Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870
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ABSTRACT |
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Phosphoenolpyruvate carboxykinase
(PEPCK) is a key regulatory enzyme in renal gluconeogenesis.
Activation of various PEPCK2300Luc reporter constructs in
LLC-PK1-F+ cells, a gluconeogenic line of
porcine renal proximal tubule-like cells, by protein kinase A (PKA) is
mediated, in part, through the cAMP-response element (CRE)-1 of the
PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with
nuclear extracts from LLC-PK1-F+ cells produced
multiple bands, all of which were blocked by antibodies that are
specific for C/EBP
but not for C/EBP
or C/EBP
. Treatment of
cells with cAMP did not affect the expression of C/EBP
, but the
observed binding activity was increased nearly threefold. Mutation of
CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of
PEPCK
2300Luc to 40% of that observed with the wild-type
construct. Coexpression of a chimeric protein containing a Gal-4
binding domain and the transactivation domain of C/EBP
, but not of
C/EBP
or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBP
functions as a dominant negative inhibitor. Thus the
binding of C/EBP
to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.
renal gluconeogenesis; adenosine-3',5'-cyclic monophosphate-response element-1; protein kinase A
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INTRODUCTION |
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THE CYTOSOLIC FORM
OF phosphoenolpyruvate carboxykinase (PEPCK) is
encoded by a single copy gene that is developmentally regulated and is
expressed in a tissue- and cell-specific manner (12). It
is expressed predominantly in liver, kidney, and adipose tissues. Transcription of PEPCK is suppressed during fetal development but is
dramatically induced at birth. Transcription of the PEPCK gene in the
postnatal liver is stimulated by glucagon via cAMP, thyroid hormone,
and glucocorticoids, whereas insulin and phorbol esters inhibit its
expression. Expression of a chimeric PEPCK-bovine growth hormone gene
in transgenic mice revealed that a relatively small region within the
PEPCK promoter (460 to +73 bp) contains most of the information
required for conferring the appropriate pattern of developmental,
tissue-specific, hormonal, and dietary regulation of the PEPCK gene
(18, 19). However, this region is highly complex. DNase I
footprinting analysis of this segment with rat liver nuclear extracts
identified at least eight protein binding domains, termed cAMP response
element (CRE)-1, CRE-2, and P1 through P6 (30).
The initial cAMP-response element (CRE-1) within the PEPCK promoter can
bind a number of transcription factors, including CRE binding protein
(CREB) (23), c-Fos/c-Jun (11), and C/EBP (23). The CRE-1 element was able to confer cAMP
responsiveness to a neutral promoter when transfected into human
choriocarcinoma cells (1). However, the extent of cAMP
induction mediated by CRE-1 alone was much less than that observed with
the intact PEPCK promoter, suggesting that other elements are also
required for full cAMP responsiveness. Transient transfection assays
performed in HepG2 liver cells using a series of deletions and block
mutations of a PEPCK490-chloroamphenicol
acetyltyransferase (CAT) construct established that both the CRE-1 and
P3(I) elements are important for cAMP induction (15).
Mutation of both the CRE-1 and P3(I) sequences resulted in the complete
loss of induction by either 8-bromo-cAMP or by the catalytic subunit of
protein kinase A (PKA).
Use of dominant negative constructs established that both CREB and
C/EBP are involved in mediating the cAMP-dependent activation of PEPCK
gene expression in liver cells (27, 28). Experiments using
Gal-4 fusion proteins (28) indicated that CREB
preferentially binds to the CRE-1 element and interacts with upstream
transcription factors to activate PEPCK transcription in HepG2 cells.
