From the Department of Biochemistry, University of Iowa,
Iowa City, Iowa 52242
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INTRODUCTION |
Malic enzyme (EC 1.1.1.40) catalyzes the oxidative decarboxylation
of malate to pyruvate and CO2, simultaneously generating NADPH from NADP+. In the livers of well fed birds, much of
the NADPH generated by this reaction is used for de novo
fatty acid synthesis. Malic enzyme activity, like that of other
lipogenic enzymes, is regulated by nutritional state (1, 2). When
starved chickens are fed or fed chickens are starved, malic enzyme
activity, malic enzyme mass, malic enzyme mRNA abundance, and
transcription of the malic enzyme gene increase or decrease by similar
relative amounts and with time courses consistent with transcription
being the primary regulated process (3, 4). These nutritionally induced
changes in the expression of the chicken malic enzyme gene can be
mimicked quantitatively in chick embryo hepatocytes in culture (5). In
the presence of insulin and triiodothyronine
(T3),1
transcription of the chicken malic enzyme gene is high, as it is in the
livers of fed chicks (6). In the absence of T3 and insulin
or in the presence of insulin and T3 plus glucagon or cAMP
or medium-chain fatty acids (MCFAs), transcription is low, as in the
livers of starved birds (6, 7). Several T3 response elements (T3REs) have been localized in the 5'-flanking DNA
of the chicken malic enzyme gene (8-10). Characterization of these T3REs indicates that each contributes differentially to the
overall response of the gene to T3 (9).
Hexanoate (C6:0) and octanoate (C8:0) inhibit the
T3-induced increase in transcription of the malic enzyme
gene in chick embryo hepatocytes within 30 min of their addition;
inhibition is reversible upon removal of fatty acid. Inhibition by
MCFAs is selective; they have no effect on total transcription in
isolated nuclei or on transcription of the genes for
glyceraldehyde-3-phosphate dehydrogenase or
-actin. Inhibition is
also specific; 4- and 10-carbon fatty acids and several modified fatty
acids have little or no effect (7). These results suggest that the MCFA
effect may be relevant biologically. What might that biological
relevance be?
Levels of MCFAs in chicken plasma that are high enough to inhibit
transcription have not been reported. During starvation, a process that
inhibits transcription of the malic enzyme gene, however, fatty acid
oxidation is increased. Concomitantly, production of hydroxylated fatty
acids similar in chain length to the inhibitory MCFAs also is increased
(11-13). In hepatocytes in culture, MCFAs may be converted to
hydroxylated fatty acids or metabolites similar thereto, and these
may be the intracellular mediators of the inhibition of transcription.
How might MCFAs inhibit transcription of the malic enzyme gene?
Hydroxylated long-chain fatty acids activate transcription of the
fungal gene for cutinase (14) by stimulating phosphorylation of a
trans-acting factor that binds to a specific cis-acting enhancer element (15, 16). Polyunsaturated long-chain fatty acids (PUFAs) stimulate transcription of the acyl-CoA oxidase gene by interacting with the trans-acting factor, peroxisomal proliferator-activated receptor (PPAR) (17), or its adipocyte counterpart, fatty
acid-activated receptor (FAAR) (18). These factors bind to peroxisomal
proliferator response elements in the acyl-CoA oxidase gene. PUFAs also
appear to inhibit expression of the S14 gene via a response element, the PUFA response element; the mechanism for this is not known (19,
20). Long-chain fatty acids inhibit binding of T3 to nuclear TR in cells in culture (21, 22). Both long-chain fatty acids
and their acyl-CoA derivatives inhibit binding of T3 to rat
liver TR in vitro (23). In intact hepatocytes, however, MCFAs fail to inhibit binding of T3 to TR (7), suggesting
that they act by some mechanism other than displacement of
T3.
We report here that inhibition of T3-stimulated
transcription of the chicken malic enzyme gene caused by MCFAs is
mediated by the receptor that binds to T3REs. This
mechanism is distinct from that of fatty acids that act through unique
sequence elements. Rather than displacing T3 from TR or
displacing TR from T3RE, MCFAs inhibit the transactivation
function of TR. MCFAs also inhibit estrogen-stimulated transcription
through an estrogen response element and the estrogen receptor (ER),
but have no effect on ligand-stimulated transcription mediated by
glucocorticoid or cAMP response elements.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes were obtained from Life
Technologies, Inc., New England Biolabs Inc., U. S. Biochemical Corp.,
or Boehringer Mannheim. Other enzymes were obtained from the indicated
sources: RQ-DNase I (Promega), T4 polynucleotide kinase and Klenow
fragment of Escherichia coli DNA polymerase I (Boehringer
Mannheim), DNA polymerase from Thermus aquaticus (Promega),
and Bst polymerase (Bio-Rad). Nucleotides were purchased
from Sigma, Amersham Pharmacia Biotech, or Life Technologies, Inc.
Radiolabeled nucleotides, Hyperfilm-HP, ECL Western blotting kits, and
donkey anti-rabbit IgG conjugated to horseradish peroxidase (secondary
antibody) were obtained from Amersham Pharmacia Biotech.
D-threo-[dichloroacetyl-1,2-14C]Chloramphenicol
was purchased from NEN Life Science Products. LipofectACE, Waymouth MD
705/1 medium, and E. coli cells of strain DH5
were
obtained from Life Technologies, Inc. SeaKem LE agarose, NuSieve GTG
agarose, and DNA isolation columns (SpinBind) were purchased from FMC
Corp. Hormones, fatty acids (as sodium salts), and heparin columns were
purchased from Sigma. Polyclonal antibody to chicken TR
was
purchased from Santa Cruz Biotechnologies (cross-reacts with chicken
TR
). Monoclonal antibody to chicken RXR was provided by Pierre
Chambon (Université Louis Pasteur/ IGBMC, Illkirch, France).
Nitrocellulose membranes were purchased from Millipore Corp.
