From the Department of Developmental Biochemistry,
Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120 and the ¶ Department of Biochemistry, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106-4935
Received for publication, January 9, 2003
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ABSTRACT |
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The cytosolic form of the phosphoenolpyruvate
carboxykinase (PEPCK-C) gene is selectively expressed in several
tissues, primarily in the liver, kidney, and adipose tissue. The
transcription of the gene is reciprocally regulated by glucocorticoids
in these tissues. It is induced in the liver and kidney but repressed
in the white adipose tissue. To elucidate which adipocyte-specific transcription factors participate in the repression of the gene, DNase
I footprinting analyses of nuclear proteins from 3T3-F442A adipocytes
and transient transfection experiments in NIH3T3 cells were utilized.
Glucocorticoid treatment slightly reduced the nuclear C/EBP Glucocorticoids play a fundamental role in the
maintenance of homeostasis in mammals. Removal of the adrenals
severely compromises the ability of animals to withstand fasting (for
reviews see Refs. 1 and 2). Glucocorticoids exert their effects via the
glucocorticoid receptor
(GR),1 predominantly by
modulating gene transcription (3-5). An attractive mode of regulation,
especially in light of the coordinated effects of glucocorticoids in
maintaining homeostasis, is the opposing control of the same
gene in different tissues by GR. PEPCK-C gene provides an optimal model
for studying this mode of regulation. The transcription of this gene is
stimulated by glucocorticoids in the liver and kidney (6, 7) but is
repressed in the adipose tissue (8).
PEPCK-C catalyzes a key reaction that determines the rates of
gluconeogenesis in the liver and kidney and glyceroneogenesis in the
adipose tissue and liver (9). Glyceroneogenesis, the de novo
synthesis of 3-glycerophosphate from pyruvate and amino acids (via an
abbreviated version of gluconeogenesis), provides this precursor for
the synthesis of triglycerides (10, 11). Recently, we have performed a
targeted mutation in the adipose tissue-specific enhancer of the
PEPCK-C gene in embryonic stem cells. The mutation ablated
PEPCK-C gene expression in white adipose tissue of mice homozygous for
this mutation and caused a decrease in the storage of triglycerides,
which in some mice developed into lipodystrophy (12). This mutation
therefore established the importance of PEPCK-C and glyceroneogenesis
in the homeostasis of triglycerides in the adipose tissue.
Because PEPCK-C is encoded by a unique copy gene, and is transcribed
from a single promoter, it is likely that tissue-specific factors are
involved in the reciprocal regulation that leads to stimulation (liver
and kidney) or repression (adipose tissue) of the gene transcription in
the presence of glucocorticoids. Yamamoto and colleagues (13) proposed
the term composite GRE to describe a nonconsensus sequence
that binds the GR with low affinity and, in turn, is capable of
mediating either repression or activation of genes.
The GRE identified in the PEPCK-C gene (14) is a nonconsensus sequence
that binds GR at a very low affinity and is not able by itself to
transmit a transcriptional response to glucocorticoids. In fact,
PEPCK-C gene promoter harbors a GR unit (GRU) containing two low
affinity, nonconsensus GR-binding sites and two accessory elements, AF1
and AF2. These elements do not bind steroid receptors, but their
occupancy is required for the response of the PEPCK-C gene to
glucocorticoids (see scheme in Fig. 2). The factors binding to the AF1 element are all nonsteroid nuclear receptors; these include
hepatocyte nuclear factor (HNF) 4, chicken ovalbumin upstream transcription factor (COUPTF), retinoic acid receptor, retinoid X
receptor (RXR), and members of the peroxisome proliferator-activated receptor (PPAR) family. The AF2 site (15) binds HNF3 To identify tissue-specific factors that are involved in the
glucocorticoid-mediated repression of PEPCK-C gene transcription in
adipocytes, we have employed a systematic DNase I footprinting analysis
of the PEPCK-C gene promoter, using adipocyte nuclear proteins. Their
functional participation in the repression has been assessed using
transient transfection experiments in PEPCK-nonexpressing NIH3T3 cells.
The results from these two independent experimental systems
consistently identified the involvement of members of the C/EBP family,
but not those of PPAR, in the GR repression of the PEPCK-C gene
promoter activity. Furthermore, experiments in NIH3T3 cells revealed a
hierarchical constraint of PEPCK-C gene promoter trans-activation by
the separate GRU elements. Both wild type and mutant GR (incapable of
binding the DNA) repress the C/EBP-mediated trans-activation by
50-60%, regardless of whether the trans-activation is low or high.
However, the repression by mutant GR critically requires an intact AF1
site in the PEPCK-C gene.
Materials--
Dulbecco's modified Eagle's medium (DMEM),
F-12, and fetal and newborn calf serum were purchased from Biological
Industries, Kibutz Beit Haemek, Israel. Biosynthetic human insulin was
obtained from Novo Nordisk (Denmark). Dexamethasone, the synthetic
glucocorticoid hormone, was purchased from Teva, Israel Pharmaceutical
Industry. Ultraspec, the commercial reagent for the preparation of
tissue RNA, was purchased from Biotecx Laboratories, Inc. (Austin, TX). Radioactive signals were quantified using a PhosphorImager (Fujix BAS
1000, Fuji, Japan). Reverse transcriptase was obtained from Invitrogen. Random hexanucleotide pd(N)6 was purchased
from Amersham Biosciences, and the ribonuclease inhibitor, RNasin,
was purchased from Promega (Madison, WI). The enzyme-linked
immunosorbent assay kit for the determination of human somatotropin was
purchased from Roche Molecular Biochemicals.
Differentiation of Adipocytes--
3T3-F442A cells, obtained
from Dr. Howard Green (19), were grown to confluence in DMEM and
supplemented with 10% newborn calf serum. For differentiation of the
cells to adipocytes, newborn calf serum was replaced by 10% fetal calf
serum; isobutylmethylxanthine was added at a final concentration of 0.2 mM, and the cells were incubated for 3 days. The cells were
further incubated for at least 8 days in a medium containing 4 milliunits/ml insulin, until ~80-90% of cells contained fat droplets.