Similar experiments have shown that the synergistic effect of the P3(I) and P4 elements can be mediated by C/EBP (27) and to a
lesser extent by C/EBP
(24). However, more recent data
obtained by constitutive expression of specific antisense RNAs indicate
that C/EBP
, and not C/EBP
, participates in cAMP-dependent
activation of PEPCK transcription in H4IIE cells (8). The
latter conclusion is supported by studies using mice homozygous for
deletions in the genes that encode either C/EBP
or C/EBP
(7). C/EBP
was found to be required for the cAMP
activation of PEPCK transcription in the neonatal liver. In contrast,
C/EBP
was normally not essential, but it could compensate for the
loss of C/EBP
if induced sufficiently. P3(II), which contains an
activator protein (AP)-1-like element, also participates in
this process, since the activity of the liver-specific region cannot be
mimicked by multiple copies of other well-characterized C/EBP binding
sites (29). Furthermore, supershift analysis suggests that
a c-Fos/c-Jun protein from HepG2 cells binds to an oligonucleotide containing the P3(II) site. Thus a c-Fos/c-Jun protein also appears to
be involved in cAMP activation of the PEPCK gene in liver cells (29).
While hepatic gluconeogenesis is essential for maintaining blood glucose levels, in kidney this process is coupled to ammoniagenesis and the maintenance of acid-base balance. The renal PEPCK is expressed solely within the proximal tubular segment of the nephron (2). Various hormones such as angiotensin II and parathyroid hormone are known to affect cAMP levels within the renal proximal tubule. The two hormones primarily regulate renal sodium, bicarbonate, and phosphate reabsorption. However, angiotensin II also stimulates renal ammoniagenesis (5), and parathyroid hormone stimulates renal gluconeogenesis (25). Previous experiments demonstrated that there are marked differences in the footprinting patterns observed with nuclear extracts prepared from rat liver and kidney (30), suggesting that kidney and liver may utilize different trans-acting factors to regulate the PEPCK gene. Thus it is physiological relevant to determine the specific cis/trans interactions that mediate the cAMP-dependent activation of the PEPCK gene in renal proximal tubule cells.
The cAMP-dependent activation of the PEPCK gene in
LLC-PK1-F+ cells, a porcine gluconeogenic
proximal tubule-like line of cells (10), mapped to only
the CRE-1 and P3(II) elements (17). Mutation of the P3(I)
and P4 elements had no effect on cAMP stimulation of the
PEPCK490CAT construct when transfected into the kidney
cells. By using dominant negative constructs, it was shown that the
renal response of the PEPCK gene to cAMP may be mediated, in part, by
an isoform of C/EBP but not by CREB (17). In the current
study, the endogenous protein in LLC-PK1-F+
cells that binds to the CRE-1 element of the PEPCK promoter was characterized. Of the various C/EBP isoforms, only antibodies against
C/EBP
were able to block the specific binding of a CRE-1 oligonucleotide to nuclear protein(s) from
LLC-PK1-F+ cells. Furthermore, luciferase
assays using various Gal-4 constructs indicated that the
transactivation domain of C/EBP
, but not that of C/EBP
or CREB,
restores full activation by PKA. Thus the binding of C/EBP
to the
CRE-1 element may contribute to the cAMP-dependent activation of the
PEPCK promoter in kidney cells.
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MATERIALS AND METHODS |
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Materials.
[14C]chloramphenicol (specific activity, 56 mCi/mmol) and
T4 polynucleotide kinase were obtained from Amersham.
[-32P]ATP (specific activity, 5,600 Ci/mmol) was
obtained from ICN. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) was purchased
from Boehringer Mannheim. The Dual-Luciferase Reporter Assay Kit
was purchased from Promega. Oligonucleotides containing consensus
sequences for CRE and AP-1 elements were purchased from Santa Cruz
Biotechnology. Antibodies specific for the
-,
-, and
-isoforms
of C/EBP used in the supershift assays were obtained from Dr. Steven L. McKnight (Tularik). The expression vector for C/EBP
was provided by
Dr. Richard Hanson (Case Western Reserve Univ., Cleveland, OH).
