this site (14).

C/EBPdelta belongs to a family of transcriptional regulators that function in tissue differentiation, metabolism, healing, and immune responses (16). Members of the C/EBP family are related structurally, each consisting of an NH2-terminal transactivation region, a central basic DNA-binding domain, and a COOH-terminal dimerization interface termed the leucine zipper segment (16). C/EBP proteins share similarities in the latter two domains with a larger group of basic-leucine zipper transcription factors (16, 17). The first C/EBP proteins to be characterized, C/EBPalpha and C/EBPbeta , have key roles in adipocyte differentiation and in gene expression in the liver and other tissues (16, 18-21). C/EBPdelta has been implicated in control of adipogenesis and in mediating the acute phase response to inflammatory stimuli (16, 18, 19). In addition, our previous work indicated a regulatory role for this protein in IGF-I gene expression in bone cells (14, 15).

The current experiments were designed to assess mechanisms of activation of C/EBPdelta in osteoblasts. We now find that a PKA-dependent pathway stimulates the rapid nuclear translocation of C/EBPdelta in the absence of ongoing protein synthesis. Continual PKA activity is required for nuclear retention of C/EBPdelta , because C/EBPdelta is quickly removed from the nucleus through an exportin-mediated pathway upon cessation of hormone action. Mutagenesis studies indicate that the basic domain of C/EBPdelta is necessary for nuclear localization and that the leucine zipper region permits full nuclear accumulation. In the aggregate, this report defines a pathway for hormone-mediated activation of C/EBPdelta through its regulated nuclear import.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Timed-pregnant Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Collagenase types 1 and 2 were obtained from Worthington Biochemical Corporation (Lakewood, NJ). Leptomycin B was a gift from Dr. Minoru Yoshida (University of Tokyo, Tokyo, Japan). PGE2, forskolin, and cycloheximide were purchased from Sigma. H-89 and KT 5720 were from Calbiochem (San Diego, CA); [gamma -32P]ATP was obtained from PerkinElmer Life Sciences. LY294002 was purchased from Biomol Research Laboratories (Plymouth Meeting, PA), and UO126 was from Promega Corporation (Madison, WI). The long lasting IGF-I analogue, R3IGF-I, was from Gropep (Adelaide, Australia), and PDGF-BB was from Life Technologies, Inc. PGE2 was reconstituted at a concentration of 1 mM in ethanol, R3IGF-I was resuspended in 10 mM HCl, and PDGF-BB was resuspended in 11.1 mM acetic acid with 10% BSA. All other drugs were dissolved in Me2SO to at least 1000 times the final concentration. The catalytic subunit of PKA was a gift from Dr. James Lundblad (Oregon Health Sciences University, Portland, OR). Recombinant His-tagged cAMP response element-binding protein (CREB) and His-tagged mutant CREB S133A were gifts from Dr. Richard A. Maurer (Oregon Health Sciences University, Portland, OR). The mutant regulatory subunit of mouse PKA (clone MtR(AB)) was a gift from Dr. G. Stanley McKnight (University of Washington, Seattle, WA). Polyclonal antibodies to C/EBPdelta were raised in chickens and purified as described previously (15). A monoclonal antibody to the flag epitope and Hoechst dye were obtained from Sigma. Other antibodies (to Akt, extracellular signal-regulated kinases 1 and 2, phospho-Akt, and phospho-extracellular signal-regulated kinase) were from New England Biolabs (Beverly, MA). Cy3-conjugated rabbit anti-chicken IgY was from Jackson ImmunoResearch Laboratories (West Grove, PA); fluorescein isothiocyanate-conjugated goat anti-mouse IgG and alkaline phosphatase-conjugated goat anti-rabbit IgG were from Southern Biotechnology Associates (Birmingham, AL); and horseradish peroxidase-coupled rabbit anti-chicken IgY was from Promega Corporation. All other reagents were purchased from commercial suppliers.

Cell Cultures and Transfections-- Osteoblast-enriched cell cultures were prepared from isolated calvarial bones of 21-day-old Sprague-Dawley rat fetuses, as previously described (22, 23). Cranial sutures were removed by dissection, and cells were dispersed by five sequential digestions with collagenase. The last three digestions, which are enriched in cells expressing the osteoblast phenotype, were pooled and plated at 8000 cells/cm2 in minimum essential medium (Life Technologies, Inc.) containing 20 mM HEPES, 10% bovine serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). Cells were incubated at 37 °C in humidified air containing 5% CO2. Human fetal osteoblast cell line hFOB 1.19 (24) was purchased from ATCC and propagated in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with 10% fetal bovine serum and 0.3 mg/ml G418 (all from Life Technologies, Inc.). These cells express a temperature-sensitive mutant of SV40 large T antigen and proliferate at the permissive temperature (34 °C) in humidified air containing 5% CO2. At confluence, T antigen expression is inhibited by shifting the cells to 39 °C, and osteoblast markers are expressed (24). Both types of cells were transfected at ~50% confluent density with 2 µg of total plasmid per 9.6 cm2 using GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) for rat osteoblasts and LipofectAMINE (Life Technologies, Inc.) for hFOB 1.19 cells, according to the manufacturers' instructions. Experiments were performed at 48-72 h after transfection, when the cells had reached confluent density.