Dexamethasone Treatment--
Dexamethasone (10 RNA Isolation and RT-PCR Analysis--
Total RNA was isolated
from a single 100-mm cultured plate of 3T3-F442A adipocytes or from 30 mg of mouse liver using the commercial reagent Ultraspec according to
the manufacturer's instructions. One µg of total RNA was
reverse-transcribed according to manufacturer's instructions, using
the Invitrogen reverse transcriptase kit in the presence of 7.5 units/ml random hexanucleotide pd(N)6 as primer and 1 unit/µl RNasin ribonuclease inhibitor, except that the incubation was
for 30 min at 42 °C.
PCR was performed using the PEPCK-C primers 5'-CTTGTCTACGAAGCTCTCAG
from exon 9 and 3'-CGTCCGAACATCCACTC from exon 10. Primers for the
aP2 gene were 5'-CCTGGAAGCTTGTCTCCAG from exon 1 and
3'-CTCTTGTGGAAGTCACGCC from exon 4. Primers for Preparation of Nuclear Proteins--
Nuclear proteins were
extracted from rat liver according to Gorski et al. (21) as
modified (22). Nuclear protein extracts from adipocytes were prepared
essentially as described previously (22), except that the sucrose
gradient step to further purify the nuclei was omitted. DNase I
footprinting assays were performed as described previously (22). The
autoradiographic density signals of specific bands in the exposed film
(see Figs. 2-4) were quantified using Fluor-STM
MultiImager with Multianalyst version 1.1 (Bio-Rad).
Western Blot Analysis--
5 µg of nuclear proteins from
adipocytes treated or untreated with dexamethasone (a synthetic
glucocorticoid) were separated on 15% SDS-PAGE and transferred to
nitrocellulose membrane (Protran BA 85, Schleicher & Schuell). C/EBP Cell Culture, Transfection Conditions, and CAT
Assays--
NIH3T3 cells were grown on 100-mm plates in DMEM
containing 10% fetal calf serum. For transfection, cells were
transferred to DMEM containing 10% newborn calf serum. Transfection
was performed essentially according to Chen and Okayama (24), as
described previously (25), 1 or 2 days after the cells reached
confluency. Supercoiled PEPCK-CAT plasmid (2 µg) and additional
carrier pBlueScript DNA (Stratagene), to make a total of 12 µg, were
used, and the transfection efficiency was monitored as described
previously (25). Where indicated, 1 µg each of the expression vectors
for C/EBP Plasmids Used in the Transfection Studies--
The previously
described plasmid 597-pck-CAT (PCK (600)-CAT) contains 597 bp of the
rat PEPCK-C gene promoter region fused to the CAT reporter gene (26).
The derived 2000-pck-CAT plasmid (PCK(2000)-CAT) contains 2000 bp of
the PEPCK-C gene promoter (27). The mutated AF1 (AF1-mut), the combined
mutation of GRE1 and GRE2 (mGRE1-2) (28), and the mutated AF2
(AF2-mut) (29) plasmids contain 2000 bp of the rat PEPCK-C gene
promoter mutated at these sites. The mouse promoters PCK(840)-CAT and
PCK(1500)-CAT contain 840 or 1500 bp upstream from the transcription
start site of PEPCK-C gene promoter, derived from a mouse genomic
clone, and fused to the CAT reporter gene as described previously (26). The PCK(1500-mut)-CAT plasmid contains a site-specific mutation of the
PPARE sequence. The mutation was generated by inserting the restriction
sites XhoI and SmaI 3' and 5', respectively, to the PPARE site. This enabled us to replace the PPARE site with 47 bp of
the pBlueScript polylinker residing between XhoI and SmaI, except that the EcoRI site of the
polylinker insert was deleted.
The DNA concentration of the constructs containing the longer PEPCK-C
gene promoters was corrected to achieve the same number of molecules as
that of the shorter PEPCK-C gene promoters. Expression vectors encoding
adipocyte-enriched transcription factors used in this work included the
following: C/EBP Repression of PEPCK-C Expression by Glucocorticoids in
Vivo--
The addition of glucocorticoids to fully differentiated
3T3-F442A adipocytes for 16-18 h caused a strong repression of PEPCK-C gene expression as shown by the absence of its RT-PCR product (Fig.
1). This repression was distinct because
glucocorticoids failed to inhibit the expression of the
adipocyte-specific aP2 gene (Fig. 1).
DNase I Footprinting Assays--
In order to assess whether GR
affects the binding of nuclear proteins to specific sites in the
PEPCK-C gene promoter, we systematically footprinted the gene promoter,
using nuclear proteins extracted from 3T3-F442A adipocyte cells that
had been incubated without or with glucocorticoids. A scheme of PEPCK-C
gene promoter depicting the binding sites of transcription factors is
shown (Fig. 2a). The figure
also includes the binding sites for nuclear proteins present in the
liver, fetal liver (where the gene for PEPCK-C is not actively
transcribed (22, 36)), kidney, and adipocytes (Fig. 2b).
Note that nuclear proteins from the liver and adipocytes bind to most
sites included within positions
Our footprinting analysis revealed that nuclear proteins extracted from
glucocorticoid-treated adipocytes failed to bind to all C/EBP
recognition sites in the PEPCK-C gene promoter: CRE-1 (Fig.
2c), P3 and P4 (Fig. 2d), and the single
C/EBP-like CRE-2 site (Fig. 2c). The prominent inhibition of
binding to all these sites occurred despite the wide spectrum of
affinities of the C/EBP recognition sites to C/EBP
The binding of adipocyte nuclear proteins to the recognition sites of
nuclear receptors in the PEPCK-C gene promoter has not been affected by
the glucocorticoid treatment. Thus, binding to AF1 site (also termed
P6) remained similar whether using nuclear proteins from adipocytes not
treated or treated with glucocorticoids (Fig. 3a). AF1 site
is a nuclear receptor recognition site that interacts with a variety of
nonsteroid nuclear receptors including HNF4, COUPTF, retinoic acid
receptor, RXR, and members of the PPAR family. Similar to adipocytes,
hepatic nuclear proteins also interact well with the AF1 site (Fig.
