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INTRODUCTION |
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
(C/EBP
) 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/EBP
to this site
(14).
C/EBP
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/EBP
and C/EBP
, have key roles in
adipocyte differentiation and in gene expression in the liver and other
tissues (16, 18-21). C/EBP
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/EBP
in osteoblasts. We now find that a
PKA-dependent pathway stimulates the rapid nuclear
translocation of C/EBP
in the absence of ongoing protein synthesis.
Continual PKA activity is required for nuclear retention of C/EBP
,
because C/EBP
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/EBP
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/EBP
through its regulated nuclear import.
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EXPERIMENTAL PROCEDURES |
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); [
-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/EBP
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/EBP
(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/EBP
(S-C/EBP
) and C/EBP
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/EBP
was translated in vitro using the
pET29a-C/EBP
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/EBP
proteins (1 µg each) were mixed on ice with 0.5 µCi of
[
-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/EBP
in pcDNA3 (pcDNA3-flag-C/EBP
) was generated by
polymerase chain reaction-mediated mutagenesis. The flag epitope tag
(codons underlined) was added to the 5' end of C/EBP
in
pBluescript-C/EBP
(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/EBP
. After DNA sequencing to
verify the intended changes, the entire C/EBP
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/EBP
. The C/EBP
deletion
plasmids diagramed in Fig. 8A were prepared using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
C/EBP
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/EBP
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/EBP
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/EBP
fusion proteins are diagramed in Fig.
8A. To generate EGFP containing the basic and leucine zipper
regions of C/EBP
(EGFP+BZip), the BZip DNA region of C/EBP
was
excised from pcDNA3-flag-C/EBP
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/EBP
(EGFP+Zip), the
EcoRV-EcoRI fragment was excised from
pcDNA3-C/EBP
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/EBP
(EGFP+B) was prepared as follows. CEBP
Zip was excised from
pcDNA3-CEBP
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/EBP
. The portion of C/EBP
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/EBP
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.
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RESULTS |
PGE2 Stimulates Nuclear Translocation of C/EBP
in
Rat Osteoblasts--
We previously identified C/EBP
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/EBP
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/EBP
was activated by PGE2 through post-translational mechanisms in osteoblasts. The current experiments were designed to determine how C/EBP
was regulated in these cells. Fig. 1 shows that incubation of
osteoblasts with PGE2 stimulated the nuclear accumulation
of C/EBP
. As seen by immunocytochemistry in Fig. 1A,
under control conditions C/EBP
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/EBP
in nuclei, indicating
that pre-existing C/EBP
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/EBP
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/EBP
in primary cultures of rat
osteoblasts.

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Fig. 1.
C/EBP
accumulates in the nucleus of rat osteoblasts upon
PGE2 treatment. A, immunocytochemistry for
C/EBP 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/EBP 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/EBP .
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We next looked at the kinetics of nuclear accumulation of C/EBP
.
Fig. 2 shows results of time course
studies. Again, under basal conditions C/EBP
was concentrated in
<1% of osteoblast nuclei. Within 15 min of PGE2
treatment, C/EBP
expression was primarily nuclear in 25.5 ± 2.0% of cells. The proportion of cells with predominantly nuclear
C/EBP
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/EBP
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/EBP
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/EBP in rat osteoblasts. The upper
panels show results of time course experiments documenting by
immunocytochemistry the appearance of C/EBP 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/EBP 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/EBP . 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.
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Nuclear Translocation of C/EBP
Is Dependent on PKA--
We next
looked at the signaling mechanisms involved in hormonal regulation of
the subcellular distribution of C/EBP
. 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/EBP
and did not
alter the primarily cytoplasmic distribution of C/EBP
under control
conditions. In contrast, the PKA inhibitors H-89 and KT5720 each
prevented the appearance of C/EBP
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/EBP in rat osteoblasts by inhibitors of PKA
but not of phosphatidylinositol 3-kinase or MEK. A,
immunocytochemistry for C/EBP 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.
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As a further test that PKA was required to stimulate the nuclear
translocation of C/EBP
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/EBP
was
assessed by immunocytochemistry (Fig. 4).
Treatment with forskolin stimulated the nuclear accumulation of
C/EBP
in nontransfected cells but did not alter the predominantly
cytoplasmic distribution of C/EBP
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/EBP
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/EBP
.

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Fig. 4.
A dominant-interfering mutant of PKA
blocks nuclear translocation of C/EBP in rat
osteoblasts. A, immunocytochemistry for C/EBP 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/EBP 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/EBP 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/EBP in their
nuclei than did cells transfected with vector (p = 0.0013).
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C/EBP
Is Not a Direct Substrate for PKA in Vitro--
The next
series of experiments was designed to determine whether C/EBP
was
phosphorylated by PKA. Other studies have shown that the related
transcription factor, C/EBP
, is a substrate for PKA (27, 28), and
inspection of the protein sequence of C/EBP
revealed several
potential PKA phosphorylation sites. To test the hypothesis that
C/EBP
is a substrate for PKA, recombinant C/EBP
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/EBP
also was not
phosphorylated by PKA, but S-C/EBP
was labeled. S-C/EBP
contains
a consensus PKA site in the NH2-terminal S-tag. These
in vitro experiments show that C/EBP
does not appear to
be a high affinity substrate for PKA.

