From the
Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme
A (HMG-CoA) reductase gene transcription in FRTL-5 rat thyroid cells,
and the effect of TSH can be mimicked by cAMP.
Sequence analysis of
the rat reductase promoter has revealed a hitherto unnoticed
cAMP-responsive element (CRE)-like octamer. This octamer is located
between 53 and 60 nucleotides downstream of the sterol regulatory
element 1; its first 6 nucleotides are identical to the consensus
somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase
CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear
extracts from FRTL-5 cells, which could be prevented by unlabeled
CRE-containing oligonucleotides whose flanking sequences were otherwise
nonidentical. The complexes were specifically supershifted by anti-CREB
antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the
bacterial chloramphenicol acetyl transferase (CAT) under the control of
the HMG-CoA reductase promoter displayed CAT activity, which was
specifically stimulated by TSH. In contrast, CAT activity in FRTL-5
cells transfected with similar constructs carrying mutations in the
reductase CRE was significantly lower and did not increase after TSH
challenge.
We suggest that the HMG-CoA reductase gene contains a
functional CRE, important for TSH regulation of transcription. The data
presented provide the molecular basis for a novel regulatory mechanism
for HMG-CoA reductase gene expression in rat thyroid cells, which
involves the direct effect of cAMP.
In mammalian cells, 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA)
So far it has been firmly believed
that, at the transcriptional level, HMG-CoA reductase is negatively
controlled by end-product (cholesterol) since the enzyme is expressed
at a relatively high rate when cells are starved for
mevalonate(5, 6, 7, 8, 9, 10, 11, 12) .
However, we have reported that in FRTL-5 rat thyroid cells, HMG-CoA
reductase is transcriptionally induced by thyrotropin (TSH), via
cAMP(4) . TSH is the physiologic mitogen in FRTL-5
cells(13, 14) . When starved of TSH, these cells become
quiescent and develop a greater than 2-fold increase in total
cholesterol, caused by the doubling of available low density
lipoprotein receptors(15) . TSH challenge increases HMG-CoA
reductase gene expression, reduces low density lipoprotein receptor
activity, decreases total cholesterol, and increases cholesterol
biosynthesis(4, 15) . The TSH-induced increase in
HMG-CoA reductase and cholesterol biosynthesis precedes the TSH-induced
increase in thymidine incorporation into DNA and cell
doubling(4) . The apparent paradox of a lower cholesterol
content in an actively dividing cell population has been explained as
follows. In order for FRTL-5 cells to proceed through the cell cycle,
active isoprenoid synthesis is a mandatory requirement; the TSH-induced
reduction in cholesterol content would maintain a high rate of HMG-CoA
reductase synthesis(4, 15) .
We have cloned and
sequenced the promoter region of the rat gene in an effort to elucidate
the mechanism by which TSH/cAMP act. Analysis of the 5`-flanking region
of the HMG-CoA reductase gene revealed the octamer, 5`-TGACGTAG-3`,
which is closely homologous to a consensus cAMP-responsive element
(CRE)(16) . In this study, we investigate the role of this
CRE-like sequence and show that it is involved in both basal and
TSH-induced HMG-CoA reductase gene expression in thyroid cells. We also
show that the CRE-binding protein, CREB(17) , modulates the
reductase CRE.
The data presented strongly support a direct cAMP
regulation of HMG-CoA reductase gene transcription in thyroid cells,
involving cis-acting CRE.
We performed competition experiments using several
synthetic oligonucleotides (Fig. 3A), which included
wild-type or mutated CRE sequences. When we used the oligonucleotide
containing the reductase CRE (self) as competitor, the retarded band
was significantly reduced by a 50-fold excess, and abolished by a
500-fold excess of the unlabeled probe (Fig. 3B, lanes3-5, and Fig. 3C, lanes2-4). Oligonucleotides containing the
major histocompatibility complex (MHC) Class I CRE (Fig. 3B, lanes6-8) and
oligonucleotides containing either somatostatin CRE (Fig. 3C, lanes5-7) or TSH
receptor CRE (Fig. 3C, lanes8-10) were as effective as the homologous RED CRE
in preventing complex formation, whereas oligonucleotides with the CRE
octamer deleted (
In a previous report (4) we showed that TSH, a
specific mitogen and physiologic regulator for thyroid cells, induces
HMG-CoA reductase and cholesterol synthesis in FRTL-5 rat thyroid
cells. In these cells, TSH induction of reductase gene transcription
precedes the increase of DNA synthesis and cell proliferation and is
mediated by cAMP(4) . In the current study, we have investigated
the molecular basis for cAMP regulation of HMG-CoA reductase gene
expression, and show that an 8-nucleotide sequence within the reductase
promoter is structurally and functionally homologous to a consensus
CRE(16) .
