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J Biol Chem, Vol. 275, Issue 8, 5804-5809, February 25, 2000
From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The CCAAT/enhancer-binding protein The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC
4.1.1.32) (PEPCK-C)1 is
generally considered to be the rate-limiting enzyme of gluconeogenesis (1), and accordingly it is expressed at highest levels in liver and
kidney (1-3). The promoter of PEPCK-C is hormonally responsive and
contains a number of cis-elements that bind
trans-acting factors capable of mediating multiple hormonal
signals (for review, see Ref. 4). In liver, the PEPCK-C promoter is
negatively regulated by insulin but induced by glucocorticoids, thyroid
hormones, and glucagon, via cAMP (2, 5, 6).
The responsiveness of the PEPCK-C promoter to cAMP is relatively
liver-specific and is mediated by a group of cis-elements collectively known as a cAMP response unit. The cAMP response unit
includes a cAMP response element (CRE), which is capable of binding
molecules of the cAMP response element-binding protein (CREB); three
binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms; and
one binding site for AP-1 (7, 8). This complex organization of DNA
sequences and DNA-binding proteins likely allows for intricate control
of tissue-specific expression and the integration of various signaling
pathways. For example, the response of the PEPCK-C promoter to thyroid
hormones is mediated not only by the binding of an activated thyroid
hormone receptor to a typical thyroid hormone response element (9, 10),
but also requires one of the C/EBP binding sites utilized in the CRU (11). Thus, there is a potential integration of the thyroid hormone
response and the cAMP response of the PEPCK-C promoter, brought about
by this C/EBP molecule, which may account for the synergistic
activation of the promoter by these two signaling pathways.
CCAAT/enhancer-binding proteins are members of the basic region-leucine
zipper (bZIP) family of transcription factors (12, 13). The two main
C/EBP isoforms expressed in liver are C/EBP Prior work by our laboratory using GAL4-C/EBP fusion proteins suggested
that either C/EBP isoform has the potential to mediate the cAMP
response of PEPCK-C in liver (11). However, to date there has been no
evidence in regards to which specific isoform mediates the cAMP
responsiveness of the endogenous PEPCK-C gene in liver cells. The goal
of this present study was to inhibit C/EBP activity in hepatoma cells
and assess what effect it had on the cAMP responsiveness of the
endogenous PEPCK-C gene.
Materials--
DNA-modifying enzymes were purchased from New
England BioLabs, Promega Corp., and U. S. Biochemical Corp.
GentacinTM and tissue culture supplies were obtained from Life
Technologies, Inc. 8-Chlorophenylthio-cAMP (8-CPT-cAMP) was obtained
from Sigma. [ Cloning of Expression Vectors--
The expression vector for
GBF-F (MSV-GBF-F), utilized for the production of the MSV-GBF-F stable
H4IIE cell line, was constructed by ligating an
EcoRI/BamHI fragment of pRGX-GBF-F (22),
corresponding to full-length GBF-F, into the
EcoRI/BamHI sites of MSV-C/EBP
The plasmid designed to express C/EBP
The plasmid designed to express C/EBP
The plasmid designed to express C/EBP Construction of Ribonuclease Protection Assay Probes--
The
plasmids designed for the production of ribonuclease protection assay
probes for detection of C/EBP Cell Culture--
Cells were grown in Dulbecco's modified
Eagle's medium/F-12 medium containing 5% fetal bovine serum and 5%
calf serum. Where indicated, cells were treated with 200 µM 8-CPT-cAMP or 1 µM dexamethasone for the
times indicated and then harvested for RNA isolation.
Preparation of Stable Cell Lines--
Rat hepatoma H4IIE cells
were transfected by the calcium phosphate precipitate method (28).
Cells were transfected with the appropriate expression vector and
pSV2neo (InforMax, Inc.) at a ratio of 10:1, respectively. The total
amount of DNA introduced per transfection was 15 µg/100-mm plate.
After 3 days, cells were placed in medium containing 400 µg/ml
gentamicin. Individual clones, once isolated and expanded, were
maintained in medium containing 100 µg/ml gentamicin.
