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J. Biol. Chem., Vol. 278, Issue 36, 34114-34118, September 5, 2003
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From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900
Received for publication, May 23, 2003 , and in revised form, June 25, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the lack of defined TR sites in the LDL receptor promoter, there are well defined functional sites through which cholesterol regulates gene expression (6). The key site binds the sterol regulatory element-binding proteins (SREBPs) (7). There are three major SREBPs that are encoded by two genes (8). SREBP-1a and -1c are from overlapping mRNAs encoded by one gene and the single SREBP-2 is encoded by a separate gene. All three are synthesized and first inserted into membranes of the endoplasmic reticulum and nuclear envelope where they cannot directly influence gene expression (9). When the sterol level of a cell falls, a multistep maturation process is initiated by a sterol-dependent alteration in membrane trafficking that culminates with the proteolytic release of SREBPs from the membrane (10). The soluble SREBPs enter the nucleus and activate overlapping sets of genes that are primarily involved in lipid metabolism (8). SREBP-2 preferentially activates the LDL receptor and other genes directly involved in cholesterol homeostasis and SREBP-1 products primarily activate genes of fatty acid metabolism (8).
The biological effects of thyroid hormone are through hormone-dependent changes in gene expression (11). TR complexes with retinoid X receptor (RXR), and the heterodimer binds to specific DNA sites in target genes (12). The liganded receptor then helps to initiate a multistep process that ultimately results in gene activation. The current studies indicate that SREBP-2 is directly regulated by TH, and we propose that SREBP-2 is an important link between TH and cholesterol metabolism.
Our studies demonstrate that SREBP-2 mRNA and nuclear protein levels are both down-regulated when mice are deprived of thyroid hormone. LDL receptor mRNA is also repressed under these conditions consistent with previous studies (3). We further show that an independent feeding regimen that increases nuclear SREBP-2 independently of TH results in both a restimulation of LDL receptor mRNA expression and a significant decline in serum cholesterol. We also demonstrate that the SREBP-2 promoter is activated directly by TR in a ligand-dependent manner and that there is at least one TR-binding site in the 5'-flanking DNA. These studies identify SREBP-2 as a direct thyroid hormone target and indicate that increased nuclear SREBP-2 can reverse the hypercholesterolemic effects of hypothyroidism independently of TH itself. Furthermore, our results indicate that genetic modifications or pharmacological treatments that lower SREBP-2 levels would result in hypercholesterolemia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Isolation and Northern Blot Analysis—Total RNA was
isolated from mouse livers using TRIzol (Invitrogen). Total RNA (20 µg)
were fractionated on 1% agarose-formaldehyde gels and transferred to Nytran
(Schleicher and Schuell BioScience). The membranes were hybridized with
32P-labeled cDNA probes (1 x 106 cpm/ml) overnight
at 42 °C. An 80-base HindIII/EcoRI fragment of rat
ribosomal protein L32 cDNA (a gift from Tom Lane, University of California,
Irvine) was used as a probe to normalize total amounts of RNA/lane. Every blot
was rehybridized with the L32 probe for normalization. After hybridization,
blots were washed and exposed to a Kodak BioMAX film followed by scanning with
an HP 7400c flat bed scanner, and mRNA levels were then quantified using the
Quantity one software (Bio-Rad). The following cDNA probes were used: HMG CoA
reductase, an 0.6-kb HindIII/BamHI fragment from pBluescript
mouse Red; PGC-1
, a 1.5-kb BglII/NcoI fragment from
the pSV-SPORT-PGC-1 (gift of Peter Tontonoz, UCLA); FAS, an 0.2-kb
EcoRI fragment from mouse FAS cDNA; LDL receptor, an 0.3-kb
PstI/BamHI fragment from pBluescript mouse LDLR;
5'-deiodinase, an 0.8-kb BamHI/EcoRI from
pCR2.1-mouse-5'DI.
RNase Protection Assay—Total RNA samples (20 µg) were subjected to the RNase protection assay using the RPAII kit (Ambion, Inc.). 32P-labeled cRNA probes were generated from pBluescript-mSREBP-1c, mSREBP-2-pGEM-T, and L32-pGEM constructs by in vitro transcription using the MAXIscript kit (Ambion, Inc.). The RNase protection assays were performed by adding a cRNA probe for either SREBP-2 or SREBP-1c mRNA (1 x 105 cpm) and a cRNA probe (1 x 105 cpm) for L32 mRNA. L32 cRNA probe was used to normalize total amounts of RNA/lane. After digestion with RNase A/RNase T1, protected fragments were resolved on 8 M urea, 5% polyacrylamide gels, dried, and processed as described above.