GST-C/EBP
, GST-C/EBP
, and GST-C/EBP
fusion constructs were
from Dr. Wen-Hwa Lee (Univ. of Texas, San Antonio, TX). The
C/EBP
Spl construct was obtained from Dr. Akira (Hyogo College of
Medicine, Hyogo, Japan). The expression vectors for Gal-4-CREB and
Gal-4-C/EBP
(G
2) were obtained from Dr. William Roesler (Univ. of
Saskatchewan, Saskatoon, SK, Canada), whereas the
Gal-4-C/EBP
-(1---138) construct was obtained from Dr. Edwards Park
(Univ. of Tennessee Health Sciences Center, Memphis, TN). Other
biochemicals were purchased from Sigma or Fluka.
Cell cultures. LLC-PK1-F+ cells were originally isolated by Gstraunthaler and Handler (10) and were obtained from Dr. Gerhard Gstraunthaler (Univ. of Innsbruck, Innsbruck, Austria). The cells were cultured on 10-cm plates in a 50:50 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 10% fetal bovine serum. The medium contained 5 mM glucose and 25 mM NaHCO3 and was adjusted to pH 7.4.
Nuclear extract isolation and electrophoretic mobility shift
assays.
Nuclear extracts were isolated from LLC-PK1-F+
cells as described by Lee et al. (14) except
that Tris · HCl (pH 8.0) was replaced with HEPES (pH 7.9), and
1 mM dithiothreitol, 10 µM antipain, 10 µg/ml aprotinin, and 1 µM
pepstatin were added to both the lysis and extraction buffers.
Double-stranded deoxyribonucleotide probes were synthesized by
Macromolecular Resources (Ft. Collins, CO) as complementary
oligonucleotides containing 5'-BamHI overhangs. They were
annealed in 50 mM NaCl, 66 mM Tris · HCl, and 6.6 mM MgCl2, pH 7.5, by heating to 85°C and cooling to 25°C.
The various oligonucleotide probes and their position in the PEPCK
promoter include the following: CRE-1, bases 99 to
77; the CRE-1
block mutation (mCRE-1), bases
99 to
77 but containing the
same 5-bp mutation as found in the CRE-1 block mutation of the
PEPCK
490CAT construct (16); and P3(II),
bases
266 to
246. The sequences of the sense strands are as
follows: CRE-1,
5'-GATCCGGCCCCTTACGTCAGAGGCGAG-3'; mCRE-1,
5'-GATCCGGCCCCTGCATGCAGAGGCGAG-3'; and P3(II), 5'-GATCTCAAAGTTTAGTCAATCAAAC-3';
where the sequences derived from the PEPCK promoter are underlined, the mutated bases are italicized, and the CRE-1 or AP-1 elements are in
bold. The sequence of the consensus CRE and AP-1 oligonucleotides with
the binding elements underlined are
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and
5'-CGCTTGATGACTCAGCCGGAA-3', respectively.
CAT assay.
LLC-PK1-F+ cells were split and replated on
10-cm plates at ~30% confluence. The cells were grown for 20 h
in culture and transfected by calcium phosphate precipitation of DNA
(4). The precipitated DNA contained 10 µg of the
PEPCK490CAT construct, 2 µg of cytomegalo virus
(CMV)-
-galactosidase, and, where indicated, 5 µg of a
C/EBP
expression vector. The total DNA in each transfection was
maintained at 40 µg by addition of salmon sperm DNA. At 20 h
posttransfection, the cells were homogenized and assayed for
-galactosidase activity. Samples of homogenate (20-100 µl)
containing equivalent units of
-galactosidase activity were used to
measure CAT activity (9). The acetylated products and the
unreacted substrate were separated by thin-layer chromatography, and
the percent conversion was quantitated using a PhosphorImager
(Molecular Dynamics).
Synthesis of luciferase reporter constructs.