RNA Analysis--
Total cellular RNA was extracted from cultured
cells using TRIzolTM reagent (Life Technologies, Inc.). RNase
protection assays and Northern analysis were conducted as described
previously (27, 29). Quantification of signals was performed by
densitometric scanning of autoradiograms using the Un-Scan-ItTM
automated digitizing system (Silk Scientific Inc.). Verification that
an equivalent amount of RNA was analyzed from each sample was
accomplished by RNA slot blot analysis using a radiolabeled 18 S rRNA
probe (30).
Western Blot Analysis--
The methods for preparation of
cellular lysates and Western blot analysis have been described
previously (31). Blots were quantified by densitometric scanning as
described above.
To address which C/EBP isoform participates in mediating the cAMP
responsiveness of the PEPCK promoter, H4IIE cell lines stably expressing either a dominant negative molecule, or antisense to C/EBP To confirm the general requirement of C/EBP isoforms for PEPCK-C basal
and cAMP inducible expression, we prepared a H4IIE cell line that
expressed a dominant negative inhibitor of C/EBP, GBF-F (22). GBF-F is
an engineered molecule consisting of the DNA-binding domain of the
plant bZIP protein GBF-1 fused to the "F" leucine zipper, a
dimerization domain designed to preferentially form inactive
heterodimers with C/EBP isoforms. High levels of GBF-F expression
within the intact cell should indiscriminately inhibit the
transcriptional activity of all C/EBP isoforms by formation of inactive
heterodimers. The level of GBF-F mRNA in the GBF-F D4 clone, and
its absence in wild-type H4IIE cells, is shown in Fig.
1A. Fig. 1B shows a
representative autoradiogram that demonstrates the effects of GBF-F
expression on C/EBP
(C/EBP) is
a transcription factor that trans-activates a number of
metabolically important genes. Previous work has demonstrated that
C/EBP and C/EBP
have the potential to mediate the cAMP
responsiveness of phosphoenolpyruvate carboxykinase (PEPCK) in liver
cells. However, these studies used GAL4 fusion proteins and artificial
promoter-reporter gene vectors in transfection experiments; as a
result, these studies only indicated that both isoforms had the
potential to mediate the hormonal response and not which isoform
actually participated in vivo. To address this issue, we
produced hepatoma cell lines that stably expressed either a dominant
negative inhibitor or antisense RNA for these two main liver C/EBP
isoforms. Inhibition of all C/EBP isoforms via expression of the
dominant negative protein eliminated cAMP responsiveness, and reduced
glucocorticoid responsiveness, of the endogenous PEPCK gene in hepatoma
cells. Antisense directed against C/EBP mRNA, which reduced
C/EBP protein levels by nearly 80%, also significantly reduced the
cAMP responsiveness of the endogenous PEPCK promoter, whereas antisense
directed against C/EBP was without effect. There was no major
alteration in cAMP signaling in the C/EBP antisense cells, as cAMP
induction of the C/EBP gene was similar to that in wild-type H4IIE
cells. These data suggest that the -isoform of C/EBP is specifically utilized for mediating the cAMP responsiveness of the PEPCK gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and C/EBP (14). The
-isoform of C/EBP is considered to be a central regulator of energy
homeostasis (15), being involved in the trans-activation of
a number of metabolically important genes (16, 17). The importance of
C/EBP in the maintenance of metabolic homeostasis has been
particularly well demonstrated by whole animal and tissue-specific
knockout studies (18, 19). The -isoform of C/EBP also appears to
have some role in metabolism (20), as well as regulating the immune
response (21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]Uridine triphosphate (3000 Ci/mmol)
was purchased from Mandel. Antisera specific for C/EBP, C/EBP,
and TFIIE were obtained from Upstate Biotechnology, Inc. TRIzolTM
reagent was obtained from Life Technologies, Inc.