3a).
Another non-C/EBP-binding site, which likewise has not been affected by
the glucocorticoid treatment, is PPARE (the recognition site of the
heterodimer nuclear receptors PPAR
Transcription of the gene for C/EBP C/EBP Transient Transfection Experiments in NIH3T3 Cells--
The
involvement of adipocyte-enriched transcription factors in the
repression of PEPCK-C gene transcription by glucocorticoids was further
assessed using transient transfection assays in NIH3T3 cells. Although
these cells do not express PEPCK-C or the adipocyte-specific transcription factors PPAR
The preferential PPAR
C/EBP
Finally, we have assessed whether the enhanced C/EBP Our results shed light on the involvement of transcription
activators from the C/EBP family, but not those from the PPAR family, in the glucocorticoid-mediated repression of PEPCK-C gene transcription in adipocytes. Glucocorticoid treatment of 3T3-F442A adipocytes led to
a reduced nuclear concentration of C/EBP Experiments using NIH3T3 cells have disclosed a hierarchical regulation
of PEPCK-C gene transcription, in particular the constraint on the gene
promoter response to C/EBP-mediated activation by the GRU element in
the PEPCK-C gene promoter. Mutating any single element within the GRU
relieves this constraint, and the trans-activation by C/EBP Is the AF1 site involved in the GR repression of the C/EBP Because the repression by GR of the C/EBP-mediated trans-activation
does not require DNA binding of the receptor, the GR probably inhibits
trans-activation of PEPCK-C gene transcription via protein-protein interactions. This notion is supported by the fact that the percent repression by GR of the C/EBP Evidence of cross-talk between members of the C/EBP family and GR has
been documented in numerous systems. Members of the C/EBP family have
been shown to bind directly to the ligand binding domain of a number of
nuclear receptors, including GR (see Refs. 48 and 49 and for review see
Refs. 5 and 50). These interactions resulted either in induction or
inhibition of the target genes and did not necessarily involve binding
of GR to the DNA (48, 49). Alternatively, binding of C/EBP Beyond the molecular aspects of the glucocorticoid repression of
PEPCK-C gene transcription in adipose tissue, its physiological significance has gained new relevance. Metabolic studies have recently
documented very substantial rates of glyceroneogenesis in the liver of
both rats and humans during fasting (52, 53) or after ingestion of a
diet high in protein but devoid of carbohydrate (52). These findings
shed new light on the metabolic significance of the reciprocal control
of PEPCK-C gene transcription by glucocorticoids. PEPCK-C activity
catalyzes the rate-limiting step of both gluconeogenesis and
glyceroneogenesis, hence regulating both pathways. This has been
verified by deletion of the PEPCK-C gene in mice, resulting in neonatal
lethality from hypoglycemia (54). In addition, a targeted mutation of
the adipose tissue-specific PPAR Lipid is released from the adipose tissue as free fatty acids and from
the liver as triglycerides. Thus, glyceroneogenesis affects lipid
metabolism in opposite ways in the two tissues; it restrains fat
release from adipose tissue (57) and enhances it from the liver (58).
It has been shown in rats that adrenalectomy enhances glyceroneogenesis
and diminishes free fatty acid release from incubated epididymal fat
pads (59, 60). In a reciprocal experiment, the addition of
dexamethasone to cultured hepatocytes stimulated the synthesis of
triglycerides and apolipoproteins E and B, as well as stimulating the
release of very low density lipoproteins to the medium (58). How then
is lipid homeostasis coordinated between the two tissues? We propose
that the reciprocal regulation of PEPCK-C gene transcription by
glucocorticoids provides a mechanism for such coordination because it
represses PEPCK-C gene transcription in the adipose tissue and
simultaneously enhances it in the liver. Experiments to further prove
this hypothesis in vivo are under way.
concentration but prominently diminished the binding of adipocyte-derived nuclear proteins to CCAAT/enhancer-binding protein (C/EBP) recognition sites, without affecting the binding to nuclear receptor sites in the PEPCK-C gene promoter. Of members of the C/EBP
family of transcription factors, C/EBP
was the strongest trans-activator of the PEPCK-C gene promoter in the NIH3T3 cell line.
The glucocorticoid receptor (GR), in the presence of its hormone
ligand, inhibited the activation of the PEPCK-C gene promoter by
C/EBP
or C/EBP
but not by the adipocyte-specific peroxisome proliferator-activated receptor
2. This inhibition effect was similar using the wild type or mutant GR and did not depend on GR
binding to the DNA. The glucocorticoid response unit (GRU) in the
PEPCK-C gene promoter (
2000 to +73) restrained
C/EBP
-mediated trans-activation, because mutation of each single GRU
element increased this activation by 3-4-fold. This series of GRU
mutations were repressed by wild type GR to the same percent as was the nonmutated PEPCK-C gene promoter. In contrast, the repression by
mutant GR depended on the intact AF1 site in the gene promoter, whereby
mutation of the AF1 element abolished the repression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(16, 17) and
has been proposed to comprise an insulin-response element as well
(18).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7
M) was added to NIH3T3 cells no later than 20 h after
transfection, when expression of the reporter gene was barely detected.
Cells were harvested no later than 24 h after addition of
dexamethasone to prevent cell lysis. 3T3-F442A adipocytes were
similarly treated with 10
7 M dexamethasone
for 16-18 h.