The various luciferase constructs were assembled in pGL2-Basic
(Promega), which contains the firefly luciferase gene preceded by a
large multicloning site. pGL2-Basic was linearized with
NheI, blunted with Klenow, and then restricted with
BglII. To obtain the 2,300-bp promoter segment of the
PEPCK gene, pPEPCK
2300CAT was partially digested with
XbaI, and the linear plasmid was isolated on an agarose gel
and purified using GeneClean (BIO 101). The XbaI overhangs
were blunted with Klenow, and the DNA was subsequently restricted with
BglII. The 2.4-kb fragment was isolated and ligated into the
restricted pGL2-Basic plasmid to produce pPEPCK
2300Luc. A
428-bp NdeI and BglII restriction fragment was
removed from pPEPCK
2300Luc. The same restriction enzymes
were used to digest the PEPCK
490CAT constructs containing
the individual block mutations in the CRE-1, P2, and P3(II) sites
(16). The isolated fragments containing the mutations were
ligated into the linearized PEPCK
2300Luc plasmid to yield
pPEPCKmCRE-1Luc, pPEPCKmP2Luc, and
pPEPCKmP3IILuc, respectively. A 1,566-bp FseI and ClaI fragment from pPEPCKmCRE-1Luc was then
cloned into pPEPCKmP3IILuc to produce a construct
containing the double mutation. The 428-bp NdeI/BglII fragment containing the mCRE-1 element
was also subcloned into pUC19. The mCRE-1 sequence contains a unique
SphI site (16). Complimentary oligonucleotides,
containing a Gal-4 binding site with SphI overhangs, were
annealed and ligated into pUC19/mCRE-1 that had been linearized with
SphI. The sequence of the sense strand is
5'-CGGGAGTACTGTCCTCCGCATG-3', where the Gal-4 binding site
is underlined. The resulting plasmid was restricted with NdeI and BglII, and the promoter fragment
containing the Gal-4 binding site was cloned into
pPEPCK
2300Luc to yield pPEPCKGal4Luc. For
each plasmid, all of the created mutations and ligated junctions were
confirmed by dideoxynucleotide sequencing.
Luciferase assay.
At 3 days postsplitting into 6-well plates, the
LLC-PK1-F+ cells were transfected by calcium
phosphate precipitation (4). Each sample contained 0.1 µg of pRL-null (Promega), 0.6 µg of a pPEPCK2300Luc
plasmid, and, where indicated, 2 µg of a chimeric Gal-4 and/or 1 µg
of PKA expression vectors. Sufficient salmon sperm DNA was added so
that all samples contained 5 µg of DNA. Approximately 24 h
later, the transfection media was removed and fresh media were added.
The cells were cultured for an additional 24 h and washed with 2 ml of phosphate-buffered saline, and cell extracts were prepared using
250 µl of passive lysis buffer (Promega). The extracts were assayed
with a Turner Design 20/20 Luminometer using the reagents contained in
the Dual-Luciferase Reporter Assay System (Promega). The firefly
luciferase activities obtained from the various
pPEPCK
2300Luc plasmids were standardized vs. the
corresponding Renilla luciferase activities to correct for differences in transfection efficiency. In a single experiment, each
transfection was performed in triplicate.
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RESULTS |
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Previous studies using a series of deletions and block
mutations of a PEPCK490CAT construct identified the CRE-1
and P3(II) sites as the primary elements within the PEPCK promoter (Fig. 1) that are essential for
cAMP-dependent activation of the PEPCK gene in
LLC-PK1-F+ cells (17). This
analysis was repeated using a more sensitive and more responsive set of
luciferase constructs (Fig. 2).