Plasmids--
Expression plasmids containing 5'-flanking DNA of
the chicken malic enzyme gene inserted upstream of the CAT gene in
pKSCAT were constructed as described (10). Synthesis of DNA constructs containing fragments of chicken malic enzyme DNA inserted into the
multiple cloning site 5' of the promoter of the herpes simplex virus
thymidine kinase (TK) gene in pBLCAT2 (24) also has been described
(10). Plasmid DR4×5-TKCAT was made by inserting five copies of
annealed oligonucleotides encoding a direct repeat element with a 4-bp
spacer (AGGTCAnnnnAGGTCA) into the multiple cloning site of pBLCAT2.
Structures of plasmid DNAs were confirmed by restriction enzyme mapping
and partial sequence analysis.
Construction of pRSV-Luc was described (10). Plasmid CMV-
GAL (25)
was obtained from Richard Maurer (Oregon Health Sciences University).
Plasmid [ME
3474/+31]CAT was provided by F. Bradley Hillgartner
(West Virginia University). Marc Montminy (Salk Institute) provided
pRSV-CREB. Bruno Luckow and Gunter Schutz (German Cancer Research
Center, Heidelberg, Germany) provided pBLCAT2. The expression vector
for GAL4, pSG424, contained sequences encoding amino acids 1-147 of
the GAL4 DNA-binding domain. Plasmid SG424 and the expression plasmid
for the GAL4-binding element, pMC110, were obtained from Mark Ptashne
(Harvard University). Herbert H. Samuels (New York University) provided
the cDNA for chicken TR
(cloned into the pET8c expression
vector) and pGAL4-cTR
(containing the sequences encoding amino acids
120-408 of the ligand-binding domain of chicken TR
cloned into
pSG424). Ronald Evans (Salk Institute) and Bert W. O'Malley (Baylor
College of Medicine) gave us pTREpal-TKCAT and pERE-TKCAT,
respectively. Expression vector for human ER (pHEO) was obtained from
Geoffrey L. Greene (University of Chicago). Expression plasmids for the
GRE linked to TKCAT DNA (p
GTCO) and rat GR (pVARO) were gifts from
Keith R. Yamamoto (University of California, San Francisco). Ganes Sen
(Cleveland Clinic Foundation) provided pCRE-TKCAT.
Cell Culture and Transient Transfection--
Livers of
19-day-old chick embryos were removed, chopped, and treated with
collagenase (26). Isolated hepatocytes were separated from red blood
cells; resuspended in Waymouth medium MD 705/1 supplemented with
penicillin (60 µg/ml), streptomycin (100 µg/ml), insulin (50 nM), and corticosterone (1 µM); and incubated
in 35-mm tissue culture dishes in an atmosphere of 5% CO2
in air at 40 °C. Twenty hours after the cells were plated, they were
transiently transfected using 40 µg of LipofectACE/well. Each 35-mm
dish was transfected with 5 µg of plasmid DNA: p[ME
5800/+31]CAT
(10.7 kilobase pairs, 2.5 µg) or a molar equivalent of other test
constructs, pCMV-
GAL or pRSV-LUC (0.5 µg), and pBluescript
KS+ (balance). Two 35-mm plates were used for each
experimental condition. After 24 h in the transfection medium, the
medium was removed by aspiration and replaced with Waymouth medium
supplemented with or without 1.6 µM T3 and
with or without 1 or 5 mM sodium hexanoate (26). Both of
these hexanoate concentrations caused similar degrees of inhibition
(data not shown).
CAT,
-Galactosidase, and Luciferase Assays--
Forty-eight
hours after adding T3, the cells were harvested; lysed by
three cycles of freezing and thawing; and analyzed for soluble protein
content (27) and
-galactosidase (28) or luciferase (29) and CAT (30)
activities.
-Galactosidase activity was determined by measuring the
absorbance at 420 nm following incubation of cell lysates with
o-nitrophenyl
-galactopyranoside. Luciferase activity was
determined by measuring the light units emitted following incubation of
cell lysates with luciferin. For CAT assays, samples of the cell
lysates were heat-treated for 30 min at 60 °C; denatured protein was
removed by centrifugation. CAT activity was determined by incubating a
portion of cell lysate with acetyl-CoA and
[14C]chloramphenicol for 15 h at 37 °C.
Incubation mixtures were then extracted with ethyl acetate and
subjected to thin-layer chromatography. Conversion to the acetylated
product was detected by liquid scintillation spectrophotometry or
direct autoradiography using the Packard InstantImager.
Gel Electrophoretic Mobility Shift Assay--
Each
oligonucleotide probe contained a 5'-extension and was labeled by a
fill-in reaction catalyzed by the Klenow fragment of E. coli
DNA polymerase I. Other procedures and the preparation of nuclear
extracts were described previously (9).
DNase I Footprint Analysis--
Nuclear extracts were prepared
from chick embryo hepatocytes incubated with insulin plus
corticosterone plus T3, with or without hexanoate (1 mM). Other procedures were as described previously (10).
Statistical Analysis--
Statistical significances of
differences between matched pairs were determined by the Wilcoxon
matched-pairs, signed-rank test (31). S.E. values are provided to
indicate the degree of variability in the data.
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RESULTS |
Identification of the Cis-acting Element Involved in Mediating
Inhibition by MCFA--
DNA constructs containing deletions from the
5'-end of the 5'-flanking DNA of the chicken malic enzyme gene were
transiently transfected into chick embryo hepatocytes in culture in an
effort to localize the inhibitory effect of MCFAs (hexanoate). Cells transfected with the longest construct (p[ME
5800/+31]CAT) responded to T3 with a 14-fold increase in CAT activity, and
hexanoate inhibited T3-stimulated CAT activity by 83%
(Fig. 1). We have previously reported
that 5800 bp of 5'-flanking DNA of the malic enzyme gene contained the
sequence element(s) required for stimulation of transcription by
T3 (10). The sequence element(s) necessary for inhibition
by hexanoate were contained in the same DNA.

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Fig. 1.