(23), thereby
displacing the full-length C/EBP insert. The GBF-F
EcoRI/BamHI fragment was obtained from pRGX-GBF-F
by polymerase chain reaction amplification utilizing a forward
polymerase chain reaction primer designed to introduce an
EcoRI site into the 5'-untranslated region of the GBF-F
cDNA sequence. The forward and reverse polymerase chain reaction
primers utilized to amplify the approximately 400-base pair GBF-F
EcoRI/BamHI fragment from pRGX-GBF-F were as
follows: GBF-F forward, 5'-TATCGAATTCATGCCAGTGAAGGAT-3' and GBF-F
reverse, 5'-TATCGGATCCAAGCTTGCCGTC-3'.
antisense RNA (MSV-C/EBP
anti) was constructed by subcloning the
PstI/BamHI fragment of pMSV-C/EBP (24),
corresponding to nucleotides +782 to +131 of the rat C/EBP cDNA
sequence, into the PstI/BamHI sites of the
multiple cloning site of pBluescript® SK+ (Stratagene) to produce
SK+-C/EBP anti. The EcoRI/BamHI fragment of
SK+-C/EBP anti was then ligated into the
EcoRI/BamHI sites of MSV-C/EBP to produce
MSV-C/EBP anti.
antisense RNA (MSV-C/EBP
anti) was constructed by subcloning the PstI fragment of pSCt-LAP (25), corresponding to nucleotides +736 to +246 of the rat
C/EBP cDNA sequence, into the PstI site of
pBluescript® SK+ to produce SK+-C/EBP anti. The
EcoRI/BamHI fragment of pBluescript® SK+-C/EBP anti was then ligated into the
EcoRI/BamHI sites of MSV-C/EBP to produce
MSV-C/EBP anti.
sense RNA (MSV-GN175) was
constructed by ligating the EcoRI/BamHI fragment
of pGN175 (26), corresponding to nucleotides +117 to +658 of the rat
C/EBP cDNA sequence (coding for amino acids 6-175 of C/EBP)
into the EcoRI/BamHI sites of MSV-C/EBP to
produce MSV-C/EBP sense.
, C/EBP, and PEPCK mRNA have
been described previously (27). The plasmid designed for the production
of ribonuclease protection assay probes for detection of C/EBP
antisense RNA was produced by ligating the EcoRI/BamHI fragment of pGN175, corresponding
to nucleotides +117 to +658 of the rat C/EBP cDNA sequence, into
the EcoRI/BamHI sites of pTZ18R (U. S.
Biochemical Corp., Inc.). This vector was linearized for probe
production by digestion with HindIII, which cuts at position
+310 of the pTZ18R multiple cloning site, producing a 573-nucleotide
probe that protects 541 nucleotides of the C/EBP antisense RNA. The
vector utilized as a template for ribonuclease protection analysis
probes for the detection of C/EBP antisense RNA (19R-LAP) was
constructed by ligating the PstI/PstI fragment of
pSCt-LAP, corresponding to nucleotides +246 to +736 of the rat C/EBP
cDNA sequence, into the PstI site of pTZ19R. This vector was linearized for probe production by digestion with
EcoRI, which cuts at nucleotide +315 of the
multiple cloning site of pTZ19R, producing a 550-nucleotide probe,
which protects 490 nucleotides of the C/EBP antisense RNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or C/EBP RNA, were prepared. A number of clones of each cell line were examined for the levels of expression (data not shown).
The highest expressing clone was selected for further characterization.
The percent or fold changes in expression levels (either in RNA or
protein) listed under "Results" are the averages ± S.E. of
data collected from at least three independent experiments.
and C/EBP mRNA levels in the GBF-F D4
cell line relative to wild-type H4IIE cells. The presence of GBF-F
resulted in a 26 ± 8% reduction in C/EBP mRNA levels and
a 60 ± 4% reduction in C/EBP mRNA levels. Despite the
repressive effect of GBF-F expression on C/EBP mRNA levels, no
alterations in C/EBP protein between GBF-F D4 cells and wild-type
H4IIE cells was detectable (Fig. 1C). However, the levels of
C/EBP protein were reduced by 59 ± 3% in GBF-F D4 cells in
comparison with wild-type H4IIE cells.