Cotransfection of an expression vector that encoded the catalytic
subunit of PKA produced a 30-fold activation of the
PEPCK
2300Luc construct. Mutation of the CRE-1 element
reduced basal activity by 20%. However, PKA activation of
PEPCKmCRE-1Luc was reduced to one-third of the wild-type
construct (11-fold). In contrast, mutation of the P2 element reduced
basal expression to one-fifth of the basal expression of
PEPCK
2300Luc, whereas PKA activation of
PEPCKmP2Luc was at least as great (64-fold) as was observed
with PEPCK
2300Luc. Mutation of the P3(II) element also
had a significant effect on basal expression without affecting PKA
activation. Basal expression of PEPCKmP3IILuc was reduced
to 65% of the wild-type vector, but coexpression of PKA again caused a
30-fold increase from the reduced basal activity. The double mutant,
PEPCKmCRE-1/mP3IILuc, exhibited properties equal to the sum
of the two individual mutants. Basal expression was equivalent to that
observed with PEPCKmP3IILuc (65%), whereas PKA activation
was equivalent to that of the PEPCKmCRE-1Luc construct
(9-fold).
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The potential role of a C/EBP protein in cAMP-dependent activation of
PEPCK transcription in LLC-PK1-F+ cells was
previously demonstrated by the finding that coexpression of either of
two dominant negative forms of C/EBP blocks the cAMP-dependent stimulation of PEPCK490CAT activity (17).
Electrophoretic mobility shift assays were used to further
characterize the protein that binds to the CRE-1 element (Fig.
3). Nuclear extracts from subconfluent
LLC-PK1-F+ cells produced multiple bands when
incubated with the CRE-1 probe (Fig. 3, lane 2). The
specificity of the apparent binding was determined from competition
experiments in which a 50- or 500-fold excess of various unlabeled
competitors was preincubated with the nuclear extract. A 500-fold
excess of unlabeled CRE-1 or an oligonucleotide containing a consensus
CRE site completely inhibited the observed binding. In contrast,
unlabeled mCRE-1, in which the CRE-1 sequence contains the same 5-bp
mutation as the CRE-1 block mutant of PEPCK
490CAT, did
not compete. Similarly, an oligonucleotide containing the P3(II) site
of the PEPCK promoter failed to inhibit the binding to the CRE-1 probe,
indicating that different proteins bind to the CRE-1 and P3(II)
elements. The oligonucleotide containing a consensus AP-1 site
exhibited only a slight competition, suggesting that c-Fos/c-Jun
proteins are not primarily responsible for the observed binding. The
cumulative data strongly suggest that the observed binding to the CRE-1
oligonucleotide is specific and represents interactions characteristic
of the CRE-1 element.
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The possibility that the protein in the nuclear extract from
subconfluent LLC-PK1-F+ cells that binds to the
CRE-1 element is an isoform of C/EBP was examined by supershift or
immunoblocking analysis (Fig. 4). A
polyclonal antibody against C/EBP was able to supershift the binding
of a GST-C/EBP
fusion protein in a concentration-dependent manner
(lanes 2-4). Preincubation with increasing
concentrations of anti-C/EBP
antibody blocked the binding of all
proteins in the nuclear extract from LLC-PK1-F+
cells to the CRE-1 probe, suggesting that differentially modified forms
of C/EBP
or heterodimers formed between C/EBP
and different partners are responsible for the multiple bands. The anti-C/EBP
antibody did not cross-react with the GST-C/EBP
or GST-C/EBP
proteins (data not shown). Furthermore, the above experiment was repeated using polyclonal antibodies specific for C/EBP
and C/EBP
(Fig. 5). The two antibodies supershifted
the respective GST-C/EBP
and GST-C/EBP
fusion proteins but
neither supershifted nor blocked the binding of nuclear proteins from
LLC-PK1-F+ cells to the CRE-1 oligonucleotide.
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Various Gal-4 constructs were used to determine the ability of
different transcription factors to restore PKA-dependent activation of
PEPCK2300Luc in LLC-PK1-F+ cells
(Fig. 6). The firefly luciferase activity
measured in LLC-PK1-F+ cells transiently
transfected with PEPCK
2300Luc is strongly activated by
cotransfection with an expression vector that encodes the catalytic
subunit of PKA. When the CRE-1 site of the PEPCK
2300Luc construct was mutated, the fold activation by PKA was reduced to 38%
of that observed with the wild-type construct. This result is similar
to that observed with the PEPCKmCRE-1Luc construct. Conversion of the mutated CRE-1 site to a Gal-4 binding site had little
effect on the fold activation by PKA. However, cotransfection of this
reporter construct with an expression vector that encoded a chimeric
protein containing a Gal-4 binding domain and the transactivation domain of C/EBP
restored the level of PKA activation to that observed with the wild-type construct. In contrast, coexpression of
Gal-4-C/EBP
or Gal-4-CREB proteins had either no effect or further
reduced PKA activation, respectively.