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Fig. 5.
PKA does not directly phosphorylate
C/EBP . 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/EBP
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.
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An Inhibitor of Nuclear Export Does Not Alter the Cytoplasmic
Distribution of C/EBP
in the Absence of Hormone Treatment--
The
results presented in Figs. 1-4 did not allow us to determine whether
C/EBP
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/EBP
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/EBP
from nuclei (Fig.
7). In vehicle-treated cells, export of
C/EBP
to the cytoplasm was rapid, being nearly complete within 30 min. By contrast, in osteoblasts incubated with leptomycin B, C/EBP
remained predominantly nuclear for up to 4 h. Based on these
results, we conclude that in the absence of hormonal stimulation,
C/EBP
is primarily cytoplasmic and that PKA induces transport of
C/EBP
into the nucleus.

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Fig. 6.
Leptomycin B does not interfere
with PKA-mediated nuclear translocation of C/EBP
in rat osteoblasts. Immunocytochemistry for C/EBP 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/EBP from the nucleus of rat
osteoblasts. Immunocytochemistry for C/EBP 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/EBP from the
nucleus was 11.8 min, and in the presence of leptomycin B,
t1/2 was >4 h.
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The Basic and Leucine Zipper Domains of C/EBP
Are Required for
Nuclear Targeting in Osteoblasts--
We next sought to identify the
domains of C/EBP
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/EBP
, each containing an
NH2-terminal flag epitope tag to distinguish them from
endogenous C/EBP
(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/EBP
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/EBP
by PKA. We instead used the various truncation mutants of C/EBP
to
establish the structural requirements for its nuclear localization. A
truncation mutant lacking the leucine zipper (C/EBP
Zip) was
primarily nuclear when expressed in hFOB 1.19 cells. In contrast,
mutant proteins lacking the basic domain (C/EBP
B) or both basic
and leucine zipper regions (C/EBP
BZip) were predominantly
cytoplasmic (Fig. 8B, top panels). These
observations are consistent with results obtained with the related
transcription factor, C/EBP
, which show that the highly conserved
basic region contains the nuclear localization sequence (34). In
C/EBP
, 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/EBP in
osteoblasts. A, schematic representations of
full-length 268-amino acid C/EBP and deletion or truncation mutants
and of fusion proteins containing different portions of C/EBP 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.
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These results were confirmed after transfection of EGFP fusion
constructs containing different segments of C/EBP
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/EBP
but that the leucine zipper contains additional determinants that
facilitate full expression within the nucleus.
One NLS Is Sufficient to Translocate a C/EBP
Dimer into the
Nucleus--
The hFOB 1.19 cell line does not produce C/EBP
(assessed by immunocytochemistry and immunoblotting; data not shown).
In these cells, transfected C/EBP
B was found exclusively in the
cytoplasm (Figs. 9, top left
panel, and 8B). However, when expressed in primary rat
osteoblasts, C/EBP
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/EBP
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/EBP
B in the cytoplasm (Fig.
9, top right panel). In confirmation of these results,
transfected C/EBP
BZip was located in the cytoplasm of both hFOB
1.19 cells and primary rat osteoblasts (Fig. 9, lower
panels). C/EBP
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/EBP
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/EBP dimer into the nucleus in
osteoblasts. Immunocytochemistry for the flag epitope tag of hFOB
1.19 cells (no endogenous C/EBP expression) and primary rat
osteoblasts (rOB) transfected with the
indicated C/EBP deletion and truncation mutants and treated with
vehicle ( ) or 10 µM H-89 for 2 h.
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DISCUSSION |
Our current studies define a mechanism for activation of the
transcription factor C/EBP
in primary rat osteoblasts through its
regulated nuclear import by a PKA-mediated pathway. In previous studies, we identified C/EBP
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/EBP
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/EBP
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/EBP
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/EBP
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/EBP
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/EBP
did not appear to be a direct substrate for PKA,
because the purified enzyme failed to phosphorylate C/EBP
in
vitro to a measurable extent. Therefore, activation of C/EBP
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/EBP
in osteoblasts.
This report presents the first example of PKA-dependent
nuclear import of C/EBP
in any cell type, although in a cultured hepatocyte cell line, treatment with tumor necrosis factor-
induced its nuclear accumulation (35). The regulated nuclear import of C/EBP
defined here appears to resemble the pathway of nuclear translocation
of the related transcription factor, C/EBP
. As shown by several
investigators, C/EBP
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/EBP
(28, 36). However, C/EBP
is a substrate for PKA, and its
phosphorylation is required for its nuclear translocation (28). In
addition, C/EBP
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/EBP
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/EBP
was a substrate
for PKA in vitro, confirming results of Kageyama et
al. (37). C/EBP
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/EBP
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/EBP
in cells. Other members of the C/EBP family,
including C/EBP
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/EBP
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/EBP
or which importins are expressed in
osteoblasts. We find that C/EBP
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/EBP
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/EBP
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/EBP
, because we find that a modified C/EBP
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/EBP
endogenously. These observations in aggregrate
indicate that dimerization may occur between transfected and endogenous C/EBP
, 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/EBP
in the nucleus
after activation by PKA or for inducing its nuclear export are
unknown. Our results show that C/EBP
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/EBP
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/EBP
involved CRM1, because inhibition of this
receptor with leptomycin B (30, 31) caused prolonged retention of
C/EBP
in nuclei after removal of hormone. The segment of C/EBP
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/EBP
. To date, however, no
functional studies have been performed to demonstrate direct
interactions of C/EBP
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/EBP
or whether other modifications of
the protein are required. As shown in this report, forced expression of
C/EBP
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/EBP
through its regulated nuclear import. Nuclear targeting required the basic region of C/EBP
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.