Immunocytochemistry-- Confluent osteoblast cultures were pre-incubated in serum-free medium for 20 h, followed by addition of drugs or vehicle (ethanol or Me2SO or both diluted 1:1000) in serum-free medium for the times specified. Where indicated, after incubation with PGE2, cultures were rinsed twice with phosphate-buffered saline (PBS), and fresh medium was added for various times. Cycloheximide and all protein kinase inhibitors were added to cells 15 min prior to addition of PGE2. Following hormone and drug treatment, cultures were rinsed with PBS, fixed in 4% paraformaldehyde, permeabilized with a 1:1 mixture of acetone and methanol, and blocked with 10% BSA in PBS. After two washes with PBS, cells were incubated with primary antibodies, either polyclonal chicken anti-C/EBPdelta (1:500) or monoclonal anti-flag (1:440) in PBS plus 3% BSA for 2 h at 25 °C. Cells were then washed three times in PBS and incubated with 100 ng/ml Hoechst dye and the appropriate labeled secondary antibodies, either Cy3-conjugated rabbit anti-chicken IgY (1:400) or fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:1000), for 1-2 h in the dark. Cells then were washed in PBS and examined by fluorescence microscopy (Nikon Eclipse TE 300). Images were captured with an Optronics CCD camera using an Apple Macintosh G3 computer and Scion Image software, version 1.62. Images were saved in Photoshop 5.5 (Adobe Systems, San Jose, CA).

Protein Extraction and Immunoblotting-- Confluent osteoblast cultures were deprived of serum for 20 h and then treated with PGE2 for varying intervals, as indicated above. For inhibitor studies, cells were pretreated with the inhibitors for 15 min followed by addition of R3IGF-I or PDGF-BB in the presence of inhibitors for 15 min. Cytoplasmic and nuclear protein extracts (13) or whole cell extracts (25) were prepared as described, and aliquots were stored at -80 °C until use. Western immunoblotting was performed as described previously (13, 25). Immunoreactive proteins were visualized by enhanced chemiluminescence, followed by exposure to x-ray film, or by enhanced chemifluorescence followed by detection and quantitation using Molecular Imager FX imaging system and Quantity One software (Bio-Rad).

Preparation of Recombinant and in Vitro Translated Proteins-- Preparation of recombinant S-tagged C/EBPdelta (S-C/EBPdelta ) and C/EBPdelta in Escherichia coli has been described (15). The 31-amino acid NH2-terminal S-tag includes a minimal consensus phosphorylation site for PKA (Arg-Gly-Ser). C/EBPdelta was translated in vitro using the pET29a-C/EBPdelta plasmid (15) and TNT coupled reticulocyte lysate system (Promega Corporation), according to the manufacturer's instructions.

PKA Assay-- The purified, recombinant catalytic subunit of PKA was diluted to 10 µg/ml in 100 µg/ml BSA. Recombinant CREB or C/EBPdelta proteins (1 µg each) were mixed on ice with 0.5 µCi of [gamma -32P]ATP in assay buffer (100 µM ATP, 10 mM MgCl2, 250 µg/ml BSA, 12.5 mM Tris-Cl, pH 7.5). PKA (10 ng) was added, and the reactions were allowed to proceed for 2 min at 30 °C. The reactions were stopped after being placed on ice by addition of EDTA to 80 mM final concentration. After boiling for 5 min in SDS sample buffer, the samples were separated by SDS-polyacrylamide gel electrophoresis. Gels were stained with Coomassie Brilliant Blue, dried, and exposed to x-ray film for 2 h at -80 °C with intensifying screens.