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Western blot analysis indicated that the level of C/EBP expressed in
LLC-PK1-F+ cells is not affected by treatment
with CPT-cAMP or forskolin (Fig.
7A). In addition, the pattern
of specific binding to the CRE-1 probe was not altered. However, the
apparent binding activity was increased significantly in nuclear
extracts prepared from cells treated with CPT-cAMP (Fig.
7B). The increase in apparent binding was specific to the
CRE-1 probe, since the apparent binding observed with an Sp-1
containing oligonucleotide was unaffected by using nuclear extracts
obtained from cAMP-treated and untreated cells (data not shown). When
normalized to the Sp-1 controls, the binding activity was increased
2.7-fold within 2 h after treatment with cAMP, and the observed
increase was sustained for 24 h.
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The importance of the activation domain of C/EBP in mediating cAMP
activation of PEPCK transcription was investigated by comparing the
activation produced by coexpression of C/EBP
or of a deletion
construct (Fig. 8). Coexpression of
C/EBP
produced a sixfold stimulation of PEPCK
490CAT
activity, whereas coexpression of
Spl significantly reduced basal
activity.
Spl is a C/EBP
construct in which amino acids
41-205 are deleted, and thus it lacks the transactivation domain
(22). Expression of the catalytic subunit of PKA produced
a fourfold activation of PEPCK
490CAT that was further
enhanced (2-fold) by the coexpression of C/EBP
. However,
coexpression of
Spl completely abolished the activation caused by
the catalytic subunit of PKA. Thus deletion of the activation domain of
C/EBP
generates a dominant negative effector.
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DISCUSSION |
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CREB was originally purified by DNA affinity chromatography using
the CRE from the somatostatin gene as the ligand (21). On
phosphorylation by PKA, CREB recruits a CREB binding protein (CBP) that
acts as an essential coactivator (13). This sequence of
events has become the paradigm to explain how cAMP-dependent activation
of transcription is mediated in a variety of systems. Roesler et al.
(28) demonstrated that cotransfection of HepG2 cells with
KCREB, a dominant negative form of CREB that contains a point mutation
in the DNA binding domain (32), blocked the PKA-dependent
activation of PEPCK490CAT. Subsequent experiments clearly
document that CREB or C/EBP
, but not C/EBP
, can interact with
CRE-1 to mediate the cAMP-dependent induction of PEPCK mRNA in various
hepatoma cells (26).
Similar experiments performed in LLC-PK1-F+
kidney cells produced very different results (17).
Cotransfection of KCREB had little effect on basal or PKA-stimulated
expression of PEPCK490CAT. However, cotransfection of
CREB inhibited the basal and PKA-stimulated activities of
PEPCK
490CAT by 70%, and these effects were reversed by
coexpression of KCREB. A similar effect was observed in the
reconstitution experiments using the Gal-4-CREB expression vector (Fig.
6). This construct resulted in further inhibition of PKA-stimulated
luciferase activity of the pPEPCKGal4Luc plasmid. However, Western blot analysis indicates that the
LLC-PK1-F+ cells express CREB at a level
comparable with that observed in various hepatoma cells (data not
shown). CREB-specific antibodies were also effective in supershifting
the complex formed between the CRE-1 oligonucleotide and recombinant
CREB. However, the same antibody failed to supershift or block the
formation of any of the bands observed when the CRE-1 probe was
incubated with the nuclear extracts of
LLC-PK1-F+ cells (data not shown). These
observations suggest that the endogenous CREB-like protein in
LLC-PK1-F+ cells may be a unique isoform.