Effects of T3 and hexanoate on
CAT activity of hepatocytes transfected with constructs containing
deletions from the 5'-end of the 5'-flanking DNA of the chicken malic
enzyme gene. Chick embryo hepatocytes were transiently transfected
using LipofectACE (40 µg/plate), p[ME 5800/+31]CAT (2.5 µg/plate
or an equimolar amount of the other constructs), pCMV- GAL (0.5 µg/plate), and pBluescript DNA (sufficient to balance DNA/plate to
5.0 µg) and treated with or without T3 and with or
without hexanoate (C6 (5 mM), except that 1 mM
C6 was used in two experiments). Each point represents the mean ± S.E. of five to eight independent sets of hepatocytes, using at least
two independently prepared batches of each plasmid. CAT and
-galactosidase activities of extracts from T3-treated
hepatocytes transfected with p[ME 5200/+31]CAT were 1.6 ± 0.5 percentage of conversion/15 h/µg of protein and (4.4 ± 1.8) × 10 4 A420 units/min/µg of
protein, respectively. Left, DNA constructs used in these
experiments; middle, effects of T3, expressed as
-fold change in CAT activity caused by T3
(+T3/ T3); right, effects of
hexanoate. Relative CAT activity of cells treated with T3
and hexanoate is expressed as a percentage of that in cells treated
with T3 alone. Statistical significance between means
within a column is indicated as follows: a,
versus p[ME 2715/+31]CAT (p < 0.05);
b, versus p[ME 2715/+31]CAT (p < 0.02); c, versus p[ME 4135/+31]CAT
(p < 0.02).
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Deletions to
5200,
4135,
3845, and
3474 bp resulted in 16-, 17-, 9-, and 7-fold stimulations by T3, respectively. The degree of inhibition by hexanoate was the same for cells transfected with each of these constructs. Cells transfected with constructs containing 5'-deletions to
2715 bp or to shorter end points did not
respond to either T3 or hexanoate. These results confirmed the location of T3 response units (T3RUs)
between
4135 and
3845 bp and between
3845 and
2715 bp (9, 10).
The upstream T3RU contains four functional direct repeat
T3REs at
3883 to
3858,
3833 to
3808,
3809 to
3784, and
3794 to
3769 bp; the 5'-most of these T3REs
conferred 90% of the responsiveness of the entire upstream
T3RU. The downstream T3RU contains a single
T3RE at
3081 to
3056 bp (9, 10). Short internal
deletions were made in two constructs (p[ME
5800/+31]CAT and
p[ME
4135/+31]CAT) that were responsive to both T3 and
hexanoate. Each deletion removed the major functional T3RE
in the upstream T3RU; the minor T3REs in the
upstream and downstream T3RUs were not deleted.
T3 responsiveness decreased by ~50% for both deletion
constructs, but the MCFA responses were unaffected. Thus, any construct
that conferred a T3 response also conferred inhibition by
hexanoate, consistent with the possibility that T3REs
themselves might confer inhibition by MCFA.
Constructs that contain the upstream T3RU linked to TKCAT
(p[ME
3903/
3617]TKCAT and p[ME
3903/
3703]TKCAT) conferred
their responses to T3 via one major and three weak
T3REs (9, 10). In cells transfected with these constructs,
CAT activity was stimulated by T3 and inhibited by
hexanoate (Fig. 2). Deletion of part of the strongest T3RE (p[ME
3868/
3617]TKCAT or
p[ME
3868/
3703]TKCAT) caused a large reduction in T3
responsiveness, but did not affect responsiveness to hexanoate.
Deletions from the 3'-end of p[ME
3903/
3703]TKCAT to
3733 or
3769 bp did not affect T3 or hexanoate responses. Deletions to
3799,
3823, and
3863 bp gradually reduced
responsiveness to T3 from >100-fold to 54-fold, consistent
with the gradual loss of weak T3REs; inhibition by
hexanoate was unaffected. In this set of experiments, the vector,
pTKCAT, conferred a 2-fold stimulation by T3 and 39%
inhibition by hexanoate; responses of cells transfected with pTKCAT
were detectable in some batches of hepatocytes, but not others
(cf. Figs. 3 and 4) and may have been transduced through a
cryptic T3RE in pTKCAT (32). In those experiments in which pTKCAT-transfected hepatocytes had small but statistically significant responses to T3 and hexanoate, cells transfected with
constructs containing the malic enzyme DNA inserted into pTKCAT were
not judged to be responsive to T3 or hexanoate unless the
responses were significantly greater than those of cells transfected
with the vector alone. In those experiments in which pTKCAT-transfected hepatocytes had no statistically significant response to
T3, hexanoate had no inhibitory effect. The results of this
set of experiments remain consistent with the hypothesis that
inhibition by hexanoate is conferred by T3REs and that
inhibition was not localized to a specific T3RE within this
region.

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Fig. 2.
Effects of T3 and hexanoate on
CAT activity of hepatocytes transfected with constructs containing 5'-
and 3'-deletions in the upstream T3 response
region of the 5'-flanking DNA of the chicken malic enzyme gene.
Chick embryo hepatocytes were transiently transfected with the
indicated constructs as described in the legend to Fig. 1 and treated
with or without T3 (1.6 µM) and with or
without hexanoate (C6, 5 mM). The results are expressed as
described in the legend to Fig. 1; each value is the mean ± S.E.
of six to eight independent experiments using at least two
independently prepared batches of each plasmid. CAT and
-galactosidase activities of extracts from T3-treated
hepatocytes transfected with p[ME 3903/ 3733]TKCAT were 4.7 ± 1.7 percentage of conversion/15 h/µg of protein and (3.9 ± 1.8) × 10 4 A420 units/min/µg of
protein, respectively. Left, DNA constructs used in these
experiments; middle, effects of T3, expressed as
-fold change in CAT activity caused by T3;
right, effects of hexanoate. Relative CAT activity of cells
treated with T3 and hexanoate is expressed as a percentage
of that in cells treated with T3 alone. Statistical
significance between means within a column is indicated as follows:
a, versus pTKCAT (p < 0.02);
b, versus pTKCAT (p < 0.05);
c, versus p[ME 3903/ 3703]TKCAT
(p < 0.05).