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Fig. 1.
Effect of the dominant negative protein GBF-F
on C/EBP isoform expression. A is a representative
Northern blot showing GBF-F expression levels in the GBF-F D4 clone in
comparison with wild-type H4IIE cells. B shows a
representative ribonuclease protection analysis of C/EBP and
C/EBP mRNA levels in the GBF-F D4 clone in comparison with H4IIE
wild-type cells. RNA isolation and analysis, and the equilibration of
RNA amounts between samples by RNA slot-blot analysis using an 18 S
rRNA probe (see A), are described under "Experimental
Procedures." C shows a representative Western blot of
C/EBP and C/EBP protein in the GBF-F D4 clone in comparison with
H4IIE wild-type cells. The equilibration of protein amount between
samples was confirmed by assessing the protein levels of the general
transcription factor TFIIE (data not shown). The preparation of
protein lysates and Western analysis are described under
"Experimental Procedures."
It should be noted that RNase protection analysis of C/EBP mRNA, shown throughout the figures of this paper, gave either one or two bands. This is a result of the inefficiency of the in vitro transcription reactions utilized to produce the antisense probes, in which two probe species of different sizes were generated. Autoradiograms showing what appears to be only one protected species simply have not been resolved to as great an extent to those displaying two protected species.
Because GBF-F inhibits the activity of all C/EBP isoforms, we next
prepared cell lines expressing antisense RNA for either the - or
-isoform. Fig. 2A shows the
levels of expression of the C/EBP antisense RNA in the A B1 H4IIE
cell line in comparison with wild-type H4IIE cells. The expression of
C/EBP antisense RNA reduced the mRNA level of C/EBP by
78 ± 5% in comparison with wild type H4IIE cells (Fig.
2B). More importantly, the levels of C/EBP protein were
also decreased in the A B1 cell line by 76 ± 9% in comparison
to wild-type cells (Fig. 2C). An interesting dichotomy was
observed regarding the effects of C/EBP antisense RNA expression on
C/EBP and C/EBP expression. The expression of C/EBP antisense
RNA caused an up-regulation of C/EBP expression, in that it was seen
to double both C/EBP mRNA (2 ± 0.3-fold) and protein
levels (1.9 ± 0.2-fold) (Fig. 2, B and C)
relative to wild-type H4IIE cells. Thus it would appear that while
antisense expression results in the expected down-regulation of
C/EBP, there is a corresponding increase in the expression of
the -isoform of C/EBP.
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In the A C4 H4IIE cell line, which expressed C/EBP antisense RNA
(Fig. 3A), the level of
C/EBP mRNA was reduced by 44 ± 2% (Fig. 3B)
and its protein levels by 71 ± 2% (Fig. 3C) relative to wild-type H4IIE cells. The levels of C/EBP protein in the A C4
cell line were increased by 6.4 ± 0.6-fold relative to wild-type H4IIE cells (Fig. 3C). Interestingly, despite the increased
levels of C/EBP protein, no corresponding increase in C/EBP
mRNA was detected in these cells (Fig. 3B).
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As a control for these experiments, a H4IIE stable cell line (GN175
A3) expressing a sense C/EBP RNA, corresponding to the coding region
for amino acids 6-175, was produced. No change in C/EBP or C/EBP
protein levels relative to wild-type H4IIE cells was detected as a
consequence of this sense C/EBP RNA expression (data not shown).
We next assessed the degree of cAMP responsiveness in the cell lines
described above. Treatment of wild-type H4IIE cells with 200 µM 8-CPT-cAMP produced a 3.5 ± 0.3-fold increase in
PEPCK-C mRNA. The responsiveness of PEPCK-C to an identical
treatment with 8-CPT-cAMP was abolished in the GBF-F expressing cell
line GBF-F D4 (1.5 ± 0.3-fold increase) (Fig.
4), confirming the involvement and
requirement of C/EBP isoforms for the induction of PEPCK-C expression
by cAMP. Furthermore, the basal expression of the PEPCK-C gene was
reduced by 35 ± 4% in the GBF-F D4 cell line relative to
wild-type H4IIE cells.