Construction of Recombinant Plasmids-- Flag-tagged rat C/EBPdelta in pcDNA3 (pcDNA3-flag-C/EBPdelta ) was generated by polymerase chain reaction-mediated mutagenesis. The flag epitope tag (codons underlined) was added to the 5' end of C/EBPdelta in pBluescript-C/EBPdelta (15) just 3' to a BamHI site and an ATG codon (bold) using the following oligonucleotides: 5'-GCGGATCCGCCACCATGGACTACAAGGACGACGATGACAAGAGCGCCGCTCTTTTCAGCCTA-3' (top strand) and 5'-CCAGTCGGGTTCGCGCTTCA-3' (bottom strand). The amplified fragment was digested with BamHI and PstI and inserted into BamHI- and PstI-digested pBluescript-C/EBPdelta . After DNA sequencing to verify the intended changes, the entire C/EBPdelta coding region was excised by digestion with BamHI and EcoRI and inserted into the corresponding sites of pcDNA3 (Invitrogen, Carlsbad, CA) to produce pcDNA3-flag- C/EBPdelta . The C/EBPdelta deletion plasmids diagramed in Fig. 8A were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). C/EBPdelta Delta Zip was prepared by introducing a point mutation (C to T) after codon 216, which places a stop codon (bold) just beyond the COOH terminus of the basic region. A KpnI site (underlined) also was added following the stop codon to aid in identifying the mutant. The oligonucleotides used were as follows: 5'-CGCCGCAACTAGGGTACCGAGATGCAGCAGA-3' (top strand) and 5'-TCTGCTGCATCTCGGTACCCTAGTTGCGG CG-3' (bottom strand). For C/EBPdelta Delta B, the basic region (amino acids 193-216) was deleted in-frame and replaced with an EcoRV site (underlined) with oligonucleotides 5'-CGGGGCAGCC CTGATATCCAGGAGATGCAG-3' (top strand) and 5'-CTGCATCTCCTGGATATCAGGGCTGCCCCG-3' (bottom strand). For C/EBPdelta Delta BZip, a stop codon (bold) followed by an XbaI site (underlined) was introduced after codon 192, immediately following the transactivation domain, with oligonucleotides 5'-CGGGGCAGCCCTTAGTCTAGATACCGGCAGC-3' (top strand) and 5'-GCTGCCGGTATCTAGACTAAGGGCTGCCCCG-3' (bottom strand). For each plasmid generated, the coding region was verified by DNA sequencing.

The EGFP·C/EBPdelta fusion proteins are diagramed in Fig. 8A. To generate EGFP containing the basic and leucine zipper regions of C/EBPdelta (EGFP+BZip), the BZip DNA region of C/EBPdelta was excised from pcDNA3-flag-C/EBPdelta Delta BZip by digestion with XbaI, followed by filling in the overhang with the Klenow fragment of DNA polymerase I, and digestion with BamHI. This DNA fragment was then inserted in-frame into EcoRI (blunted with Klenow) and BamHI-digested pEGFP-C1 (CLONTECH Laboratories, Palo Alto, CA). To produce EGFP with the leucine zipper region of C/EBPdelta (EGFP+Zip), the EcoRV-EcoRI fragment was excised from pcDNA3-C/EBPdelta Delta B and inserted into pEGFP-C1 that had been digested with HindIII (blunted with Klenow) and EcoRI. EGFP containing just the basic segment of C/EBPdelta (EGFP+B) was prepared as follows. CEBPdelta Delta Zip was excised from pcDNA3-CEBPdelta Delta Zip by digestion with BamHI and EcoRI and ligated into corresponding sites in the polylinker of pEGFP-C1. In this construct there is a stop codon after the basic region of C/EBPdelta . The portion of C/EBPdelta 5' to the basic region was then eliminated by mutagenesis using oligonucleotides that overlapped the 3' end of the EGFP coding region (5'-TCCGGACTTGTACAGCTCGTCCATGCCGAGTG-3' (top strand)) and the 5' end of the C/EBPdelta basic domain (5'-GAGTACCGGCAGCGACGCGAGCGCAACAACATC-3' (bottom strand)). The amplified region was verified by sequencing.

Statistical Analysis-- Data are presented as the means ± S.E. Statistical significance was determined using the Student's t test for paired samples. Results were considered statistically different when p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PGE2 Stimulates Nuclear Translocation of C/EBPdelta in Rat Osteoblasts-- We previously identified C/EBPdelta as the key transcription factor mediating cAMP-activated IGF-I gene transcription in primary rat osteoblasts (14, 15). We showed that stimulation of DNA binding of C/EBPdelta to its critical recognition site in the major IGF-I gene promoter and the subsequent induction of IGF-I gene expression were independent of the new protein synthesis (13). These results indicated that C/EBPdelta was activated by PGE2 through post-translational mechanisms in osteoblasts. The current experiments were designed to determine how C/EBPdelta was regulated in these cells. Fig. 1 shows that incubation of osteoblasts with PGE2 stimulated the nuclear accumulation of C/EBPdelta . As seen by immunocytochemistry in Fig. 1A, under control conditions C/EBPdelta was diffusely distributed within the cell, but after 4 h of incubation with 1 µM PGE2 the protein was predominantly nuclear. Pre-incubation with cycloheximide at a concentration (2 µM) found previously to block >90% of ongoing protein synthesis in osteoblasts (13) did not prevent accumulation of C/EBPdelta in nuclei, indicating that pre-existing C/EBPdelta was translocated to the nucleus in a protein synthesis-independent manner. This interpretation was validated by Western immunoblotting of osteoblast protein extracts (Fig. 1B). In control cells, C/EBPdelta was detected in cytoplasmic but not nuclear extracts. However, within 2 h of hormone treatment, it was found predominantly among soluble nuclear proteins and was depleted from the cytoplasm. Thus, PGE2 induces nuclear translocation of C/EBPdelta in primary cultures of rat osteoblasts.