The rat reductase promoter is nearly 90% homologous
with the hamster promoter between nucleotides -283 and +14
(see Fig. 1) and identical in long stretches of nucleotides, most
notably around the major transcription start site and the sequence
known as footprint 4, which is required for reductase gene
transcription(6) . This particular region includes the CRE-like
octamer 5`-TGACGTAG-3`. We have focused on this sequence as a potential
target for cAMP regulation and provide evidences indicating that it is
indeed a functional CRE involved in TSH regulation of reductase gene
expression. These can be summarized as follows.
Oligonucleotides
containing the CRE-like sequence and nuclear extracts from FRTL-5 cells
formed specific protein-DNA complexes that reacted with antibodies to
CREB but failed to react with antibodies to ATF-2 or preimmune
serum(16, 17, 30, 31) .
TSH or
forskolin could stimulate CAT activity in FRTL-5 cells in which the
reporter gene was under the control of the reductase promoter.
Stimulation was significant also under conditions of
cholesterol/mevalonate down-regulation. In FRTL-5 cells transfected
with a construct in which the reductase CRE was mutated to
nonpalindromic sequence but was otherwise identical, TSH or forskolin
failed to affect the activity of the reporter gene.
Thus, in FRTL-5
cells, the HMG-CoA reductase gene is positively regulated by cAMP
through modulation of a cis-acting element on its promoter, a process
that has not been reported so far. The role of cAMP regulation of the
reductase gene expression has never been addressed in detail. We have
previously reported that in rat thyroid cells, cAMP has a key role in
regulating sterol metabolism by down-regulating LDL receptor activity
and inducing HMG-CoA reductase gene transcription. Studies on different
cell lines should tell us whether this is a general phenomenon rather
than a unique feature of thyroid epithelial cells; we suggest, at this
point, that it may well be applicable to all cells where the cAMP
signal plays a role in the growth process. The identification of a
CRE-like sequence as a functional CRE in the reductase promoter
indicates direct transcriptional activation through cAMP signaling;
however, we are still unable to resolve quantitatively the contribution
of cAMP-mediated cholesterol depletion(4, 15) , since
CAT assays on cells transfected with chimeric constructs of the
reporter gene and the reductase promoter are affected by cholesterol
and mevalonate incubation under conditions of high intracellular cAMP
(see Fig. 5).
In summary, this study has identified and
characterized a CRE in the HMG-CoA reductase promoter and in FRTL-5
cells and has demonstrated that, at least in this particular cell line,
the gene can be positively regulated by cAMP. The relevance of this
regulatory mechanism, which has been ignored so far, is still a matter
of investigation.
We thank M. Berardone, F. D'Agnello, and F.
Moscato for the art work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)reductase (mevalonate:NADP
oxidoreductase CoA-acylating, EC 1.1.1.34) is the rate-limiting
enzyme for the synthesis of mevalonic acid, the precursor common to
many isoprenoids, including cholesterol (1). Because of its involvement
in cholesterol biosynthesis, HMG-CoA reductase has become the target of
many drugs aimed at lowering threatening levels of serum
cholesterol(2) . An additional interest in this enzyme has
mounted in recent years, since mevalonate is also the precursor of the
donor isoprenoids, farnesyl and geranyl-geranyl diphosphate, for a
growing class of biologically relevant proteins, which include
monomeric G proteins, most notably the product of the ras gene, p21, a key transducer of mitogenic signals(3) . As
the rate-limiting enzyme for synthesis of mevalonate, the precursor of
isoprenes, HMG-CoA reductase is, therefore, a critical regulator of
cell proliferation in normal as well as in tumor
cells(3, 4) .
Materials
TSH was a highly purified preparation
from bovine pituitary extracts(18) . Insulin, cristalline bovine
serum albumin, phenylmethylsulfonyl fluoride, leupeptin, pepstatin A,
and other reagents were obtained from Sigma. Trypticase peptone and
yeast extract were from Beckton Dickinson. M13-mp18 DNA was purchased
from Life Technologies, Inc. Restriction enzymes, T4 DNA ligase, DNA
polymerase I (Klenow fragment), T4 polynucleotide kinase, and Random
primed DNA labeling kits were obtained from Boehringer Mannheim.