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The specific inhibition of C/EBP activity by antisense in the A
B1 cell line also abolished PEPCK-C cAMP responsiveness (1.2 ± 0.3-fold increase) (Fig. 4). The basal levels of PEPCK-C mRNA were
also reduced in the A B1 cell line by 70 ± 8% compared with
wild-type cells. No alteration in PEPCK-C responsiveness to 8-CPT-cAMP
was observed in C/EBP antisense-expressing A C4 cells (Fig. 4).
In fact, the 5 ± 0.5-fold induction of PEPCK-C gene expression
levels by cAMP in A C4 cells was consistently greater than that seen
in wild-type H4IIE cells. No notable difference in PEPCK-C basal
expression levels was observed in A C4 cells relative to wild-type cells.
No inhibition of cAMP induction of PEPCK mRNA levels was observed
in the C/EBP sense RNA expressing cell line GN175 A3 (3.7 ± 0.2-fold increase) (Fig. 4). This result indicates that the processes
of stable integration and selection themselves did not interfere with
the molecular mechanisms of cAMP induction of endogenous PEPCK-C.
It was possible that the effects of C/EBP antisense were exerted
through an indirect mechanism, perhaps by decreasing the levels and/or
activity of a protein necessary for cAMP responsiveness, such as CREB.
Thus, it was necessary to determine whether the C/EBP antisense
cells had a general defect in cAMP signaling. We assessed the effects
of GBF-F and C/EBP antisense RNA expression on the cAMP
responsiveness of an endogenous H4IIE gene other than PEPCK-C. The
expression of C/EBP is induced by cAMP (27), by a transcriptional
mechanism that is CREB-mediated (32). As shown in Fig.
5, the levels of C/EBP protein were
induced by 3.1 ± 0.2-fold in A B1 cells and by 3.4 ± 0.2-fold in GBF-F D4 cells by 8-CPT-cAMP, which is comparable with the
3.8 ± 0.3-fold induction observed in wild type H4IIE cells.
Furthermore, the levels of CREB protein were not significantly altered
relative to wild-type cells in either GBF-F D4 or A B1 cells (data
not shown). Thus, it appeared that general cAMP signaling in the
C/EBP antisense RNA and GBF-F expressing lines was not altered,
supporting the hypothesis that the impairment in cAMP responsiveness of
the PEPCK-C gene was due to a specific inhibition of C/EBP activity
in these cells.
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We also utilized these stable cell lines to assess the requirements of
C/EBP for the glucocorticoid responsiveness of the PEPCK-C gene. As
shown in Fig. 6, the 4.1 ± 0.5-fold
induction of PEPCK-C mRNA obtained by dexamethasone treatment of
wild-type H4IIE cells was not affected by antisense RNA for either
C/EBP (5.3 ± 0.5-fold) or C/EBP (4.9 ± 0.9-fold).
However, in cells expressing GBF-F, which simultaneously inhibits
the activity of all C/EBP isoforms, the level of induction of PEPCK-C
mRNA in response to dexamethasone was reduced to 2.6 ± 0.1-fold (averaged from four independent experiments).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The data presented in this paper identify C/EBP as the specific
C/EBP isoform that participates in the cAMP responsiveness of the
PEPCK-C gene. This observation is supported by the lack of cAMP
induction of PEPCK-C in both the GBF-F D4 and A B1 cell lines, and
the robust cAMP response that still occurs in the A C4 cell line.
Further support for this hypothesis is derived from the observation
that the levels of C/EBP protein are reduced in the GBF-F D4 cell
line (Fig. 1), in which PEPCK-C cAMP responsiveness is abolished.
Moreover, while the levels of C/EBP protein are increased in the
A B1 cell line (Fig. 2), the cAMP responsiveness of the PEPCK-C gene
was lost (Fig. 4). Furthermore, the levels of C/EBP protein are
considerably elevated in the A C4 cell line (Fig. 3), which
consistently display an enhanced response of the PEPCK-C gene to cAMP.