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Cells were transfected with constructs containing block or deletion
mutations to verify that the T3 and hexanoate effects were
inseparable. In two sets of experiments, p[ME
3903/
3703]TKCAT was
transfected into hepatocytes; T3 caused 65- and 113-fold
increases in CAT activity, and hexanoate reduced the
T3-induced activity by 67 and 65%, respectively (Figs. 3
and 4). Mutation of the first half-site (pMUT1[ME
3903/
3703]TKCAT)
did not alter significantly either stimulation by T3 or
inhibition by hexanoate, compared with that of the nonmutated parent
construct (Fig. 3). Cells transfected with constructs containing block mutations in sites 2, 3, 4, 5, or 6 (individually) exhibited significantly reduced responsiveness to
T3, but no change in inhibition by hexanoate (Fig. 3).
Block mutations in either the upstream
(pMUTB[ME
3903/
3703]TKCAT) or downstream
(pMUTC[ME
3903/
3703]TKCAT) half-site of T3RE2 or both (pMUTD[ME
3903/
3703]TKCAT) significantly reduced responsiveness to
T3, but did not significantly alter responsiveness to
hexanoate (Fig. 4). Similarly, deletion
of T3RE2 decreased the T3 response by 95%, but
inhibition by hexanoate was still 49% (Fig. 4). In all of the
experiments reported thus far, the effects of T3 and hexanoate were inseparable, suggesting that any T3RE was
sufficient for the hexanoate effect. Cells transfected with a single
copy of the strong T3RE, p[ME
3883/
3858]TKCAT,
responded to both T3 and hexanoate (Figs. 4 and
5), indicating that T3RE2 by
itself is sufficient to confer both stimulation by T3 and
inhibition by hexanoate.

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Fig. 3.
Effects of T3 and hexanoate on
CAT activity in hepatocytes transfected with DNA constructs containing
block mutations in T3RE half-sites. A,
natural and mutant sequences of the T3RE half-sites. The
numbers refer to the half-sites identified in the T3RU of
the chicken malic enzyme gene (10). MUT1 to MUT6 refer to the number of
the half-site that was mutated within the 5'-flanking DNA of each
construct. B, chick embryo hepatocytes were transiently
transfected as described in the legend to Fig. 1 and treated with or
without T3 (1.6 mM) and with or without
hexanoate (C6, 5 mM). The results are expressed as
described in the legend to Fig. 1; each value is the mean ± S.E.
of seven to nine independent experiments using at least two
independently prepared batches of each plasmid. CAT and
-galactosidase activities of extracts from T3-treated
hepatocytes transfected with p[ME 3903/ 3703]TKCAT were 8.8 ± 2.6 percentage of conversion/15 h/µg of protein and (4.4 ± 1.4) × 10 4 A420 units/min/µg of
protein, respectively. Left, DNA constructs used in these
experiments; middle, effects of T3, expressed as
-fold change in CAT activity caused by T3;
right, effects of hexanoate. Relative CAT activity of cells
treated with T3 and C6 is expressed as a percentage of that
in cells treated with T3 alone. Statistical significance
between means within a column is indicated as follows: a,
versus pTKCAT (p < 0.01); b,
versus pTKCAT (p < 0.05); c,
versus p[ME 3903/ 3703]TKCAT (p < 0.01); d, versus p[ME 3903/ 3703]TKCAT
(p < 0.05).
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Fig. 4.
Effects of T3 and hexanoate on
CAT activity in hepatocytes transfected with constructs containing
wild-type and mutant versions of a T3 response region of
the chicken malic enzyme gene linked to TKCAT. A, block
mutations in half-sites 2U and 2D. Fragment A has the wild-type
sequence; fragments B, C, and D contain block mutations in positions
II-IV of one (B and C) or both (D) half-sites. B,
chick embryo hepatocytes were transiently transfected as described in
the legend to Fig. 1 and treated with or without T3 (1.6 µM) and with or without hexanoate (C6, 5 mM).
Left, DNA constructs used in these experiments.
Middle, effects of T3, expressed as -fold change
in CAT activity caused by T3. The results are expressed as
described in the legend to Fig. 1 and represent the mean ± S.E.
of four to eight independent experiments using at least two
independently prepared batches of each plasmid. CAT and
-galactosidase activities of extracts from T3-treated
hepatocytes transfected with p[ME 3903/ 3703]TKCAT were 8.1 ± 2.7 percentage of conversion/15 h/µg of protein and (5.7 ± 1.8) × 10 4 A420 units/min/µg of
protein, respectively. Right, effects of hexanoate. Relative
CAT activity of cells treated with T3 and C6 is expressed
as a percentage of that in cells treated with T3 alone.
Statistical significance between means within a column is indicated as
follows: a, versus pTKCAT (p < 0.01); b, versus pTKCAT (p < 0.02); c, versus p[ME 3883/ 3858]TKCAT
(p < 0.02); d, versus
p[ME 3883/ 3858]TKCAT (p < 0.01).
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Fig. 5.
Effects of T3 and hexanoate on
CAT activity in hepatocytes transfected with constructs containing
different kinds and numbers of T3REs. Chick embryo
hepatocytes were transiently transfected as described in the legend to
Fig. 1 and treated with or without T3 and with or without
hexanoate (C6, 1 mM). Left, DNA constructs used
in these experiments. First four columns,
relative CAT activities are expressed as described in the legend
to Fig. 1; each value is the mean ± S.E. of six to nine
independent experiments using at least two independently prepared
batches of each plasmid. Relative CAT activities were calculated by
setting the CAT activities for T3-treated hepatocytes
transfected with pTKCAT to 1.0 and adjusting all other activities
proportionately. CAT and -galactosidase activities of extracts from
T3-treated hepatocytes transfected with pTKCAT were
0.21 ± 0.04 percentage of conversion/15 h/µg of protein and
(7.1 ± 1.3) × 10 3 A420
units/min/µg of protein, respectively. Fifth
column, effects of T3 are expressed as -fold
change in CAT activity caused by T3. Sixth
column, the effect of hexanoate is expressed as CAT activity in
cells treated with T3 plus hexanoate divided by that in
cells treated with T3 alone × 100. The
boxed region A is wild-type T3RE2. TREpal
contains two copies of a palindromic T3RE, and DR4 contains
five copies of a DR4 T3RE, both linked to TKCAT.