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Fig. 1.   C/EBPdelta accumulates in the nucleus of rat osteoblasts upon PGE2 treatment. A, immunocytochemistry for C/EBPdelta of primary rat osteoblasts after incubation with vehicle (con) or 1 µM PGE2 for 4 h in the absence (-Chx) or presence (+Chx) of 2 µM cycloheximide. Panels on the right show nuclei identified with Hoechst dye. B, Western immunoblot for C/EBPdelta of nuclear (nuc; 20 µg) and cytoplasmic (cyto; 50 µg) protein extracts of primary rat osteoblasts treated with vehicle (0) or 1 µM PGE2 for 2 or 4 h. The control lane (C) contains 5 µl of in vitro translated C/EBPdelta .

We next looked at the kinetics of nuclear accumulation of C/EBPdelta . Fig. 2 shows results of time course studies. Again, under basal conditions C/EBPdelta was concentrated in <1% of osteoblast nuclei. Within 15 min of PGE2 treatment, C/EBPdelta expression was primarily nuclear in 25.5 ± 2.0% of cells. The proportion of cells with predominantly nuclear C/EBPdelta increased to 64% by 30 min and to ~94% at 1 and 2 h (t1/2 of nuclear accumulation = 27.1 min). Upon removal of hormone, C/EBPdelta rapidly disappeared from nuclei and reaccumulated in the cytoplasm (t1/2 = 11.6 min). Only 20.0 ± 2.2% of cells retained prominent nuclear expression by 15 min (the earliest time point examined), and <1% retained prominent expression by 1 h. Thus, in response to PGE2, C/EBPdelta was rapidly redistributed from cytoplasm to nucleus and returned to the cytoplasm quickly after termination of the hormonal stimulus.


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Fig. 2.   Kinetics of nuclear accumulation of C/EBPdelta in rat osteoblasts. The upper panels show results of time course experiments documenting by immunocytochemistry the appearance of C/EBPdelta in nuclei of primary rat osteoblasts after incubation with 1 µM PGE2 or vehicle (con) for the indicated times. For washout studies, cells were treated with PGE2 for 2 h, washed twice with PBS, and incubated in fresh medium without PGE2 for the times indicated. In the lower panels, nuclei stained with Hoechst dye are indicated in blue. The bar graphs below the fluorescence micrographs show the percentage of cells with nuclear C/EBPdelta at each time point (mean ± S.E. of four fields of cells (40-100 cells/field) from two experiments). Under control conditions, fewer than 1% of cells had predominantly nuclear C/EBPdelta . The process of nuclear accumulation fitted a linear progression curve with t1/2 = 27.1 min; nuclear disappearance was found to be an exponential function with t1/2 = 11.6 min.

Nuclear Translocation of C/EBPdelta Is Dependent on PKA-- We next looked at the signaling mechanisms involved in hormonal regulation of the subcellular distribution of C/EBPdelta . Primary osteoblasts were treated with PGE2 after pre-incubation with specific protein kinase inhibitors (Fig. 3A). The drugs LY294002 (phosphatidylinositol 3-kinase) or UO126 (MEK1 and 2) did not prevent PGE2-induced nuclear translocation of C/EBPdelta and did not alter the primarily cytoplasmic distribution of C/EBPdelta under control conditions. In contrast, the PKA inhibitors H-89 and KT5720 each prevented the appearance of C/EBPdelta in nuclei after PGE2 treatment. To demonstrate that LY294002 and UO126 were effective in osteoblasts, cells were treated with either IGF-I or PDGF in either the absence or the presence of the two inhibitors. As shown in Fig. 3B, pre-incubation with LY294002 inhibited IGF-mediated phosphorylation of the phosphatidylinositol 3-kinase target, Akt, and UO126 prevented phosphorylation of the MEK targets, extracellular signal-regulated kinases 1 and 2, by PDGF.


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Fig. 3.   Prevention of nuclear translocation of C/EBPdelta in rat osteoblasts by inhibitors of PKA but not of phosphatidylinositol 3-kinase or MEK. A, immunocytochemistry for C/EBPdelta of primary rat osteoblasts after incubation with vehicle (con) or 1 µM PGE2 for 4 h in the absence or presence of the inhibitors listed below the fluorescence micrographs. B, inhibition of target kinases by LY294002 and UO126. The graph shows the percent inhibition of IGF-I-stimulated phosphorylation of Akt by LY294002 (LY) and PDGF-stimulated phosphorylation of extracellular signal-regulated kinases 1 and 2 by UO126 (UO). The IGF-I analogue, R3IGF-I (2 nM) was used to activate the phosphatidylinositol 3-kinase-Akt pathway, and PDGF-BB (0.4 nM) was used for the MEK-extracellular signal-regulated kinase pathway. Results are presented as the mean of two experiments. Inhibitors were used at the following concentrations: H-89, 10 µM; KT5720, 10 µM; LY294002, 30 µM; and UO126, 10 µM.