Further support for our hypothesis comes from the observation that
C/EBP knockout mice show no alteration in the basal expression or
cAMP inducibility of the PEPCK-C gene (20). Collectively, these data
indicate that C/EBP participates in this hormonal response, whereas
C/EBP does not. It should be noted that while C/EBP knockout mice
have been generated, assessing the cAMP responsiveness of the PEPCK-C
gene has proven difficult, since the mice die shortly after birth (18).
It is interesting to note, however, the newborn mice show an impairment in gluconeogenesis, for which PEPCK is the rate-limiting enzyme.
The observation that GBF-F inhibited expression of C/EBP expression
(Fig. 1) is consistent with the autoregulatory nature of the C/EBP
gene (33, 34). The murine C/EBP promoter is known to bind C/EBP
directly, and in transient transfection studies, overexpression of
C/EBP trans-activated the C/EBP promoter (33). The
work of Timchenko et al. (34) also suggested autoregulation of the human C/EBP gene.
Selective inhibition of either C/EBP or C/EBP in hepatoma cells
by antisense affected the expression of both main hepatic C/EBP
isoforms. In hepatoma cells expressing C/EBP antisense, not only was
the expression of C/EBP affected as expected, there was also a
corresponding increase in the expression of C/EBP (Fig. 2).
Conversely, cells expressing C/EBP antisense displayed the expected
reduction in C/EBP along with an up-regulation of C/EBP protein,
which, curiously, was not accompanied by elevations in C/EBP
mRNA (Fig. 3). These results suggest that there might again be some
compensatory mechanism in place which functions to maintain a certain
level of C/EBP protein in the liver cell. While there is mounting
evidence that these two isoforms have some distinct properties, and
roles in specific gene promoters, it is also clear that these two
isoforms can and do bind to the same sequences in many genes, providing
trans-activation. Thus, when the level of one isoform
declines and a compensatory increase in the concentration of the other
occurs, genes that are dependent on C/EBPs for their constitutive level
of activity could be expected to maintain their expression at or near
ambient levels.
The results presented in Fig. 6 suggest a requirement for C/EBP
isoforms in the mediation of the glucocorticoid responsiveness of the
endogenous PEPCK-C gene. The fact that inhibition of any one isoform by
antisense had no affect on glucocorticoid responsiveness, whereas GBF-F
expression reduced the fold responsiveness, is consistent with the
hypothesis that either isoform can participate in this hormonal
response. This conclusion is different from that made by Yamada
et al. (36), who specifically assigned C/EBP as the accessory factor participating in the glucocorticoid responsiveness. However, it should be noted that again this conclusion was based on
experiments using promoter-reporter gene constructs and GAL4-C/EBP proteins. Moreover, overexpression of the C/EBP fusion protein was
not able to fully restore glucocorticoid responsiveness, which had been
reduced due to the substitution of the C/EBP binding site for a GAL4
site. These differences in experimental design could explain the
different conclusions between our study and that of Yamada et
al. (36). However, they are consistent in that they both point to
a C/EBP protein as an important accessory factor in the glucocorticoid response.
The data presented in this study offer further evidence that C/EBP
proteins, particularly the -isoform, function in mediating the
effects of several hormones on gene transcription. In the case of the
PEPCK promoter, C/EBPs assist in mediating the transcriptional effects
of cAMP (26, 37, this study), glucocorticoids (36, this study), and T3
(9, 11). The production of stable rat hepatoma H4IIE cell lines,
expressing molecules designed to inhibit the activity of C/EBP
isoforms, have allowed us to assess the requirement of these
transcription factors in various cellular processes. This work has also
demonstrated the need to build upon promoter-reporter gene/overexpression studies with experiments that can characterize the
role that individual C/EBP isoforms play in the regulation of specific,
endogenous genes that lie within a chromatin environment.
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ACKNOWLEDGEMENTS |
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We thank Uehli Schibler, Steve McKnight, Richard Hanson, Yaacov Hod, and Charles Vinson for plasmids. We also thank Maria Hatzoglou for helpful advice for the H4IIE stable transfection protocol.