Statistical significance between means within a column
(p < 0.05) is indicated as follows: a,
versus pTKCAT; b, versus
pDR4-TKCAT.
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We next tested constructs containing artificial
T3REs, the malic enzyme gene T3RU, and
T3RE2 to determine if the response to hexanoate was
specific to natural T3REs of the malic enzyme gene or could
be mediated by any element that responded to T3 (Fig. 5).
Constructs containing T3RE2
(p[ME
3883/
3858]TKCAT) or T3RE2 within the
entire T3RU (p[ME
3903/
3703]TKCAT) bestowed 120- and
130-fold responses to T3, respectively. These
T3-induced activities were decreased to 35 and 37%,
respectively, by hexanoate. Transfection of constructs containing
either two copies of the artificial palindromic T3RE or
five copies of a consensus direct repeat T3RE linked to
TKCAT DNA conferred robust responsiveness to T3 and
inhibition by hexanoate (Fig. 5). These results suggest that the
inhibitory effect of hexanoate is not limited to sequences from the
malic enzyme gene; inhibition by hexanoate is transduced by several
kinds of T3REs. Moreover, when sequences containing the
T3RUs were inserted into TKCAT in reverse orientation or
were linked to the minimal promoter of the malic enzyme gene (
147 to
+31 bp) in either orientation and transfected into hepatocytes, T3 stimulation and hexanoate inhibition were conferred
(data not shown). The minimal promoter of the malic enzyme gene did not confer responsiveness to T3 or MCFA. Therefore, the
T3REs that act as cis-acting elements for the effect of
hexanoate can vary greatly in their distance from the start site of
transcription, gene of origin, and orientation with respect to the
direction of transcription.
Identification of the Trans-acting Factor(s) Involved in Mediating
Inhibition by MCFA --
TR is the trans-acting factor common to all
the T3REs that conferred MCFA inhibition in transfected
cells, suggesting that it also may be the trans-acting factor that
mediates the effects of MCFAs. TR acts as a silencer of transcription
in the absence of T3 (33). Mutation or deletion of the
major functional T3RE (T3RE2) from sequences
linked to either a natural or a heterologous promoter causes an
increase in basal activity in transfected cells (10). This increase is
consistent with loss of the silencing action of TR. Nuclear receptors
such as PPAR and chicken ovalbumin upstream promoter transcription
factor inhibit T3-stimulated transactivation by
T3; they also inhibit TR-mediated silencing of basal
activity (20, 34-36). Three constructs, each of which contained the
major functional T3RE of the malic enzyme gene, were tested
for an effect of MCFA on basal activity (Fig.
6). Plasmid [ME
3903/
3703]TKCAT contains the entire T3RU; pMUT1[ME
3903/
3703]TKCAT
contains a block mutation in a nonfunctional direct repeat upstream of
the major T3RE of the upstream T3RU; and
p[ME
3883/
3858]TKCAT contains a single copy of the major
T3RE. When transfected into hepatocytes, all three
constructs conferred robust responses to T3 and strong inhibitory responses to hexanoate (Fig. 6). None of these constructs nor the vector itself conferred responsiveness to hexanoate in the
absence of T3. This result suggests that the factor that
mediates responsiveness to hexanoate does not mediate the silencing
action of unliganded TR.

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Fig. 6.
Effects of T3 and hexanoate on
CAT activity in hepatocytes transfected with constructs containing
wild-type and mutant versions of a T3 response region and
wild-type T3RE2. Chick embryo hepatocytes were
transiently transfected as described in the legend to Fig. 1 and
treated with or without T3 (1.6 µM) and with
or without hexanoate (C6, 1 mM). Left, DNA
constructs used in these experiments. First four
columns, relative CAT activities are expressed as described
in the legend to Fig. 1; each value is the mean ± S.E. of seven
independent experiments using at least two independently prepared
batches of each plasmid. Relative CAT activities were calculated by
setting the CAT activities for T3-treated hepatocytes
transfected with p[ME 3903/ 3703]TKCAT to 1.0 and adjusting all
other activities proportionately. CAT and -galactosidase activities
of extracts from T3-treated hepatocytes transfected with
p[ME 3903/ 3703]TKCAT were 11.1 ± 3.3 percentage of
conversion/15 h/µg of protein and (9.5 ± 3.9) × 10 3 A420 units/min/µg of
protein, respectively. Fifth column, effects of
T3 are expressed as -fold change in CAT activity caused by
T3. Sixth column, the effect of
hexanoate on CAT activity is expressed as CAT activity in cells treated
with T3 plus hexanoate divided by that in cells treated
with T3 alone × 100. The boxed region A is
wild-type T3RE2; x indicates a mutation upstream
of T3RE2 (MUT1; see Fig. 3) that has little or no effect on
T3 responsiveness. No statistically significant differences
were observed between means for samples lacking T3, with or
without hexanoate.