As a further test that PKA was required to stimulate the nuclear translocation of C/EBPdelta in osteoblasts, cells were co-transfected with expression plasmids for the marker protein, EGFP, and for a modified regulatory subunit of PKA that cannot bind cAMP. This latter protein thus acts to block endogenous enzyme activity (26). Following incubation with forskolin (10 µM for 2 h) to activate adenylate cyclase, the subcellular location of C/EBPdelta was assessed by immunocytochemistry (Fig. 4). Treatment with forskolin stimulated the nuclear accumulation of C/EBPdelta in nontransfected cells but did not alter the predominantly cytoplasmic distribution of C/EBPdelta in osteoblasts expressing the dominant-interfering PKA regulatory subunit (Fig. 4A, left panels). As shown in Fig. 4B, forskolin treatment induced nuclear translocation of C/EBPdelta in 91.7 ± 3.5% of cells transfected with the empty expression plasmid but only in 11.2 ± 1.7% of cells transfected with the dominant-interfering regulatory subunit of PKA (p = 0.0013). Thus, PKA activity is required for hormone-regulated nuclear translocation of C/EBPdelta .


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Fig. 4.   A dominant-interfering mutant of PKA blocks nuclear translocation of C/EBPdelta in rat osteoblasts. A, immunocytochemistry for C/EBPdelta of primary rat osteoblasts after transient co-transfection with EGFP and a dominant-interfering mutant of the PKA regulatory subunit and treatment with forskolin (10 µM) for 2 h. C/EBPdelta is shown in red (left panels), EGFP is shown in green (center panels), and nuclei are shown in blue (right panels). B, the graph shows the percentage of transfected cells with C/EBPdelta in the nucleus after treatment with forskolin (forsk) for 2 h (mean ± S.E. of three experiments with no fewer than 100 cells counted per treatment). The asterisk indicates that significantly fewer cells transfected with the dominant-interfering PKA regulatory subunit (dnPKA) expressed C/EBPdelta in their nuclei than did cells transfected with vector (p = 0.0013).

C/EBPdelta Is Not a Direct Substrate for PKA in Vitro-- The next series of experiments was designed to determine whether C/EBPdelta was phosphorylated by PKA. Other studies have shown that the related transcription factor, C/EBPbeta , is a substrate for PKA (27, 28), and inspection of the protein sequence of C/EBPdelta revealed several potential PKA phosphorylation sites. To test the hypothesis that C/EBPdelta is a substrate for PKA, recombinant C/EBPdelta was generated in E. coli, purified, and used in in vitro kinase assays with the purified, recombinant catalytic subunit of PKA. As shown in Fig. 5, under the conditions described under "Experimental Procedures," the cAMP-regulated transcription factor, CREB, was readily phosphorylated by PKA, whereas a mutant CREB lacking the PKA phosphorylation site at serine residue 133 was not labeled (29). These results demonstrate the specificity of the in vitro kinase assay. C/EBPdelta also was not phosphorylated by PKA, but S-C/EBPdelta was labeled. S-C/EBPdelta contains a consensus PKA site in the NH2-terminal S-tag. These in vitro experiments show that C/EBPdelta does not appear to be a high affinity substrate for PKA.


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Fig. 5.   PKA does not directly phosphorylate C/EBPdelta . The top panel shows results of in vitro kinase assays performed as described under "Experimental Procedures." The bottom panel shows the dried gel stained with Coomassie Brilliant Blue. Bovine albumin was added as a carrier to all samples, as indicated on the gel. S-C/EBPdelta contained an NH2-terminal "S-tag" as described under "Experimental Procedures" and included a consensus phosphorylation site for PKA. The mutant CREB (m-CREB) contained a substitution of alanine for serine at residue 133 within the PKA phosphorylation site.

An Inhibitor of Nuclear Export Does Not Alter the Cytoplasmic Distribution of C/EBPdelta in the Absence of Hormone Treatment-- The results presented in Figs. 1-4 did not allow us to determine whether C/EBPdelta was constitutively cytoplasmic under basal conditions or whether it rapidly shuttled between cytoplasmic and nuclear compartments. To distinguish between these possibilities, we employed the antibiotic leptomycin B (30, 31), which specifically inhibits chromosome region maintenance 1 (CRM1), the receptor that functions to export proteins from the nucleus (32, 33). Fig. 6 shows that leptomycin B did not alter the subcellular distribution of C/EBPdelta under basal conditions and did not influence the ability of PGE2 to stimulate its nuclear translocation or of H-89 to prevent it. To demonstrate that leptomycin B was effective in osteoblasts, cells were pre-treated with PGE2 for 2 h, washed with PBS, and then incubated with leptomycin B or with vehicle. Under these experimental conditions, leptomycin B inhibited the exit of C/EBPdelta from nuclei (Fig. 7). In vehicle-treated cells, export of C/EBPdelta to the cytoplasm was rapid, being nearly complete within 30 min. By contrast, in osteoblasts incubated with leptomycin B, C/EBPdelta remained predominantly nuclear for up to 4 h. Based on these results, we conclude that in the absence of hormonal stimulation, C/EBPdelta is primarily cytoplasmic and that PKA induces transport of C/EBPdelta into the nucleus.