-actin
were the same as published previously (20). PCR was performed in the
presence of a trace of [32P]dCTP (0.5 × 106 dpm) to allow semi-quantification (20). The PCR program
included denaturation at 94 °C for 1 min, annealing at 55 °C for
1 min, and elongation at 72 °C for 2 min, 20 cycles each consisting
of denaturation for 30 s, annealing for 1 min, and elongation for 1 min. The PCR product was separated by electrophoresis on 8% polyacrylamide gel and quantified using a PhosphorImager apparatus and
visualized by its exposure to autoradiographic film.
was probed with rabbit polyclonal anti-C/EBP
antibody c100 diluted
1:1000 (a gift from Dr. Steven McKnight) and was detected using
horseradish peroxidase-conjugated goat anti-rabbit antibody diluted
1:4000. Nuclear Y12 protein was probed with mouse anti-sm
monoclonal antibody Y12 (23) (a gift from Dr. Ruth Sperling), diluted
1:10, and detected using horseradish peroxidase conjugated to
affinity-purified goat anti-mouse IgG F(ab)' fragment diluted 1:3000
(23). Secondary antibodies were visualized with SuperSignal West Pico
chemiluminescent substrate (Pierce).
and PPAR
2 together with RXR
or GR was used. The
optimal quantity of C/EBP
or C/EBP
expression vectors added to
the transfection mixture was 0.5 µg each. In all cases, titration
test of various concentrations of the expression vectors allowed us to
choose the amounts that yielded optimal effects. Assays for
chloramphenicol acetyltransferase (CAT) activity were determined
44 h after transfection, as described (25), and quantified using a
PhosphorImager apparatus. The S.E. was calculated as the S.D. divided
by the square root of the number of experiments.
(30) obtained from Dr. Steven McKnight; C/EBP
(31) from Dr. David Ron; C/EBP
(32) from Dr. Daniel Lane; PPAR
2
(33) from Dr. Bruce Spiegelman; RXR
(34) and human GR (35) from the
laboratory of Dr. Ron Evans; and rat wild type and mutant GR were a
gift from Dr. Keith Yamamoto (13).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of the glucocorticoids on PEPCK-C gene
expression in adipocytes. cDNA made from 1 µg of total RNA,
using random hexamer primers, was amplified by PCR (1 µl out of the
total 30 µl of RT reaction mix), using PEPCK-C and -actin primers.
The PCR products from adipocytes treated with dexamethasone (A + Dex) compared with the nontreated cells (A
Dex),
were identified by size separation as indicated on the right-hand
side of the figure. The RT-PCR products of liver PEPCK-C RNA
(L) and of the adipocyte-specific aP2 gene are
shown as controls.
70 to
1200 of the transcription
start site of the PEPCK-C gene promoter. Adipocyte nuclear proteins do
not bind to the P2 site (HNF-1 recognition motif) in the PEPCK-C gene
promoter (37), whereas nuclear proteins from the liver bind poorly to
PPARE (the PPAR recognition site (see Fig.
3b)). Unlike the kidney and
fetal liver, nuclear proteins from adipocytes and liver bind all C/EBP
recognition sites. These include the CRE-1 (cAMP-response element),
P3I, P4, and CRE-2 sites (CRE-2 is a CRE-like sequence). Of these, P3I
binds exclusively isoforms of the C/EBP family (38). CRE-1 and P4 sites
bind AP1 as well, and CRE-1 also binds cAMP-response
element-binding protein, ATF-2 (39), and ATF-3 (40). CRE-2 binds
C/EBP
with very low affinity but binds a nonidentified
temperature-labile protein in the liver nuclear proteins (22).
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Fig. 2.
DNase I footprint analysis of the PEPCK-C
gene promoter. a, scheme of the gene promoter. The entire
GRU in the proximal region of the gene promoter includes two GR-binding
sites (GR) and two accessory sites, out of which the more 5'
is AF1 and the more 3' is AF2 (IRS, insulin response signal)
(14). A distal element (PPARE) is homologous to the AF1
element except that it binds only PPAR and RXR (33). Note that four
structures (CRE-1, CRE-2, P3, and P4) contain
C/EBP recognition sequences (22, 41). b, binding (+) and
absence of binding ( ) of nuclear proteins from the indicated tissues.
DNase I footprinting data of adult liver (61), fetal liver (22), kidney
(61), and adipocytes (37) are indicated. c, a 390-bp DNA
fragment, spanning positions
8 to
390 of the rat PEPCK-C gene
promoter was 32P-end-labeled at the 3' site (position
8)
of the fragment. 50,000 cpm per reaction of the labeled fragment were
incubated without proteins (0') or with 15 µg of nuclear
proteins from 3T3-F442A adipocytes not treated (A
Dex) or
treated overnight with Dexamethasone (A + Dex). The
lane on the left (seq) designates sequencing of A + G, which enabled the identification of the regions protected by
protein binding to CRE-2 and P1/CRE-1 sites. The binding regions are
marked by the positions 5' to the transcription start site. Bands used
for quantification of the signals (see Fig. 3c), a band at
position
88 (within the CRE-1 protected area) and at position
160
(outside the protected area) (b), are marked by
arrows on the right. d, the
same probe as described in c was used, except that a longer
separation enabled the identification of regions protected by protein
binding to the C/EBP recognition sites P3 and P4 indicated in the
figure by their positions 5' to the transcription start site.
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Fig. 3.