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To transactivate, liganded TR must bind to a T3RE. Previous
work suggested that MCFAs do not inhibit transactivation by disrupting binding of T3 to TR (7), suggesting that TR binding to the T3RE or transactivation may be disrupted by MCFAs. DNase I
footprint and gel mobility shift analyses were used to test the effect
of hexanoate on binding of TR to the T3REs in the upstream
T3RU of the malic enzyme gene. Bacterially expressed (37)
and partially purified (0.3 µg) TR bound to regions of the
T3RU corresponding to T3RE2 and an extension of
T3RE-3 (10). Proteins present in nuclear extracts prepared
from chick embryo hepatocytes in culture treated with T3
and/or hexanoate for 24 h showed the same protection in these two
regions (data not shown). Hexanoate added directly to binding reactions
had no effect on binding of TR or other nuclear proteins, indicating
that these fatty acids did not have a direct effect on binding of TR to
the T3RE. Gel mobility shift analyses indicated that, in
nuclear extracts from cells treated with T3, RXR/TR
heterodimers bound to the major functional T3RE in a manner similar to that of those in nuclear extracts made from hepatocytes treated with T3 plus hexanoate (data not shown). Thus,
hexanoate does not disrupt binding of TR to the T3RE, nor
does it disrupt the interaction between TR and its heterodimerization
partner, RXR.
If inhibition by hexanoate were mediated by a factor that competed with
TR or RXR/TR dimers for binding to the T3RE, inhibition by
hexanoate should be decreased in cells that overexpress TR. We tested
p[ME
3868/
3703]TKCAT and p[ME
3474/+31]CAT for responsiveness to T3 and hexanoate in the absence and presence of
overexpressed TR
(Fig. 7). Cells
transfected with these constructs have higher basal CAT activities than
those transfected with constructs containing an intact
T3RE2 or the entire upstream T3RU.
Overexpression of chicken TR
caused 62- and 100-fold increases in
T3 responsiveness of cells transfected with
p[ME
3868/
3703]TKCAT and p[ME
3474/+31]CAT, respectively.
Hexanoate inhibited the response to T3 to a similar extent
whether or not chicken TR
was overexpressed. As noted earlier,
hexanoate had no effect on CAT activity in the absence of
T3. Thus, the factor that mediates the MCFA response may
not compete with TR for binding to the T3RE.

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Fig. 7.
Effects of T3 and hexanoate on
CAT activity of hepatocytes transfected with constructs containing part
of the T3RU linked to TKCAT or part of the 5'-flanking DNA
containing the downstream T3RU linked to ME 147/+31CAT,
with and without overexpression of chicken TR . Chick embryo
hepatocytes were transiently transfected as described in the legend to
Fig. 1 with or without overexpression of pRSV-cTR (0.2 µg/plate)
and treated with or without T3 and with or without
hexanoate (C6, 1 mM). Left, DNA constructs
used in these experiments. First four
columns, relative CAT activities are expressed as described
in the legend to Fig. 1; each value is the mean ± S.E. of six to
seven independent experiments using at least two independently prepared
batches of each plasmid. Relative CAT activities were calculated by
setting CAT activities for T3-treated hepatocytes
transfected with p[ME 3868/ 3703]TKCAT DNA minus overexpressed TR
equal to 100 and adjusting all other activities proportionately. CAT
and -galactosidase activities of extracts from
T3-treated hepatocytes transfected with
p[ME 3868/ 3703]TKCAT were 4.9 ± 0.8 percentage of
conversion/15 h/µg of protein and (2.0 ± 0.3) × 10 4 A420 units/min/µg of
protein, respectively. Fifth column, effects of
T3 are expressed as -fold change in CAT activity caused by
T3. Sixth column, the effect of
hexanoate on CAT activity is expressed as CAT activity in cells treated
with T3 plus hexanoate divided by that in cells treated
with T3 alone × 100. Statistical significance between
means within a column (p < 0.05) is indicated as
follows: a, versus pTKCAT; b, versus
p[ME 147/+31]CAT.
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Many TR-associated proteins regulate transactivation by liganded TR
(38-40); MCFAs could inhibit transactivation by interacting with such
TR-associated proteins. Some of these proteins also interact with ER,
but do not interact with GR. To gain insight into the specificity of
action by MCFA and to narrow the potential list of factors that might
mediate inhibition by MCFA, we tested the effects of hexanoate on cells
transfected with constructs that bestow responsiveness to estrogen and
glucocorticoids. Single copies of elements that bind these receptors
were linked to TKCAT DNA and transfected into hepatocytes with and
without overexpression of the cognate receptor. Plasmid ERE-TKCAT
conferred 30- and 37-fold responses to
-estradiol in the absence and
presence of overexpressed ER, respectively (Fig.
8). Plasmid GRE-TKCAT conferred 220- and 160-fold responses to corticosterone without and with overexpression of
GR, respectively. Therefore, endogenous forms of both ER and GR were
functional on these cis-acting elements. Hexanoate inhibited function
of the ERE, but not the GRE, with or without overexpression of the
cognate receptors. A hormone-activated transcription factor that does
not belong to the steroid/thyroid hormone receptor superfamily also was
tested. The cis-acting element was a CRE linked to TKCAT DNA. Cells
transfected with the CRE construct showed 10- and 23-fold responses to
CPT-cAMP (a nonmetabolizable analog of cAMP) with and without
overexpressed CRE-binding protein (CREB ), respectively; they did not
respond to hexanoate. These results suggest that inhibition by
hexanoate has specificity with respect to the involved transcription
factor. The actions of TR and ER are inhibited; those of GR and CREB
(or other CRE-binding protein) are not inhibited. This distinction may
be due to the interaction of TR and ER with a factor that mediates the
effect of MCFA; GR and CREB may not interact with that factor.

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Fig. 8.
Effects of estradiol, corticosterone, cyclic
AMP, and hexanoate on CAT activity of hepatocytes transfected with
constructs containing single copies of the estrogen, glucocorticoid,
and cyclic AMP response elements linked to TKCAT, with or without
overexpression of the respective receptor or binding protein.
Chick embryo hepatocytes were transiently transfected as described in
the legend to Fig. 1 with or without overexpression of pHEO (ER), pVARO
(GR), and pRSV-CREB (0.5, 1, and 0.5 µg/plate, respectively).