[P]dATP (3,000 Ci/mmol),
[
P]dGTP (3,000 Ci/mmol),
[
P]dATP (6,000 Ci/mmol), and nylon filters
were from Amity. Sephadex nick columns and DEAE-dextran were obtained
from Pharmacia Biotech Inc. BA-85 nitrocellulose filters were purchased
from Schleicher & Schuell. For autoradiography, Kodak XAR-5 films
have been used with DuPont Cronex intensifying screens (Du Pont). All
plasmids have been purified by Qiagen plasmid kits purchased from
Qiagen Inc. Synthetic oligonucleotides were purchased from Operon Inc.
For sequencing, the Sequenase kit``from U. S. Biochemical Corp.
has been used. Plasmid pRED-227 was supplied by the American Type
Culture Collection, Rockville, MD. The pCAT-promoter plasmid was
purchased from Promega Corp. Antiserum to CREB or activating
transcription factor-2 (ATF-2), each with its respective control
preimmune counterpart, were kindly provided by Dr. J. P. Hoeffler,
University of Colorado Health Center, Denver, CO. pEMBL-8-CAT vector
was kindly provided by Dr. R. Cortese, EMBL, Heidelberg, Germany.
Luciferase expression vector (pRSV-Luc) was kindly provided by Dr. S.
Subramani, Stanford University, Stanford, CA.
Cells
FRTL-5 cells (ATCC CRL 8305) are a strain of
rat thyroid cells whose characteristics and culture conditions have
been extensively described(13, 14, 19) . They
were grown at 37 °C in a humidified atmosphere composed of 95% air
and 5% CO, in Coon's modified F-12 medium
supplemented with 5% calf serum and a six-hormone mixture of TSH,
insulin, hydrocortisone, transferrin, somatostatin, and
glycil-L-histidyl-L-lysine acetate. The medium
containing the complete mixture will be referred to as 6H. Depending on
experimental needs, a similar mixture, but lacking TSH (referred to as
5H), was used. Unless otherwise stated, cells were provided fresh
medium every 3 days.
Screening of a FRTL-5 Genomic Library and Cloning the Rat
HMG-CoA Reductase Promoter
A FRTL-5 genomic library was screened
with the XhoI-XbaI fragment (referred to as probe 5`)
and the BstEII-XbaI fragment (referred to as probe
3`) of plasmid pRED-227 containing the full-length hamster HMG-CoA
reductase cDNA(20) . The screening was accomplished using in
situ plaque hybridization according to Maniatis(21) . For
labeling of cDNA probes, random primed DNA labeling kits were used. The
clone that hybridized only with probe 5` was further characterized.
Digestion of this clone with BamHI-EcoRI gave rise to
three fragments that were transferred to nylon filters. The fragment
that still hybridized with probe 5`, approximately 4.5 kilobase pairs,
was further analyzed by restriction enzymes. This analysis revealed the
presence of five PstI sites. The one positive PstI
fragment was subcloned into M13-mp18 bacteriophage or pCAT plasmid for
sequencing.
Restriction Enzyme Digestions, Nucleic Acid
Hybridizations, and DNA Sequencing
Digestions of DNA with
restriction endonucleases, electrophoresis on agarose gels, transfer of
DNA fragments from gels to nylon filters, and hybridizations were
performed using standard procedures(21) . DNA restriction
fragments, cloned into M13-mp18 bacteriophage or pCAT-promoter plasmid,
were sequenced by the method of Sanger(22) .
Nuclear Extracts
Nuclear extracts from quiescent
or TSH-challenged FRTL-5 cells were prepared as described
previously(23, 24) . Cells were washed with
Dulbecco's modified phosphate-buffered saline without
Mg and Ca
at pH 7.4, harvested by
scraping, and, after centrifugation at 500
g,
suspended in 5-pellet volumes of 0.3 M sucrose and 2% Tween 40
in buffer A (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5
mM MgCl
, 0.1 mM EGTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2
µg/ml leupeptin, and 2 µg/ml pepstatin A). After freezing in
liquid nitrogen, the cells were thawed and gently homogenized using
hand homogenizer pestel B; the suspension was layered onto 1.5 M sucrose in buffer A and centrifuged at 25,000
g in a swinging bucket rotor. Nuclei were washed with 0.3 M sucrose in buffer A, and nuclear proteins were extracted with 2.5
volumes of buffer B (10 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl
, 0.1 mM EGTA, 10%
glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2
µg/ml pepstatin A). Extracts were centrifuged at 100,000
g for 1 h; the supernatant was dialyzed for 4 h at 4 °C
against buffer C (20 mM HEPES-KOH, 100 mM KCl, 0.1
mM EDTA, and 30% glycerol), and precipitated materials were
discarded by centrifigation at 25,000
g for 15 min
prior to use in electrophoretic mobility shift assays.