DNase I footprint analysis of the nuclear
receptor-binding sites in the PEPCK-C gene promoter. a, a
128-bp DNA fragment, spanning positions 490 to
362 of the rat
PEPCK-C gene, was 32P-end-labeled at the 5' site (position
490) of the fragment. 50,000 cpm of the labeled probe was added per
reaction. Incubation without (0'), with 15 µg of nuclear
proteins from 3T3-F442A adipocytes not treated (A
Dex),
or treated overnight (A + Dex) with dexamethasone or with 10 µg of rat liver nuclear proteins (L) are indicated. The
lane on the right (seq) designates sequencing of
A + G, which enabled the identification of the region protected by
proteins binding to the AF1 site. The protected region is marked by the
positions 5' to the transcription start site. b, a 160-bp
DNA fragment, spanning positions
950 to
1110 of the rat PEPCK-C
promoter, was 32P-end-labeled at the 3' site (position
950) of the fragment. 50,000 cpm per reaction of the labeled fragment
were incubated without proteins (0') or with 15 µg of
nuclear proteins from 3T3-F442A adipocytes not treated (A
Dex) or treated overnight (A + Dex) with dexamethasone
or with 10 µg of rat liver nuclear proteins (L). The
lane on the right (seq) designates sequencing of
A + G, which enabled the identification of the region protected by
proteins binding to PPARE site, indicated by the positions 5' to the
transcription start site. c, the ratio between the density
signal of a distinct band within the protected CRE-1 site (position
88) and a band outside (position
160) (marked by arrows
in Fig. 2b) are shown in the histogram. The ratio
in the absence of proteins was set at 10. d, the ratio
between the density signals of two distinct bands within the AF1
site-protected region (positions
445 and
446) and a band outside
(position
457) (marked by arrows in a) is shown
by the histogram. The ratio in the absence of proteins was
set at 10.
, which gradually
decreases from the highest affinity (CRE-1 site) to the lowest (CRE-2
site) (22, 41). In addition, binding to the P1 site (nuclear factor 1 recognition site) in the PEPCK-C gene promoter was also inhibited (Fig.
2c), although this is not a C/EBP recognition site. In
previous studies (38) the binding of C/EBP
to CRE-1 has been shown
to cooperate with the binding to the P1 site. Therefore, a reduced
binding to CRE-1 site might have led to a secondary effect on the
binding to the P1 site.
2/RXR) (Fig. 3b). In
contrast to the adipocyte nuclear proteins, binding of hepatic nuclear
proteins to the PPARE site could barely be detected (Fig. 3b), unlike their efficient binding to the AF1 site (Fig.
3a). Furthermore, the hypersensitive site (position
985)
at the 3' end of the protected region appeared only in the presence of
adipocyte nuclear proteins. It was undetectable both in the absence of
nuclear proteins and in the presence of liver nuclear proteins (Fig.
3b). Therefore, PPAR isoforms seem less enriched in the
liver (from nonfasted rat) than they are in adipocyte nuclear proteins.
To gain quantitative estimation of the glucocorticoid effect, we measured the density signals of specific bands inside and outside the
protected regions of the CRE-1 (Fig. 2a) and AF1 (Fig.
3a) sites. The footprinting intensity of the CRE-1 site was
quantified from the autoradiographic films by measuring the density
signal of a band within the CRE-1 site (position
88 from the
transcription start site of the PEPCK-C gene) and a band outside, at
position
160 (both marked by arrows). Likewise, the
footprinting intensity of the AF1 site was quantified by measuring the
density signals of two bands inside (positions
445 and
446) and a
band outside (position
457) the AF1 site. The ratios between the
density signals inside and outside the protected region were computed.
The ratios obtained from the footprinting done without nuclear proteins
were arbitrarily set at 10 and used to normalize other ratios of the footprinting done in the presence of nuclear proteins. These
measurements have clearly assessed that dexamethasone treatment
interfered with the adipocyte nuclear protein footprinting of the CRE-1
site (Fig. 3c) but not with the footprinting of the AF1 site
(Fig. 3d).
is also repressed by
glucocorticoids but only for a few hours (32). Yet this repression might lead to a longer lasting reduction of C/EBP
protein
concentration in adipocyte nuclei (32), resulting in an apparent,
rather than real, interference of binding to its recognition sites.
Therefore, we determined whether the concentration of C/EBP
in
nuclei corresponded to the observed diminished footprinting of the
PEPCK-C gene promoter.
Level in Extracted Adipocyte Nuclear
Proteins--
The concentration of C/EBP
in the adipocyte extracts
of nuclear proteins used for footprinting was determined by Western blot assay. The hormonal treatment diminished the nuclear concentration of C/EBP
by 30% when normalized to the level of the Y12 nuclear protein (Fig. 4a). Whether
this reduced concentration of C/EBP
accounted for the inhibited
footprinting of nuclear proteins from glucocorticoid-treated adipocytes
was assessed. Thus, we used lower amounts of nuclear proteins extracted
from untreated adipocytes (10 (2/3) and 7.5 µg (1/2)), compared with
the whole amount (15 µg), to footprint the gene promoter region
containing the CRE-1, P1, and CRE-2 sites. This region enabled us to
assay the site with the highest affinity for C/EBP binding (CRE-1) and
the site with the lowest affinity for C/EBP binding (CRE-2). The
analysis showed that 10 µg of nuclear proteins footprinted all three
sites (Fig. 4b). Footprinting of the CRE-1 site was
quantified as detailed above for Fig. 2c. Thus, the ratio in
the absence of proteins was set at 10, relative to a ratio of 2.5 obtained with 10 µg of protein and a ratio of 8.5 obtained with 7.5 µg of protein (Fig. 4c). The dexamethasone effect on CRE-1
footprinting (Fig. 2c) that was likewise quantified yielded
a ratio of 1.7 in the presence of 15 µg of nuclear proteins from
untreated adipocytes, and 15.8 in the presence of 15 µg of nuclear
proteins from dexamethasone-treated adipocytes (Fig. 3c). In
contrast, footprinting the AF1 site yielded similar ratios (2.7 and
3.3, respectively) using 15 µg of nuclear proteins from untreated or
dexamethasone-treated adipocytes (Fig. 3d). We therefore
conclude that in addition to lowering the level of C/EBP
in the
nucleus, the hormonal treatment interfered with the binding to their
recognition sites in the PEPCK-C gene promoter.
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Fig. 4.