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FOOTNOTES |
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* This work was supported by an operating grant from the Medical Research Council of Canada (to W. J. R.) and by an Arthur Smythe Graduate Scholarship and University of Saskatchewan Graduate Teaching Fellowship (to S. C.).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: Dept. of Biochemistry,
University of Saskatchewan, 107 Wiggins Rd., Saskatoon SK S7N 5E5,
Canada. Tel.: 306-966-4375; Fax: 306-966-4390; E-mail: roesler@duke.usask.ca.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PEPCK-C, cytosolic form of PEPCK; CRE, cAMP response element; CREB, cAMP response element-binding protein; 8-CPT-cAMP, 8-chlorophenylthio-cAMP.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Rognstad, R.
(1979)
J. Biol. Chem.
254,
1875-1878 |
| 2. | Alleyne, G. A. O., and Scullard, G. H. (1969) J. Clin. Invest. 48, 364-370 |
| 3. | Hanson, R. W., and Reshef, L. (1997) Annu. Rev. Biochem. 66, 581-611[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Hanson, R. W., and Patel, Y. M. (1994) Adv. Enzymol. Relat. Areas Mol. Biol. 69, 203-281[Medline] [Order article via Infotrieve] |
| 5. |
Tilghman, S. M.,
Hanson, R. W.,
Reshef, L.,
Hopgood, M. F.,
and Ballard, F. J.
(1974)
Proc. Natl. Acad. Sci. U. S. A.
71,
1304-1308 |
| 6. | Loose, D. S., Cameron, D. K., Short, H. P., and Hanson, R. W. (1985) Biochemistry 24, 4509-4512[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Roesler, W. J.,
Simard, J.,
Graham, J. G.,
and McFie, P. J.
(1994)
J. Biol. Chem.
269,
14276-14283 |
| 8. |
Yeagley, D.,
Agati, J. M.,
and Quinn, P. G.
(1998)
J. Biol. Chem.
273,
18743-18750 |
| 9. |
Giralt, M.,
Park, E. A.,
Liu, J. S.,
Gurney, A. L.,
Hakimi, P.,
and Hanson, R. W.
(1991)
J. Biol. Chem.
266,
21991-21996 |
| 10. | Schmidt, E. D. L, van Beeren, M., Glass, C. K., Wiersinga, W. M., and Lamers, W. H. (1993) Biochim. Biophys. Acta 1172, 82-88[Medline] [Order article via Infotrieve] |
| 11. |
Park, E. A.,
Song, S.,
Vinson, C.,
and Roesler, W. J.
(1999)
J. Biol. Chem.
274,
211-217 |
| 12. |
Landshultz, W. H.,
Johnson, P. F.,
Adashi, E. Y.,
Graves, B. J.,
and McKnight, S. L.
(1988)
Genes Dev.
2,
786-800 |
| 13. |
Landschultz, W. H.,
Johnson, P. F.,
and McKnight, S. L.
(1988)
Science
240,
1759-1763 |
| 14. |
Alam, T.,
An, M. R.,
and Papaconstantinou, J.
(1992)
J. Biol. Chem.
267,
5021-5024 |
| 15. |
McKnight, S. L.,
Lane, M. D.,
and Gluecksohn-Waelsch, S.
(1989)
Genes Dev.
3,
2021-2024 |
| 16. |
Park, E. A.,
Gurney, A. L.,
Nizielski, S. E.,
Hakimi, P.,
Cao, Z.,
Moorman, A.,
and Hanson, R. W.
(1993)
J. Biol. Chem.
268,
613-619 |
| 17. |
Park, E. A.,
Roesler, W. J.,
Liu, J. S.,
Klemm, D. J.,
Gurney, A. L.,
Thatcher, J. D.,
Shuman, J.,
Friedman, A.,
and Hanson, R. W.
(1990)
Mol. Cell. Biol.