Electrophoretic Mobility Shift
Assays
Electrophoretic mobility shift assays were performed as
described previously(25) . The 18-base pair (bp) RED CRE
synthetic double-stranded oligonucleotide was labeled with
[-
P]ATP and T4 polynucleotide kinase and
then purified on an 8% native polyacrylamide gel. The nuclear extract
(3 µg) was incubated for 20 min on ice in a 20-µl reaction
volume containing 10 mM Tris-HCl, pH 7.6, 50 mM KCl,
5 mM MgCl
, 1 mM dithiothreitol, 1 mM EDTA, 12.5% glycerol, 0.1% Triton X-100, and 0.5 µg of
poly(dI-dC). Approximately 1.5 fmol of
P-labeled probe
(approximately 50,000 cpm) were added, and the incubation was continued
for an additional 20 min at room temperature. Where indicated,
unlabeled double-stranded oligonucleotides were also added to the
binding reaction as competitors and incubated with the extract for 20
min, prior the addition of labeled DNA. Protein-DNA complexes were
subjected to electrophoresis on 5% native polyacrylamide gels at 160 V
in 1
Tris borate/EDTA buffer at 4 °C. In experiments using
antiserum to CREB or ATF-2, nuclear extracts were incubated with the
antiserum or its control counterpart in the same buffer for 20 min on
ice before adding the labeled probe and processing as above. Gels were
dried and autoradiographed.
Promoter CAT Chimeric Plasmids
The 765-nucleotide PstI fragment (from -323 to +442, see Fig. 1) (10) spanning the entire reductase promoter was cloned into the
pEMBL-8-CAT vector (26) upstream to the bacterial gene for CAT.
Mutations of the HMG-CoA reductase promoter sequence were created by
two-step, recombinant polymerase chain reaction methods(27) . In
the first step, two polymerase chain reaction products were generated
using the 5` or 3` end of the final products and overlapping sequence
containing the same mutation. The second step was performed using the
overlapping polymerase chain reaction products as templates, and DNA
sequences of the 5` or 3` end of the final products as primers.
Amplified fragments were ligated to pCAT-promoter plasmid and sequenced
to verify the predicted sequence. Similar to the wild-type HMG-CoA
reductase promoter, the CRE-mutated reductase fragment (765 bp) was
cloned into the pEMBL-8-CAT vector. pRSV-Luc was used as positive
control to evaluate transfection efficiency(28) .
Figure 1:
Sequence of the rat HMG-CoA reductase
promoter. The sterol regulatory element 1 site is underlined,
the CRE octamer is boxed. The asterisk denotes the
major transcription start site, as previously reported in the hamster
reductase promoter (10). The entire fragment will be cloned 5` to the
CAT gene in pEMBL-8-CAT plasmid to be used in transfection assays (see
below).
Transient Expression Analysis
FRTL-5 cells were
grown to 50-60% confluence and then shifted to 5H medium (without
TSH) for 3-5 days. Transfection used a DEAE-dextran
protocol(21) , with minor modifications. Four hours before
transfection, the 5H medium was changed. Then, medium was removed, and
cells were washed once with phosphate-buffered saline and incubated at
37 °C with 0.25 ml of DEAE-dextran (2 mg/ml) containing 20 µg
of pEMBL-8-CAT under the control of wild-type or mutated HMG-CoA
reductase promoter, and 2 µg of pRSV-Luc/dish was added to 4.75 ml
of 5H medium containing no calf serum. After a 1-h incubation, medium
was aspirated, and the cells were treated for 3 min with 2.5 ml of 10%
dimethyl sulfoxide in phosphate-buffered saline, washed once with
phosphate-buffered saline, and then incubated in 5H medium. After 24 h,
medium was changed, and cells were exposed to either 5H medium
(control) or 6H medium (TSH challenge), both in the presence or in the
absence of 25-hydroxycholesterol (25 µM) and mevalonate
(0.8 mM). After 16-18 h, cells were harvested. Where
indicated, forskolin (10 µM) was added to the cells during
the last 6 h of incubation. CAT assays were performed as described
previously(29) . Luciferase activity was measured using a
luminometer.
Other Assays
Protein concentration was determined
using a Bio-Rad kit; recrystallized bovine serum albumin was the
standard.