The glucocorticoid inhibition of the binding
to C/EBP recognition sites of the PEPCK-C gene promoter cannot be
accounted for by a simple reduction of C/EBP
nuclear level. a, Western blot analysis of
C/EBP
concentration in adipocyte nuclear proteins: 5 µg of nuclear
proteins extracted from adipocytes untreated (control) or
treated (+Dex) with 10
7 M
dexamethasone. The equally blotted amounts of proteins were verified
after stripping the membrane and re-probing with anti-Y12 antibody. The
percent reduction of C/EBP
nuclear level by dexamethasone treatment
is indicated above. b, footprinting analysis of
the same probe as described in Fig. 2c. The probe was
incubated without proteins (0') or with the indicated
amounts (15, 10, or 7.5 µg) of nuclear proteins extracted from
adipocytes not treated with dexamethasone. c, the ratio
between the density signal of a band at position
88 (within the CRE-1
protected region) and a band at position
160 outside the protected
area (marked by arrows on the right of the
footprinting in b) was plotted against the amounts of
nuclear protein used. The ratio in the absence of proteins was set at
10.
2 or C/EBP
(42), co-transfecting expression vectors coding for PPAR
2 and RXR
(PPAR
2/RXR) were reported to stimulate transcription from the PEPCK-C gene promoter in
these cells (33). We have compared the stimulation of the PEPCK-C gene
promoter activity by PPAR
2/RXR and by C/EBP
expression vectors
using PEPCK-CAT chimeric genes driven either by 600 (PCK(600)-CAT) or
2000 bp (PCK(2000)-CAT) of the PEPCK-C gene promoter (27). PPAR
2/RXR
preferentially stimulated the transcription from PCK(2000)-CAT PEPCK-C
gene promoter, whereas C/EBP
preferentially and markedly stimulated
transcription from the PCK(600)-CAT gene promoter (Fig. 5a). The latter is most likely
because of the localization of a cluster of C/EBP recognition sites in
the proximal region of the PEPCK-C gene promoter (9). For orientation
see the scheme of the PEPCK-C gene promoter (Fig. 2a). In
addition to C/EBP
, other members of the C/EBP family also
trans-activated the PEPCK-C gene promoter but to a lesser extent.
C/EBP
was the most effective; C/EBP
was half as effective, and
C/EBP
was the least effective. There was a 5-fold difference in the
magnitude of stimulation between C/EBP
and C/EBP
(Fig.
5b). We then determined the effect of GR and its hormone
(dexamethasone) on the level of trans-activation of the PCK(600)-CAT
gene promoter by co-transfected C/EBP
, C/EBP
, or PPAR
2/RXR.
Co-transfection of GR expression vector and the addition of
dexamethasone for the last 24 h after transfection inhibited (by
about 60%) the activation of transcription from the PEPCK-C gene
promoter either by C/EBP
or by C/EBP
. In contrast, there was no
effect of GR and dexamethasone on the trans-activation by PPAR
2/RXR
of either PCK(600)-CAT (Fig. 5c) or PCK(2000)-CAT (Fig.
5d) gene promoters.
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Fig. 5.
Effect of PPAR 2 and
C/EBP
on transcription from the PEPCK-C gene
promoter in NIH3T3 cells. a, trans-activation by
PPAR
2/RXR (dark bars) or C/EBP
(hatched
bars) over basal activity (open bars) of PCK(600)-CAT
(600) and PCK(2000)-CAT (2000) rat PEPCK-C gene promoters is shown. The
fold stimulation by the transcription factors over basal transcription
activity of the PCK(600)-CAT gene promoter, taken as one, represents
the mean ± S.E. for at least five independent experiments.
b, comparison of the fold stimulation by isoforms of C/EBP.
Expression vectors encoding C/EBP
(1 µg), C/EBP
, or C/EBP
(0.5 µg each) were co-transfected with the PCK(600)-CAT rat PEPCK-C
gene promoter as described under "Experimental Procedures." The
fold stimulation by members of the C/EBP family, over basal
transcription activity of the PCK(600)-CAT promoter, taken as one,
represents the mean ± S.E. for at least six independent
experiments. c, effect of GR and dexamethasone on the
activation of transcription from the PEPCK-C gene promoter by C/EBP
(
), C/EBP
(
), and PPAR
2/RXR (PPAR
2). Details
as described in a, without GR (
GR, open
bars), except that 1 µg of the human GR expression vector was
included in the transfection mix and dexamethasone (10
7
M, final concentration) was added 19 h later for
24 h (+GR, dark bars). The fold stimulation
over basal transcription activity of the PCK(600)-CAT promoter, taken
as one, represents the mean ± S.E. for six independent
experiments. d, effect of GR and dexamethasone on the
activation of transcription from the PCK(2000)-CAT gene promoter by
PPAR
2/RXR (PPAR
2). Details as described in
c are as follows: without GR (
GR, open
bars) and with GR (+GR, dark bars). The fold
stimulation over basal transcription activity, taken as one, represents
the mean ± S.E. for four independent experiments.
2/RXR trans-activation of the longer region of
the PEPCK-C gene promoter, either PCK(2000)-CAT (rat) or PCK(1500)-CAT
(mouse) gene promoters compared with PCK(600)-CAT (rat) and
PCK(840)-CAT (mouse) (Figs. 5a and 6a), is due to
the two PPAR
2/RXR recognition sites (a proximal AF1 side and a
distal PPARE site). Moreover, mutation of the PPARE sequence in
PCK(1500)-CAT gene promoter (mouse PCK(1500-mut)-CAT) abolished its
response to PPAR
2/RXR, although it contained the proximal
AF1-binding site (Fig. 6a).
Note that the sequence of PEPCK-C gene promoter is highly preserved
between the rat (43) and the mouse (44). Furthermore, mutation of the
PPARE sequence specifically ablated PEPCK-C gene expression in the
adipose tissue in vivo as shown with the mutated rat PEPCK-C
transgene in transgenic mice (45) and the targeted mutation in the
mouse endogenous PEPCK-C gene (12). In contrast, mutation of the
proximal PPAR
2/RXR-binding site (AF1 site) in the context of
PCK(2000)-CAT gene promoter markedly increased the PPAR
2/RXR
trans-activation of the gene promoter (Fig. 6b). The
constraining features of the wild type AF1 site of the PEPCK-C gene
promoter in NIH3T3 cells have been studied recently in detail by Eubank
et al. (46). These authors showed that the AF1 site tightly
binds the nuclear receptor COUPTF II whose expression vector inhibited
the PPAR
2/RXR-mediated trans-activation of the PEPCK-C gene promoter
via the AF1-binding site (46). These features of AF1 site are not
shared by other GRU elements, because mutation of the AF2 site had no
effect on the trans-activation of PCK(2000)-CAT by PPAR
2/RXR (Fig.