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Fig. 6.   Leptomycin B does not interfere with PKA-mediated nuclear translocation of C/EBPdelta in rat osteoblasts. Immunocytochemistry for C/EBPdelta of primary rat osteoblasts after incubation with vehicle (con), 1 µM PGE2, or 10 µM H-89 in the absence or presence of 10 ng/ml of leptomycin B (lept B). Nuclei stained with Hoechst dye are blue.


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Fig. 7.   Treatment with leptomycin B prevents the exit of C/EBPdelta from the nucleus of rat osteoblasts. Immunocytochemistry for C/EBPdelta of primary rat osteoblasts after incubation with 1 µM PGE2 for 2 h followed by washes with PBS and addition of vehicle or 10 ng/ml leptomycin B (- lept B or + lept B, respectively) for the times indicated. In the absence of leptomycin B, the t1/2 for the disappearance of C/EBPdelta from the nucleus was 11.8 min, and in the presence of leptomycin B, t1/2 was >4 h.

The Basic and Leucine Zipper Domains of C/EBPdelta Are Required for Nuclear Targeting in Osteoblasts-- We next sought to identify the domains of C/EBPdelta that were required for its nuclear localization. The full-length protein contains three major functional segments: an NH2-terminal transcriptional activation domain, a basic region that mediates DNA binding, and a leucine zipper segment that is responsible for dimerization (16). We generated expression plasmids for full-length and truncated rat C/EBPdelta , each containing an NH2-terminal flag epitope tag to distinguish them from endogenous C/EBPdelta (Fig. 8A). Upon transient transfection into primary rat osteoblasts (data not shown) or into the human osteoblast cell line hFOB 1.19, full-length C/EBPdelta was found in the nucleus even in the absence of hormone treatment (Fig. 8B, left top panel). This precluded us from investigating the regulation of transfected C/EBPdelta by PKA. We instead used the various truncation mutants of C/EBPdelta to establish the structural requirements for its nuclear localization. A truncation mutant lacking the leucine zipper (C/EBPdelta Delta Zip) was primarily nuclear when expressed in hFOB 1.19 cells. In contrast, mutant proteins lacking the basic domain (C/EBPdelta Delta B) or both basic and leucine zipper regions (C/EBPdelta Delta BZip) were predominantly cytoplasmic (Fig. 8B, top panels). These observations are consistent with results obtained with the related transcription factor, C/EBPbeta , which show that the highly conserved basic region contains the nuclear localization sequence (34). In C/EBPdelta , however, removal of the leucine zipper led to partial expression in the cytoplasm (Fig. 8B, top, compare the two left panels), indicating that this latter region also contributes to nuclear localization.


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Fig. 8.   The basic and leucine zipper domains are required for nuclear targeting of C/EBPdelta in osteoblasts. A, schematic representations of full-length 268-amino acid C/EBPdelta and deletion or truncation mutants and of fusion proteins containing different portions of C/EBPdelta joined to the COOH terminus of EGFP. F, flag epitope tag; TA, transactivation domain; B, basic region; Zip, leucine zipper segment. B, immunocytochemistry for the flag epitope tag (upper panels) or immunofluorescence for EGFP (lower panels) after transient transfections of the indicated expression plasmids into fetal human osteoblast cell line hFOB 1.19.

These results were confirmed after transfection of EGFP fusion constructs containing different segments of C/EBPdelta at their COOH termini (diagrammed in Fig. 8A). EGFP fused to the basic and leucine zipper regions (EGFP+BZip) was exclusively nuclear when expressed in hFOB 1.19 cells. EGFP plus the basic domain (EGFP+B) was predominantly nuclear, and EGFP plus the leucine zipper (EGFP+Zip) was diffusely distributed, as was EGFP (Fig. 8B, lower panels). We interpret these experiments to indicate that a nuclear localization sequence (NLS) resides within the basic region of C/EBPdelta but that the leucine zipper contains additional determinants that facilitate full expression within the nucleus.

One NLS Is Sufficient to Translocate a C/EBPdelta Dimer into the Nucleus-- The hFOB 1.19 cell line does not produce C/EBPdelta (assessed by immunocytochemistry and immunoblotting; data not shown). In these cells, transfected C/EBPdelta Delta B was found exclusively in the cytoplasm (Figs. 9, top left panel, and 8B). However, when expressed in primary rat osteoblasts, C/EBPdelta Delta B was concentrated in the nucleus (Fig. 9, top row, third panel from left). These results suggested the possibility that the transfected protein dimerized with endogenous C/EBPdelta and used its NLS for translocation into the nucleus. Consistent with this hypothesis, blocking basal PKA activity with H-89 resulted in retention of C/EBPdelta Delta B in the cytoplasm (Fig. 9, top right panel). In confirmation of these results, transfected C/EBPdelta Delta BZip was located in the cytoplasm of both hFOB 1.19 cells and primary rat osteoblasts (Fig. 9, lower panels). C/EBPdelta Delta BZip lacks both the basic and leucine zipper domains and can neither dimerize nor translocate to the nucleus by itself. We interpret these observations to indicate that C/EBPdelta forms dimers in the cytoplasm and that one NLS is sufficient for nuclear localization of the dimer when at least basal PKA activity is present in the cells.