The Rat HMG-CoA Reductase Promoter
A PstI fragment of 765 bp, a candidate to contain the complete
rat reductase promoter, was sequenced. The fragment spans 323
nucleotides upstream and 442 nucleotides downstream of the major
transcription start site, which is identical to the hamster promoter (Fig. 1). Comparison with the hamster sequence revealed nearly
90% homology in the region -283 to +14 (data not shown).
Long stretches of identity included the sterol regulatory element 1 and
the major transcription start site. Ninety-five nucleotides upstream of
the major transcription start site we noticed an 8-nucleotide sequence
closely homologous to a CRE. The first 6 nucleotides of this octamer
are identical to a consensus CRE (Fig. 2). We questioned whether
this element, if a functionally active CRE, could be involved in the
direct cAMP regulation of HMG-CoA reductase gene transcription.
Figure 2:
The
ability of a radiolabeled oligonucleotide containing the HMG-CoA
reductase CRE-like box (RED CRE) to form protein-DNA complexes
with nuclear extracts from FRTL-5 cells, not exposed (5H) or
exposed (6H) to TSH. The sequences of the oligonucleotide
probe and, for comparison, the consensus (somatostatin) CRE are
reported. The two nucleotides, which make the reductase and
somatostatin CRE motif different, are underlined.
Characterization of the HMG-CoA Reductase CRE-like
Sequence
We synthesized an 18-bp double-stranded oligonucleotide
that includes the octamer identified as a potential CRE in the HMG-CoA
reductase promoter (RED CRE). The CRE octamer was flanked 5` by 6 bp
and 3` by 4 bp (Fig. 2). The oligonucleotide was end-labeled and
used as a probe to detect protein-DNA complexes with FRTL-5 cell
nuclear extracts. When reacted with extracts from either quiescent (5H)
or chronically TSH-stimulated (6H) FRTL-5 cells, protein-DNA complexes
were formed that gave rise to a broad retarded band in both cases (Fig. 2).
CRE) (Fig. 3B, lanes9-11) or mutated to a nonpalindromic sequence (NPCRE) (Fig. 3Clanes12-14) did not behave as competitors, even at a
500-fold excess concentration.
Figure 3:
The ability of unlabeled oligonucleotides
containing the CRE motif (sequences reported in panel A) to
prevent the formation of protein-DNA complexes with a radiolabeled
oligonucleotide containing the HMG-CoA reductase CRE-like box (RED
CRE). Equal amounts of the unlabeled oligonucleotides containing
RED CRE (Self) (panelB, lanes3-5 and panelC, lanes2-4), somatostatin CRE (panelC, lanes5-7), TSH receptor CRE (panelC, lanes8-10), and MHC class I
CRE (panelB, lanes6-8); and
of oligonucleotides harboring a deletion (CRE) (panelB, lanes9-11) or a
nonpalindromic (NPCRE) MHC Class I CRE (panelB, lanes12-14) or the sequence
5` to the MHC Class I CRE (panelB, lane15 and panelC, lanes11-13) were incubated with FRTL-5 nuclear extracts
and the radiolabeled RED CRE probe and analyzed by electromobility
shift assays, as detailed under ``Experimental
Procedures.''
The addition of an antibody to CREB,
to the incubation mixture containing the end-labeled synthetic RED CRE
and FRTL-5 nuclear extracts resulted in a supershift of the protein-DNA
complex (Fig. 4, lane3). In contrast, antisera
to the trans-ATF-2 did not cause supershift (Fig. 4, lane4), nor did the preimmune serum (Fig. 4, lane5). The specificity of the reaction was further
indicated by the ability of a 500-fold excess of the unlabeled RED CRE
to eliminate the supershifted complex (Fig. 4, lane6) but not of an unlabeled oligonucleotide containing
identical flanking regions and a mutated CRE resembling the MHC Class I
nonpalindromic CRE (data not shown).
Figure 4:
The effect of anti-CREB or anti-ATF-2
antibodies or preimmune serum on the migration of protein-DNA complexes
formed by nuclear extracts from TSH-exposed (6H) FRTL-5 cells with the
RED CRE radiolabeled probe. Samples in individual lanes are as follows:
no extract (lane1); 6H extract (lane2); 6H extract plus antibody to CREB (lane3); 6H extract plus anti-ATF-2 antibody (lane4); 6H extract plus preimmune serum (lane5); 6H extract plus antibody to CREB in the presence of a
500-fold excess of cold RED CRE (lane6). The
migration of the protein-DNA complex was only affected by the anti-CREB
antibody (lane3).