Alternatively, the binding of CREB to CRE-1 may be inhibited by the
binding of other transcription factors. For example, NF1-C, which binds
effectively to the adjacent P1 site and is highly expressed in kidney,
abrogates both CREB and PKA stimulation of PEPCK
490CAT
activity when expressed in HepG2 cells (6). The presence
of a unique coactivator could also affect CREB interactions with the
CRE-1. The coexpression of CBP partially reversed the CREB-dependent
inhibition of the PKA-stimulated PEPCK
490CAT activity in
LLC-PK1-F+ cells (17). This
finding was previously interpreted to indicate that the
LLC-PK1-F+ cells lack CBP and express an
alternative coactivator. Western blot analysis also indicated that
LLC-PK1-F+ cells express very little, if any,
CBP (data not shown). Thus cAMP activation of the PEPCK gene in kidney
cells must differ significantly from the mechanism characterized in
liver cells.
PEPCK2300Luc, when expressed in subconfluent
LLC-PK1-F+ cells, is stimulated 30-fold by
cotransfection of the catalytic subunit of PKA. The observed activation
was significantly greater than previously reported for the
PEPCK
490CAT construct (17). Part of the
greater response was due to the incorporation of the longer segment of
promoter sequence, since the corresponding PEPCK
490Luc
construct is activated only 15-fold by PKA (data not shown). As
observed with the previous CAT assays (17), mutation of
the CRE-1 element had a slight effect on basal activity (decreased by
20%) but a greater effect on PKA activation. In both sets of
experiments, mutation of the CRE-1 reduced the fold activation by PKA
of the reporter activities to about one-third of the wild type. The
data obtained with the CAT assays suggested that the remaining
activation may be due to AP-1 binding to the P3(II) element
(17). However, assays with the more responsive luciferase
constructs suggest that the P3(II) site contributes to basal expression
and does not mediate the effect of PKA. Thus some element in addition
to the CRE-1 must also contribute to the PKA-dependent activation of
the PEPCK promoter in LLC-PK1-F+ cells.
Nuclear extracts of HepG2 cells contain at least two proteins that form
specific complexes with the CRE-1 oligonucleotide (28).