Transfected cells were treated with or without -estradiol (10 µM), corticosterone (1 µM), and CPT-cAMP
(10 µM), respectively, with or without hexanoate (C6, 1 mM). Left, DNA constructs used in these
experiments. First four columns,
relative CAT activities are expressed as described in the legend to
Fig. 1; each value is the mean ± S.E. of six to seven independent
experiments using at least two independently prepared batches of each
plasmid. Relative CAT activities were calculated by setting CAT
activities for individual ligand-treated hepatocytes transfected with
the respective element plus or minus overexpressed binding protein
equal to 100 and adjusting all other activities proportionately. CAT
activities of extracts from ligand-treated hepatocytes were, in order
of presentation in the figure, 3.2 ± 0.5, 1.7 ± 0.5, 9.8 ± 0.9, 4.3 ± 1.0, 16.1 ± 2.4, and 23.3 ± 1.7 percentage of conversion/15 h/µg of protein. -Galactosidase
activities of extracts from ligand-treated hepatocytes were, in order
of presentation in the figure, (7.9 ± 1.2), (13.5 ± 2.3),
(3.8 ± 1.1), (6.4 ± 1.3), (8.6 ± 1.1), and (2.1 ± 0.6) × 10 3 A420 units/min/µg
of protein. Fifth column, effects of ligand are
expressed as -fold change in CAT activity caused by ligand.
Sixth column, the effect of hexanoate on CAT
activity is expressed as CAT activity in cells treated with ligand plus
hexanoate divided by that in cells treated with ligand alone × 100. Constructs containing single copies of the estrogen,
glucocorticoid, and cAMP response elements were obtained as described
under "Experimental Procedures." Statistical significance between
means within a row is indicated as follows: a,
versus control without hexanoate (p < 0.05).
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Members of the family of proteins that interact with TR and ER, but not
GR, bind to the ligand-binding domains of these receptors (40). These
proteins were identified in yeast two-hybrid screens using the
ligand-binding domain (with no N-terminal or DNA-binding domains) of TR
or ER as "bait." The ligand-binding domain of TR, fused to the
DNA-binding domain of GAL4, was transfected into hepatocytes with a
plasmid that encodes GAL4-binding sites (Fig. 9). In this model,
T3-stimulated CAT activity is due to binding of
T3 to the hybrid GAL4-TR and not to endogenous TR (Fig. 9). We used p[ME
3903/
3703]TKCAT as a positive control; it conferred both T3 and hexanoate responsiveness. In cells
cotransfected with the negative control, pGAL4 (pSG424), and GAL4-CAT,
CAT activity was unaffected by T3 or hexanoate.
Cotransfection of pGAL4-cTR
, the ligand-binding domain of chicken
TR
fused to pGAL4, with pGAL4-CAT resulted in a robust response to
T3, indicating that the truncated TR was capable of binding
T3 and transactivating the linked promoter. Cells
transfected with the GAL4-TR fusion did not respond to hexanoate. These
results suggest that the N-terminal sequence of TR is required for
inhibition by hexanoate. They also confirm that binding of
T3 to TR is not disrupted by hexanoate.

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Fig. 9.
Effects of T3 and hexanoate on
CAT activity in hepatocytes transfected with chimeric GAL4-cTR and a
construct containing a GAL4-binding site. Chimeric GAL4-cTR and
the construct containing the GAL4-binding site were obtained as
described under "Experimental Procedures." Chick embryo hepatocytes
were transiently transfected as described in the legend to Fig. 1 and
treated with or without T3 (1.6 µM) and with
or without hexanoate (C6, 1 mM). pGAL4-DBD (pSG424) and
pGAL4-DBD/TR-LBD were cotransfected (0.2 µg each per plate) with the
pGAL4-CAT (pMC110) reporter construct. Left, DNA
constructs used in these experiments. First four
columns, relative CAT activities are expressed as described
in the legend to Fig. 1; each value is the mean ± S.E. of six
independent experiments using at least two independently prepared
batches of each plasmid. Relative CAT activities were calculated by
setting CAT activities for T3-treated hepatocytes
transfected with p[ME 3903/ 3703]TKCAT (T3RUTKCAT) to
100 and adjusting all other activities proportionately. CAT and
-galactosidase activities of extracts from T3-treated
hepatocytes transfected with p[ME 3903/ 3703]TKCAT were 22.7 ± 2.3 percentage of conversion/15 h/µg of protein and (6.5 ± 1.3) × 10 3 A420 units/min/µg of
protein, respectively. Fifth column, the effect
of hexanoate on relative CAT activity is expressed as relative CAT
activity in cells treated with T3 plus hexanoate divided by
that in cells treated with T3 alone × 100.
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DISCUSSION |
The cis-acting elements in the 5'-flanking DNA of the chicken
malic enzyme gene that confer inhibition by MCFAs co-localized with the
T3REs in this DNA. Furthermore, artificial palindromic and
direct repeat T3REs also conferred inhibition by MCFA.
MCFAs also inhibited transactivation from an ERE, but had no effect on
basal activity in the absence of T3 or estradiol. Thus, the trans-acting factor that binds to T3REs or EREs and
participates in the inhibitory effect of MCFA is TR or ER,
respectively. The MCFA-regulated factor did not compete with TR for
binding to the T3RE, but probably interacted with TR to
influence its ability to transactivate linked promoters. Sequences
N-terminal to the ligand-binding domain of TR are required for the
action of MCFA, presumably because they interact with a MCFA-regulated
factor. T3REs are found in many nutritionally regulated
genes; T3 stimulates and MCFAs inhibit transcription of
both malic enzyme and fatty acid synthase (7). Modulation of the
transactivation function of TR bound to a T3RE may
represent an effective mechanism for modulation of
T3-stimulated transcription of lipogenic genes. MCFAs
themselves are unlikely to be physiological regulators of the lipogenic
genes in chicks because there is no evidence that plasma concentrations
of MCFA in chicks ever reach the required levels. Nevertheless, the
specificity of the response with respect to chemical structure, its
selectivity with respect to the genes affected, and the rapid onset and
reversal of its effects (Ref. 9 and data not shown) are consistent with
physiological importance. MCFAs or a compound derived therefrom may be
similar in structure to the true inhibitor, the production of which may
signal the starved state.