10,
6264-6272 |
| 18. |
Wang, N.,
Finegold, M. J.,
Bradley, A.,
Ou, C. N.,
Abdelsayed, S. V.,
Wilde, N. M.,
Taylor, L. R.,
Wilson, D. R.,
and Darlington, G. J.
(1995)
Science
269,
1108-1112 |
| 19. | Lee, Y., Sauer, B., Johnson, P. F., and Gonzalez, F. J. (1997) Mol. Cell. Biol. 17, 6014-6022[Abstract] |
| 20. | Liu, S., Croniger, C., Arizmendi, C., Harada-Shiba, M., Ren, J., Poli, V., Hanson, R. W., and Friedman, J. E. (1999) J. Clin. Invest. 103, 207-213[Medline] [Order article via Infotrieve] |
| 21. | Tanaka, T., Akira, S., Yoshida, K., Umemoto, M., Yoneda, Y., Shirafuji, N., Fujiwara, H., Suematsu, S., Yoshida, N., and Kishimoto, T. (1995) Cell 80, 353-361[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Olive, M.,
Williams, S. C.,
Dezan, C.,
Johnson, P. F.,
and Vinson, C.
(1996)
J. Biol. Chem.
271,
2040-2047 |
| 23. |
Cao, Z.,
Umek, R. M.,
and McKnight, S. L.
(1991)
Genes Dev.
5,
1538-1552 |
| 24. |
Friedman, A. D.,
Landshultz, W. H.,
and McKnight, S. L.
(1989)
Genes Dev.
3,
1314-1322 |
| 25. |
Descombes, P.,
Chojkier, M.,
Lichtsteiner, S.,
Falvey, E.,
and Schibler, U.
(1990)
Genes Dev.
4,
1541-1551 |
| 26. |
Roesler, W. J.,
Park, E. A.,
and McFie, P. J.
(1998)
J. Biol. Chem.
273,
14950-14957 |
| 27. | Crosson, S. M., Davies, G. F., and Roesler, W. J. (1997) Diabetologia 40, 1117-1124[CrossRef][Medline] [Order article via Infotrieve] |
| 28. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 16.32-16.38, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 29. | Davies, G. F., Khandelwal, R. L., and Roesler, W. J. (1999) Biochim. Biophys. Acta 1451, 122-131[Medline] [Order article via Infotrieve] |
| 30. | Katz, R. A., Erlanger, B. F, and Guntaka, R. V. (1983) Biochim. Biophys. Acta 73, 9258-9264 |
| 31. | Davies, G. F., Crosson, S. M., Khandelwal, R. L., and Roesler, W. J. (1995) Arch. Biochem. Biophys. 323, 477-483[CrossRef][Medline] [Order article via Infotrieve] |
| 32. | Niehof, M., Manns, N. W., and Trautwein, C. (1997) Mol. Cell. Biol. 17, 3600-3613[Abstract] |
| 33. |
Legraverend, C.,
Antonson, P.,
Flodby, P.,
and Xanthopoulos, K. G.
(1993)
Nucleic Acids Res.
21,
1735-1742 |
| 34. | Timchenko, N., Wilson, D. R., Taylor, L. R., Abdelsayed, S., Wilde, M., Sawadogo, M., and Darlington, G. J. (1995) Mol. Cell. Biol. 15, 1192-1202[Abstract] |
| 35. | Deleted in proof |
| 36. |
Yamada, K.,
Duong, D. T.,
Scott, D. K.,
Wang, J.,
and Granner, D. K.
(1999)
J. Biol. Chem.
274,
5880-5887 |
| 37. |
Roesler, W. J.,
Crosson, S. M.,
Vinson, C.,
and McFie, P. J.
(1996)
J. Biol. Chem.
271,
8068-8075 |
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. The upper panel shows representative
autoradiograms of ribonuclease protection analysis, while the
lower panel shows a graphical representation of the results
of three independent experiments (means ± S.E.). The isolation of
RNA, ribonuclease protection analysis, and the equilibration of RNA
amounts between samples by RNA slot-blot analysis using an 18 S rRNA
probe (data not shown) are described under "Experimental
Procedures."