6b).
View larger version (17K):
[in a new window]
Fig. 6.
Effects of site-specific mutations on the
trans-activation of PCK(2000)-CAT rat PEPCK-C gene promoter.
a, fold trans-activation by PPAR 2/RXR (dark
bars) over basal activity (open bars) of the mouse
PCK(840)-CAT (840) and PCK(1500)-CAT (1500)
PEPCK-C gene promoters and of the PCK(1500)-CAT PEPCK-C gene promoter
containing a mutation of the PPARE site (1500-mut) is shown.
The fold stimulation by PPAR
2/RXR over basal transcription activity
of the PCK(840)-CAT gene promoter, taken as one, represents the
mean ± S.E. for at least nine independent experiments.
b, effect of mutations of the AF1 (AF1-mut) and
AF2 (AF2-mut) sites in the context of the PCK(2000)-CAT rat
PEPCK-C gene promoter on its trans-activation by PPAR
2/RXR
(dark bars) over basal activity (open bars). The
fold stimulation by PPAR
2/RXR over basal transcription activity of
the PCK(2000)-CAT gene promoter (2000), taken as one,
represents the mean ± S.E. for at least six independent
experiments. c, effect of mutations of GRE1 and GRE2
(mGRE1-2), AF1 (AF1-mut), and AF2
(AF2-mut) sites in the context of the PCK(2000)-CAT rat
PEPCK-C gene promoter on its trans-activation by C/EBP
(hatched bars) over basal activity (open bars).
The fold stimulation by C/EBP
over basal transcription activity from
the PCK(2000)-CAT chimeric gene (2000), taken as one,
represents the mean ± S.E. for at least four independent
experiments.
-mediated trans-activation of the PCK(2000)-CAT chimeric gene
is also constrained (Fig. 6c). However, unlike the
trans-activation by PPAR
2/RXR, in this case mutations of any single
element of the GRU (14), AF1, AF2, and GRE1-2 of PCK(2000)-CAT
chimeric gene, enhanced the C/EBP
-mediated trans-activation of the
gene promoter by 3-4-fold (Fig. 6c). Therefore, unlike
PPAR
2/RXR, not only the AF1 site but the entire GRU domain
constrains the C/EBP
trans-activation of the PCK(2000)-CAT chimeric
gene. Because each of the elements comprising the GRU constrained the
C/EBP
trans-activation of the PCK(2000)-CAT chimeric gene, we asked whether-binding of GR to the PEPCK-C gene promoter is required for its
mediated repression. To this end, the capacity of the wild type rat GR
was compared with its counterpart rat mutant GR that is incapable of
binding to the DNA (13, 47) to inhibit the C/EBP
-mediated
trans-activation of PCK(2000)-CAT. The data showed that either wild
type or mutant rat GR equally repressed the stimulation of
transcription from the PEPCK-C gene promoter by C/EBP
(Fig.
7a).
View larger version (11K):
[in a new window]
Fig. 7.
Effect of rat wild type and mutant GR on the
trans-activation of the PCK(2000)-CAT rat PEPCK-C gene promoter by
C/EBP . a, effect of wild type rat GR
(wt GR) and rat mutant GR (mut GR) in the
presence of dexamethasone (as described in the legend of Fig.
5c) on the trans-activation of the PCK(2000)-CAT rat PEPCK-C
gene promoter by C/EBP
. The fold stimulation by C/EBP
over basal
transcription activity of the PCK(2000)-CAT promoter, taken as one,
represents the mean ± S.E. for at least eight independent
experiments. b, effect of wild type GR (wt GR)
and mutant GR (mut GR) on the trans-activation of the
PCK(2000)-CAT rat PEPCK-C gene promoter and its derived series of the
GRU mutants described in Fig. 6c. The values are expressed
as percent repression by GR from the C/EBP
-stimulated activity of
each gene promoter. These represent the mean ± S.E. of at least
four independent experiments.
-mediated
transcriptional stimulation of the GRU-mutated series of PCK(2000)-CAT could be repressed by GR and whether the mutant GR was also effective. The wild type GR repressed the C/EBP
-mediated stimulation of the GRU
series of PCK(2000)-CAT mutants to a similar extent as that of the wild
type (about 50%) (Fig. 7b). The mutant GR repressed the
stimulation of the wild type and GRE1-2 mutant of PCK(2000)-CAT to a
similar extent, less so the AF2 mutant, but completely failed to
repress the stimulation of PCK(2000)-CAT-AF1 mutant (Fig.
7b). Therefore, the AF1 site is required for the repression
by mutant GR but not by the wild type GR (Fig. 7b). In fact,
the AF1 site emerges as an inherent constraining element on
transcription from the PEPCK-C gene promoter because it markedly
restrains the trans-activation of PCK(2000)-CAT gene promoter activity
either by PPAR
2 or by C/EBP
(Fig. 6, b and
c). Moreover, intact AF1 is also required for the mutant
GR-mediated repression of C/EBP
trans-activation of the PEPCK-C gene
promoter. When AF1 is mutated, the repression by mutant GR is abolished
(Fig. 7b).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, in addition to a markedly
diminished binding of nuclear proteins to the C/EBP recognition sites
(but not to the PPAR/RXR recognition sites) in the PEPCK-C gene
promoter. Previous studies from our laboratory (26) documented a
requirement for the C/EBP recognition sites of the PEPCK-C gene
promoter that were crucial for its basal activity in adipocytes.