Modulation of the transactivation function of TR without displacement
of T3 from TR or of TR from the T3RE has not
been described previously as a mechanism by which fatty acids regulate
transcription. Long-chain fatty acids, specifically polyunsaturated
fatty acids, regulate transcription of some eucaryotic genes by binding
to or modifying regulatory proteins, which, in turn, bind to unique DNA
elements in the 5'-flanking regions of these genes (17-20). Hydroxy
fatty acids also regulate transcription through a unique cis-acting
element (14-16). By contrast, our results suggest that MCFAs differ
from these other fatty acids in that their inhibitory actions are
transduced through the T3RE·TR·T3 complex,
rather than by a complex unique to MCFAs.
Proximal promoter elements may play important roles in
T3-regulated expression of some genes (41). This is
unlikely to confound the interpretation of our results. First,
T3REs linked to both TK and minimal malic enzyme promoters
are inhibited by hexanoate; the two promoters have quite different
structures (10). Second, both estrogen- and T3-stimulated
transcription is inhibited by MCFA. Third, MCFAs have no effect on
promoter activity in the basal state. Fourth, if hexanoate did regulate
activity of a factor bound at a site common to both TK and malic enzyme
promoters and that factor mediated both estrogen- and
T3-regulated gene transcription, then one would expect
fractional inhibition by hexanoate to decrease as T3
responsiveness decreased; it did not.
The superfamily of steroid/thyroid hormone receptors is subdivided into
two major classes. Class I includes GR and ER; these receptors are
cytosolic until bound by ligand. Class II includes TR, RXR, and PPAR,
receptors that are nuclear in the absence or presence of ligand (42).
The interaction of estrogen with ER triggers the translocation of the
complex to the nucleus, where estrogen-bound ER binds to an ERE present
in the DNA (43). Because both TR and ER are affected by MCFA, it seems
unlikely that MCFA acts cytosolically to disrupt the interaction of
estrogen and ER, preventing ER from reaching the nucleus. The
mechanisms by which MCFAs inhibit estrogen- and T3-induced
transcription are probably the same.
Inhibition of the action of ER by MCFA is consistent with the
hypothesis that RXR and PPAR, a known heterodimerization partner for TR
at T3REs of the malic enzyme gene and a potential partner for TR, respectively, are not targets for MCFA because ER dimerizes with itself and not RXR or PPAR (42, 43). Overexpression of TR should
have increased the number of TR/TR dimers bound to T3REs. Overexpression of TR, however, failed to alter inhibition by MCFA, additional evidence that potential heterodimerization partners such as
RXR and PPAR did not mediate the inhibitory action of MCFA.
Furthermore, fatty acids with carbon chain lengths shorter than 10 are
poor activators of PPAR (44). The ligand-binding domains of both ER and
TR interact with TR-interacting proteins (TRIPs); TRIPs do not interact
with GR (41). The specificity of the inhibitory action of MCFAs for ER
or TR, but not GR, suggested that one or more of these TRIPs might be
the target for MCFA; modification or displacement of a TRIP could
decrease transactivation by ER or TR. TRIPs, however, bind to the
ligand-binding domain of TR or ER, and this part of TR was not
sufficient to confer inhibition by MCFA even though it was sufficient
for T3 responsiveness. TRIPs thus seem unlikely to be the
MCFA-regulated factor that interacts with TR or ER.
T3 responsiveness of hepatocytes transfected with
constructs containing all or parts of the T3RU, mutant
versions of T3REs, or artificial T3REs varied
from <5-fold to >1500-fold. These differences in responsiveness are
due to novel proteins that bind to the different T3REs and
differentially influence responsiveness (9), differences in binding of
RXR/TR to T3REs of different sequences (42), and artificial
overexpression of TR. Despite the resulting broad range of promoter
activities, MCFA always resulted in inhibitions of 50-80%. Within
individual sets of experiments, the range for inhibition was even
narrower, despite a wide range in T3 responsiveness
(e.g. Fig. 2). Thus, MCFAs may modify function of a factor
that has the same relative regulatory effect on RXR/TR heterodimers
bound at any T3RE, whether the intrinsic ability of that
T3RE to transactivate a linked promoter is large or
small.
MCFA action appears to require that part of TR that is N-terminal to
the ligand-binding domain because the N-terminal (A/B) and DNA-binding
domains were absent from the GAL4-TR chimera that responded to
T3, but not MCFA. MCFA did not interfere with the T3RE-TR or RXR-TR interactions, suggesting that the
DNA-binding domain and the N- and C-terminal regions required for
heterodimerization were not targets for interaction with the factor
that mediates the effect of MCFA. This leaves the N-terminal A/B domain
of TR as a putative interaction site for the factor that mediates
action of MCFA. A number of TR accessory factors have been identified and are required for full transactivation of the T3RE-bound
receptors with which they interact. The TR accessory factors that have
been characterized interact with activation function domains in the ligand-binding region of TR and other members of the superfamily of
steroid/thyroid hormone receptors (45-50). Because the MCFA effect
does not appear to be mediated by this region of TR, the known TR
accessory factors are not promising candidates for factors regulated by
MCFA. Analogous factors may interact with the N terminus, and MCFAs may
target one or more of these factors, thus eliciting its inhibitory
response.
We are indebted to Drs. Richard Maurer
(pCMV-
GAL), Ronald Evans (pTREpal-TKCAT), Bert W. O'Malley
(pERE-TKCAT), Geoffrey L. Greene (pHEO), F. Bradley Hillgartner
(pRSV-CREB), p[ME
3474/+31]CAT), Marc Montminy, Mark Ptashne (pSG424
and pMC110), Herbert H. Samuels (pET8cTR
and pGAL4-cTR
), Keith R. Yamamoto (p
GTCO and pVARO), Ganes Sen (pCRE-TKCAT), and Pierre
Chambon (monoclonal antibody raised against chicken RXR) for the
indicated plasmids and antibody. We thank Drs. Catherine Mounier and
Thomas Carlisle for helpful discussions.