HMG-CoA Reductase CRE Is Involved in cAMP-mediated TSH
Induction of HMG-CoA Reductase Transcription
To address the
question as to whether the stimulatory effect of TSH on HMG-CoA
reductase gene expression involves CRE transactivation, we transfected
FRTL-5 cells with a chimeric plasmid containing the coding sequence of
the bacterial enzyme CAT linked to the rat PstI fragment (765
bp, between nucleotides -323 and +442, see Fig. 1),
which comprised the HMG-CoA reductase promoter. Assays for CAT activity
were performed in cells maintained in the absence or in the presence of
TSH (1 10
M) for 16-18 h, i.e. the time required for the maximal hormone-induced HMG-CoA
reductase gene transcription, or treated with forskolin (10
µM) for 6 h. Separate sets of cells were also exposed to
25-hydroxycholesterol (25 µM) and mevalonate (0.8
mM). Fig. 5shows that TSH exposure increased CAT
activity and that forskolin (10 µM) mimicked the TSH
effect; it also shows that the effects of TSH and forskolin were
decreased, but not abated, when cells were exposed to
25-hydroxycholesterol and mevalonate. These results indicate that the
rat HMG-CoA reductase promoter-CAT construct is sensitive to sterol
feedback regulation and that the TSH/cAMP effect is measurable
independent of sterol regulatory element-mediated repression.
Figure 5:
CAT activity in FRTL-5 cells
co-transfected with 20 µg of a chimeric plasmid containing the CAT
gene under the control of the wild-type rat HMG-CoA reductase promoter (i.e. the 765-bp fragment shown in Fig. 1) and 2 µg of the
pRSV-Luc expression vector. Transfection was performed as described
under ``Experimental Procedures.'' CAT activity in FRTL-5
cells exposed to 5H medium (without TSH) is reported as Control, and given the arbitrary value of 1 after evaluation
of transfection efficiency with pRSV-Luc used as positive control.
Other bars are as follows: TSH and CM/TSH,
FRTL-5 cells challenged with TSH (1 10
M) for 18 h, in the absence (TSH) or in the
presence (CM/TSH) of 25-hydroxycholesterol (25
µM) and mevalonate (0.8 mM); FSK and CM/FSK, FRTL-5 cells challenged with forskolin (10
µM) for 6 h, in the absence (FSK) or in the
presence (CM/FSK) of 25-hydroxycholesterol (25
µM) and mevalonate (0.8 mM); CM, FRTL-5
cells in 5H medium, exposed to 25-hydroxycholesterol (25
µM) and mevalonate (0.8 mM) for 18 h. The errorbars reflect the standard error of three
separate assays.
In
order to prove that the reductase CRE octamer was involved in
cAMP-mediated TSH or forskolin effects, experiments were performed
using the same PstI fragment of the reductase promoter linked
5` to the CAT gene but carrying the CRE octamer mutated from
5`-TGACGTAG-3` to the nonpalindromic sequence, 5`-CGACACGA-3` (Fig. 6). TSH or forskolin failed to increase CAT activity in
FRTL-5 cells transfected with the CAT gene under the control of the
mutated HMG-CoA reductase promoter. It should be noted that in cells
maintained in the absence of TSH (control), CAT activity was still
measurable ( Fig. 5and Fig. 6); this activity is likely to
be sustained by residual cAMP in TSH-starved cells.
Figure 6:
CAT activity in FRTL-5 cells
co-transfected with 20 µg of chimeric plasmids carrying the CAT
gene under the control of either wild-type (Wild Type CRE) or
mutated (Non Palindromic CRE) HMG-CoA reductase promoter (the
765-bp fragment shown in Fig. 1) and 2 µg of pRSV-Luc. Mutation of
the reductase CRE was performed as detailed under ``Experimental
Procedures.'' CAT activity in FRTL-5 cells transfected with
wild-type reductase promoter, exposed to 5H medium (without TSH) is
reported as Control and given the arbitrary value of 1. Other
bars are as follows: TSH, FRTL-5 cells exposed to TSH (1
10
M) for 18 h; FSK,
FRTL-5 cells exposed to forskolin (10 µM) for 6 h. The errorbars reflect the standard error of three
separate assays. Normalization has been accomplished using pRSV-Luc as
positive control to evaluate transfection
efficiency.
These findings
indicate that the CRE plays a functional role in the TSH/cAMP
regulation of HMG-CoA reductase transcription in FRTL-5 cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.