Transient transfection in NIH3T3 cells revealed that GR together with
glucocorticoids partially inhibited the C/EBP-mediated, but not
PPAR
2-mediated, trans-activation of PEPCK-C gene promoter. We have
thus described the involvement of members of the C/EBP family in the
adipocyte-specific glucocorticoid repression of PEPCK-C gene
expression. This occurs in at least two ways: (a) by
reducing the concentration of C/EBP
in adipocyte nuclei, and
(b) interfering with the binding of nuclear proteins to the C/EBP recognition sites in the DNA and as a consequence of, or in
addition to, inhibiting the trans-activation of PEPCK-C gene promoter
by C/EBP isoforms. Clearly, using nuclear proteins from the
PEPCK-expressing adipocytes and transient transfection in the
PEPCK-nonexpressing NIH3T3 cells comprised two independent experimental
approaches and systems, both of which consistently revealed the
involvement of C/EBP but not PPAR activators in the glucocorticoid
repression of PEPCK-C gene transcription. However, because GR succeeded
to inhibit the C/EBP trans-activation only by 60%, it is likely that
additional factors beside members of C/EBP family participate in
PEPCK-C gene transcription in the adipocytes.
is
markedly elevated. This is different from trans-activation by
PPAR
2/RXR, which is also constrained, but in this case, it is not
exerted via each element of the GRU but exclusively by the AF1 site. In
that sense, the AF1 site has emerged as a unique element whose mutation
elevates by 4-fold the response of PCK(2000)-CAT to either PPAR
2/RXR
or C/EBP
. These constraining features of AF1 are accentuated by the
very modest response of this site to PPAR
2/RXR activation of
transcription from the PEPCK-C gene promoter (Fig. 6a). The
main PPAR
2/RXR-responsive element is PPARE (Fig. 6a) that
comprises an adipose tissue-specific enhancer, as has been shown
previously in transgenic mice (45). Furthermore, a targeted mutation of
PPARE in embryonic stem cells resulted in ablation of PEPCK-C
gene expression in the white adipose tissue of offspring mice
homozygous for the mutation (12).
-mediated
trans-activation of PCK(2000)-CAT? The present data seem to support
such involvement. Initially, our results have established that the GR
repression does not require binding to the DNA. A GR mutated in the
zinc finger (13), making it incapable of binding the DNA, was as active
as wild type in repressing the C/EBP
stimulation. Subsequently, we
have assessed that the wild type GR equally repressed the wild type
PCK(2000)-CAT and derived mutated GRU series, whereas the mutant GR
repressed the mutated GRE1-2 sites and, to a lesser extent, the
mutated AF2 site of PCK(2000)-CAT. However, mutation of the AF1 site
abolished the capability of the mutant GR to repress the C/EBP-mediated
trans-activation. Because the mutated AF1 site did not hinder the
repression by wild type GR, we suggest that the AF1 site undertakes a
docking role to facilitate the mutant GR-mediated repression of the
PCK(2000)-CAT gene promoter. Thus, these observations expand the
hierarchy of regulation to strongly suggest that, in addition to its
constraint features, the wild type AF1 site is crucial for the hormonal repression.
-mediated activation of transcription remained 50-60% whether the activation was low (wild type
PCK(2000)-CAT gene promoter) or elevated by 3-4-fold (GRU-mutated
series of PCK(2000)-CAT gene promoter).
to the
DNA binding domain of GR has been implicated recently in the GR
repression of the vitellogenin gene transcription in hepatocytes from
the rainbow trout. The vitellogenin gene promoter lacks a GR-binding
site (51).
2-binding site of the PEPCK-C gene
promoter, which selectively ablates gene expression in white adipose
tissue, caused a marked diminution of glyceroneogenesis in this tissue.
Mice homozygous for this mutation lost lipid from adipose tissue even
to the extent of lipodystrophy, attesting to the metabolic significance
of PEPCK-C and glyceroneogenesis in the adipose tissue (12). Moreover, a recent adipocyte-specific knockout of glucose transporter 4, required
for glucose metabolism in the adipose tissue, generated mice exhibiting
features of noninsulin-dependent diabetes mellitus without
a loss of triglycerides from adipose tissue (55). Finally, Franckhauser
et al. (56) overexpressed a chimeric gene containing the
PEPCK-C structural gene linked to the aP2 promoter in mice resulting in obesity in adult mice. Taken together, these results further emphasize the crucial role of PEPCK-C and glyceroneogenesis in
maintaining lipid homeostasis in the adipose tissue. Therefore, it is
conceivable that hormonally mediated alterations of PEPCK-C gene
expression ultimately regulate glyceroneogenesis in both liver and
adipose tissue.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Keith Yamamoto for the
wild type and mutant rat GR, to Dr. Bruce Spiegelman for supplying the
PPAR expression vector, to Dr. Daniel Lane for the C/EBP
expression vector, and to Dr. Ron Evans for the human GR and mouse
RXR
expression vectors. We especially appreciate the gift from Dr.
Steven L. McKnight of the anti-C/EBP
antiserum, the valuable advice
during the transfection experiments in confluent NIH3T3 given by Dr. Peter Tontonoz, and the many fruitful discussions with Dr. Oded Meyuhas.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants 1999346 and 9600117 from the United States-Israel Binational Science Foundation, Grant 540197-19 from the Israel Science Foundation, a grant from the Israel Ministry of Health, and by Grant DK22541 from the National Institutes of Health.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Developmental Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120. Fax: 972-2-675-7379; E-mail:
reshef@cc.huji.ac.il.
Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M300263200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GR, glucocorticoid receptor; PEPCK-C, phosphoenolpyruvate carboxykinase-C; C/EBP, CCAAT/enhancer-binding protein; GRE, glucocorticoid-response element; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; CRE, cyclic AMP-response element; PPARE, PPAR-response element; RT, reverse transcriptase; CAT, chloramphenicol acetyltransferase; GRU, glucocorticoid response unit; DMEM, Dulbecco's modified Eagle's medium; HNF, hepatocyte nuclear factor; COUPTF, chicken ovalbumin upstream transcription factor.
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