Formation of one of the observed complexes was blocked by preincubation
with antibodies to CREB, the other by antibodies to C/EBP. Neither
antibody formed a supershift of the respective complex. In contrast,
all of the specific binding interactions observed with the nuclear
proteins from LLC-PK1-F+ cells were blocked
with antibodies specific for C/EBP (Figs. 4 and 5). However, this
polyclonal antibody supershifted the complex formed between the
GST-C/EBP
fusion protein and the CRE-1 element. Thus the added
domain may either block or alter the conformation of an epitope that is
adjacent to the DNA binding domain of C/EBP
. The binding of an
antibody to this epitope could block DNA binding of the wild-type
C/EBP
. Alternatively, the binding of the antibodies to C/EBP
could produce conformational changes that reduce its affinity for the
CRE-1 element, and the additional domain of the GST-C/EBP
fusion
protein may restrict the conformation of the C/EBP
DNA binding
domain sufficiently to prevent such changes. Curiously, two other
C/EBP
-specific antibodies obtained from Santa Cruz Biotechnology
also blocked binding of the nuclear protein from
LLC-PK1-F+ cells without forming a supershift
(data not shown). Thus the current experiments identify C/EBP
as one
isoform of C/EBP that is expressed in
LLC-PK1-F+ cells. This conclusion was confirmed
by Western blot analysis (Fig. 7A). Furthermore, the Gal-4
experiments indicate that C/EBP
can contribute to the cAMP-dependent
activation of transcription in the LLC-PK1-F+
cells by binding to the CRE-1 region of the PEPCK promoter. Only the
Gal-4-C/EBP
chimera was able to restore full PKA activation to the
PEPCKGal4Luc construct (Fig. 6). In this experiment,
Gal-4-C/EBP
or Gal-4-CREB chimeras had little effect or were
inhibitory, respectively. These results are the complete opposite of
data previously reported with HepG2 cells (27, 28). To
verify this difference, the plasmids used in the current experiments
were transfected into HepG2 cells and shown to produce the previously
reported results (unpublished data of M. Mallozzi, Q.-T. Wall, and
N. P. Curthoys).
Electrophoretic mobility shift assays using nuclear extracts from
LLC-PK1-F+ cells (Fig. 3) produced multiple
bands that may represent homodimeric and heterodimeric forms
(3) of C/EBP. For example, the upper band may contain a
heterodimer of C/EBP
and activating transcription factor (ATF)-2,
since it is selectively supershifted with antibodies specific
for ATF-2 (unpublished data of A. Tang, L. Taylor, and N. P. Curthoys). C/EBP
was previously shown to be an in vitro substrate
for PKA (20). Furthermore, on phosphorylation by PKA, C/EBP
was translocated into the nucleus where it then activated transcription of c-Fos (20). Nuclear extracts prepared
from cAMP-treated LLC-PK1-F+ cells exhibited
about a threefold higher binding activity than those obtained from
untreated cells. The kinetics of the observed increase in binding
activity and the absence of a corresponding increase in the level of
C/EBP
protein suggest that phosphorylation of C/EBP
may
contribute to the observed response. In addition, deletion of the
activation domain of C/EBP
produced a dominant negative inhibitor
that reduced both the basal and the PKA-stimulated activity of
PEPCK
490CAT (Fig. 8). All of these observations are
consistent with the conclusion that C/EBP
contributes to the
cAMP-dependent activation of PEPCK transcription in
LLC-PK1-F+ cells.
Mutation of the CRE-1 element produces a slight, but highly
reproducible, decrease (20%) in either CAT (17) or
luciferase basal reporter activities. In contrast, addition of the
dominant negative Spl construct causes a fourfold reduction in
basal CAT activity (Fig. 8). Identical results were obtained with
the PEPCK
2300Luc reporter construct (data
not shown). Therefore, the binding of C/EBP
to the CRE-1 element and
to a second site may also contribute to the basal activity of the PEPCK
promoter in LLC-PK1-F+ cells. Furthermore, PKA
activation is only partially inhibited by mutation of the CRE-1
element, but it is completely reversed by addition of the dominant
negative
Spl construct. Thus the postulated second binding site for
C/EBP
may also contribute to the PKA-dependent activation in
LLC-PK1-F+ cells. The identification and
characterization of this putative site will require further analysis.
Tissue-specific regulation of a gene may be achieved by utilizing
different combinations of cis-elements and
trans-acting factors. In the case of cAMP activation of the
PEPCK gene, liver cells use the CRE-1 element and a "liver-specific
region" containing the P3(I), P4, and P3(II) elements. The individual
elements apparently bind CREB, a C/EBP protein, and c-Fos/c-Jun,
respectively (26). In contrast, in kidney cells, binding
of C/EBP to the CRE-1 element may contribute to the cAMP-dependent
activation of PEPCK transcription.
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ACKNOWLEDGEMENTS |
---|
The assistance of Dr. Richard W. Hanson, Dr. William Roesler, Dr. Edwards Park, Dr. Steven McKnight, Dr. Wen-Hwa Lee, and Dr. Shizuo Akira, who provided various plasmids or antibodies used in this study, is greatly appreciated.
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FOOTNOTES |
---|
This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43704 awarded to N. P. Curthoys.
Address for reprint requests and other correspondence: N. P. Curthoys, Dept. of Biochemistry and Molecular Biology, Colorado State Univ., Ft. Collins, CO 80523-1870 (E-mail: NCurth{at}lamar.ColoState.edu).
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.
Received 18 July 2000; accepted in final form 21 May 2001.
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