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Fig. 9.   One NLS is sufficient to translocate a C/EBPdelta dimer into the nucleus in osteoblasts. Immunocytochemistry for the flag epitope tag of hFOB 1.19 cells (no endogenous C/EBPdelta expression) and primary rat osteoblasts (rOB) transfected with the indicated C/EBPdelta deletion and truncation mutants and treated with vehicle (-) or 10 µM H-89 for 2 h.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our current studies define a mechanism for activation of the transcription factor C/EBPdelta in primary rat osteoblasts through its regulated nuclear import by a PKA-mediated pathway. In previous studies, we identified C/EBPdelta as the critical transcription factor for induction of IGF-I gene expression in response to PGE2 (14, 15). We showed that PGE2 stimulated PKA in osteoblasts (12) and that PKA induced C/EBPdelta to bind to a site termed HS3D located within the major IGF-I gene promoter, leading to activation of IGF-I gene transcription (10, 13, 14). This pathway of hormonal stimulation of gene expression also has been shown to be independent of new protein synthesis (13). Using a combination of experimental approaches we now demonstrate that activation of PKA by forskolin or PGE2 leads to the rapid accumulation of C/EBPdelta in osteoblast nuclei. Nuclear translocation was induced within 15 min of hormone treatment (the earliest time point examined), was detected in the majority of osteoblasts by 60 min, and persisted for at least 4 h when cells were continually incubated with PGE2. Translocation of C/EBPdelta into osteoblast nuclei occurred in the presence of the protein synthesis inhibitor cycloheximide, indicating that it was mediated by post-translational mechanisms. Upon removal of hormone, C/EBPdelta exited the nucleus rapidly (t1/2 < 12 min), suggesting that a continuous stimulus was needed to maintain its nuclear localization. Regulated nuclear translocation of C/EBPdelta was blocked by the specific PKA inhibitors H-89 and KT5720 and by forced expression of a dominant-interfering regulatory subunit of PKA, indicating that nuclear translocation was stimulated by PKA. Surprisingly, C/EBPdelta did not appear to be a direct substrate for PKA, because the purified enzyme failed to phosphorylate C/EBPdelta in vitro to a measurable extent. Therefore, activation of C/EBPdelta by PKA occurs through an indirect mechanism, perhaps by a PKA-initiated signaling cascade. However, attempts to block this postulated pathway with LY294002 or UO126 were unsuccessful. These latter results may be interpreted to indicate that neither phosphatidylinositol 3-kinase-Akt nor MEK-extracellular signal-regulated kinase pathways control the activity of C/EBPdelta in osteoblasts.

This report presents the first example of PKA-dependent nuclear import of C/EBPdelta in any cell type, although in a cultured hepatocyte cell line, treatment with tumor necrosis factor-alpha induced its nuclear accumulation (35). The regulated nuclear import of C/EBPdelta defined here appears to resemble the pathway of nuclear translocation of the related transcription factor, C/EBPbeta . As shown by several investigators, C/EBPbeta resided in the cytoplasm in unstimulated cells and accumulated in the nucleus after treatment with agents that activated PKA with kinetics similar to those observed here for C/EBPdelta (28, 36). However, C/EBPbeta is a substrate for PKA, and its phosphorylation is required for its nuclear translocation (28). In addition, C/EBPbeta can be phosphorylated in vitro by PKA on serines 277 and 299 (27) and in cells on serine 299 (28). An alanine substitution at residue 299 blocked regulated nuclear translocation of C/EBPbeta in DKO-1 colon carcinoma cells (28), providing clear evidence for control of nuclear localization by direct phosphorylation. In contrast, we were unable to demonstrate that C/EBPdelta was a substrate for PKA in vitro, confirming results of Kageyama et al. (37). C/EBPdelta has been shown to become phosphorylated after treatment of HepG2 cells with interleukin-1 (38), and changes in phosphorylation induced by other cytokines have been implicated in its transcriptional activation (38, 39). DNA binding of C/EBPdelta to a consensus site also has been shown to be increased ~3-fold after phosphorylation in vitro by casein kinase II (40). No information is available on a role for casein kinase II in modulating the function of C/EBPdelta in cells. Other members of the C/EBP family, including C/EBPbeta and CHOP, undergo regulated phosphorylation by several different protein kinases. These modifications result in either altered DNA binding (27, 41) or transcriptional activity (28, 42-47). However, in preliminary experiments we were unable to detect an increase in phosphorylated C/EBPdelta in primary rat osteoblasts after incubation with PGE2 (data not shown), indicating that this modification may not be part of the mechanism of nuclear translocation induced by PKA in these cells.

Very little is known about the cellular steps controlling nuclear import of members of the C/EBP family of transcription factors. Morever, no information is available regarding which nuclear import receptors interact with C/EBPdelta or which importins are expressed in osteoblasts. We find that C/EBPdelta is exclusively cytoplasmic when PKA activity is suppressed by H-89 or KT5720, providing more evidence for a key role for PKA in promoting nuclear translocation, but not further identifying cellular mechanisms. Our transfection experiments show that the basic region of C/EBPdelta is essential for nuclear import, as has been described for other C/EBPs (34) and other members of the basic-leucine zipper transcription factor family (48-50). Our results additionally suggest a secondary function for the leucine zipper domain of C/EBPdelta in maintaining full nuclear expression, because proteins lacking this domain were partially distributed in the cytoplasm. The leucine zipper also may play a role in regulated nuclear expression of C/EBPdelta , because we find that a modified C/EBPdelta lacking the basic segment is located in the nucleus in rat osteoblasts under basal conditions but in the cytoplasm after treatment of cells with H-89. The same protein is exclusively cytoplasmic in hFOB 1.19 cells that do not express C/EBPdelta endogenously. These observations in aggregrate indicate that dimerization may occur between transfected and endogenous C/EBPdelta , potentially through the leucine zipper regions, and that a single NLS within the dimer was sufficient for nuclear localization.

The mechanisms responsible for maintaining C/EBPdelta in the nucleus after activation by PKA or for inducing its nuclear export are unknown. Our results show that C/EBPdelta did not undergo continual shuttling between subcellular compartments under basal conditions or after hormone treatment. Rather, continuous activity of PKA was required to retain C/EBPdelta in the nucleus of primary rat osteoblasts, because removal of hormonal stimulus led to rapid redistribution into the cytoplasm (t1/2 < 12 min). The pathways of nuclear export of C/EBPdelta involved CRM1, because inhibition of this receptor with leptomycin B (30, 31) caused prolonged retention of C/EBPdelta in nuclei after removal of hormone. The segment of C/EBPdelta that interacts with CRM1 is not known. The currently recognized consensus sequences for binding to the nuclear export receptor include closely spaced short stretches of leucine residues (51, 52). No typical consensus sequence is found in C/EBPdelta . To date, however, no functional studies have been performed to demonstrate direct interactions of C/EBPdelta with CRM1.

An intriguing question arising from our current and previous results is whether nuclear localization alone is sufficient for full transcriptional activity of C/EBPdelta or whether other modifications of the protein are required. As shown in this report, forced expression of C/EBPdelta in osteoblasts resulted in its accumulation in the nucleus and is sufficient to transactivate an IGF-I promoter-reporter gene, although treatment of cells with PGE2 further increases the level of IGF-I promoter function (14, 15). Thus, potentially more than one mechanism controls the transcriptional response of the IGF-I gene to PKA.

In summary, we have shown that stimulation of PKA in primary rat osteoblasts led to the rapid activation of the transcription factor C/EBPdelta through its regulated nuclear import. Nuclear targeting required the basic region of C/EBPdelta and was enhanced by the presence of the leucine zipper motif. Our results provide a framework for defining the specific cellular machinery and molecular mechanisms by which hormones that activate cAMP induce the nuclear translocation of a critical transcription factor that regulates expression of IGF-I, a key gene product for bone growth and maturation.

    ACKNOWLEDGEMENTS

We thank the following individuals for reagents: Dr. James Lundblad for the catalytic subunit of PKA, Dr. Richard Maurer for CREB and mutant CREB, Dr. G. Stanley McKnight for the expression plasmid encoding the dominant-interfering subunit of PKA, and Dr. Minoru Yoshida for leptomycin B. We are grateful to Dr. Hong Ma for help in constructing recombinant plasmids and Anne Evans for assistance in establishing the primary rat osteoblast cultures.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants 5-RO1-DK37449 (to P. R.), 1-RO1-DK56310 (to T. L. M.), and 5F32-DK09802 (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Oregon Health Sciences University, Molecular Medicine Division, 3181 S.W. Sam Jackson Park Rd., Mail code NRC3, Portland, OR 97201-3098. Tel.: 503-494-0536; Fax: 503-494-7368; E-mail: rotweinp@ohsu.edu.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009973200

    ABBREVIATIONS

The abbreviations used are: IGF-I, insulin-like growth factor-I; PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase; C/EBPdelta , CCAAT/enhancer-binding protein delta ; PDGF, platelet-derived growth factor; BSA, bovine serum albumin; CREB, cAMP response element-binding protein; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CRM1, chromosome region maintenance 1; NLS, nuclear localization sequence.

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