Protein Isolation and Blotting—Fresh slices of the same livers used above for RNA analysis were processed for nuclear proteins (13). Nuclear extracts were fractionated on 8% SDS-PAGE, transferred to nitrocellulose, and analyzed first by Ponceau S staining to confirm that equal amounts of total protein were both loaded and transferred in each lane. Then, the blot was incubated with a rabbit polyclonal antisera directed against mouse SREBP-2 (from Jay Horton, University of Texas, South-western) followed by a secondary antibody conjugated to horseradish peroxidase. Reactivity was then detected with the ECL kit (Pierce).
Cell Culture and Transient Transfection Assay—HepG2 cells
were maintained in minimum essential medium supplemented with 10% fetal bovine
serum at 37 °C and 5% CO2. One day before transfection, cells
were seeded in 6-well plates at 350,000 cells/well. After incubation for 16 h,
cells were transfected by the calcium phosphate co-precipitation method as
described (14) with 2
µg/well of the indicated SREBP-2 promoter-luciferase reporter, 2 µg/well
of the cytomegalovirus 
-galactosidase plasmid as an internal control for
transfection efficiency, and indicated expression vectors for CMX-hTR-
(2 µg) or CMX-hRXR- (0.5 µg). Salmon sperm DNA was added to
equalize total amounts of DNA. After 4–6 h, cells were treated with 10%
glycerol, washed three times with phosphate-buffered saline, and refed with a
serum-free minimum essential medium supplemented with 5 µg/ml insulin, 5
µg/ml transferrin, 5 ng/ml selenite, 5 µl/ml defined lipid mix
(Invitrogen), and 0.11% de-lipidated bovine serum albumin (Sigma) in the
presence or absence of 1 µM 3,3', T3. The cells were
incubated for an additional 40 h before harvest. Luciferase and
-galactosidase assays and normalization were described previously
(13). Values represent the
mean of duplicate ± S.D. Each experiment was repeated at least three
times.
Plasmids—The 5'-deletion mutants of the SREBP-2
promoter were constructed by PCR-based amplification and cloning upstream of
the luciferase gene in pGL3-Basic. Mouse genomic DNA isolated from B6129 mouse
liver was used as a template for PCR. The following forward primers were used:
–1019/SREBP-2.Luc, 5'-ACGATGCCCTGATATTCTACTGTTTG-3';
–1509/SREBP-2.Luc, 5'-AGAAACCCTGTCTCTTGCTCAGTTCT-3';
–2053/SREBP-2.Luc, 5'-CACCAGAAGAGGGCATCAGATCCTGTTACA-3'. The
reverse primer for all was 5'-GCTTGGCGCAGAGTCCCCCGTGAGGTCCAG-3'.
SacI and HindIII were added to the forward and reverse
primers, respectively, for cloning purposes. The PCR fragments were cloned
into SacI/HindIII sites of pGL3-Basic.
–4316/SREBP-2.Luc was generated by cloning of a PCR fragment from
–4316 to –1704 into SacI/EcoRV sites of
–2053/SREBP-2.Luc. The primers were as follows: forward primer,
5'-ATCTGTTCCCAGCATCCATATGGTAGCTT-3'; reverse primer,
5'-AGACTAACAAGGGGCTGGATATCACGAC-3'. Mouse cDNA was produced from
total RNA using cDNA Cycle kit (Invitrogen) and used to generate cDNA
fragments for type I 5'-deiodinase and SREBP-1c by PCR. A
5'-deiodinase PCR fragment was ligated into pCR2.1. pBluescript mouse
SREBP-1c was constructed by blunt-end ligation of a PCR fragment into the
SmaI site of pBluescript KS. The following primer pairs were used:
5'deiodinase, 5'-GCAAGGTGCTAATGACGCTGTTC-3' and
5'-AGACATCTGTACTGGTGTGGGAAAG-3'; mSREBP-1c,
5'-ATCGGCGCGGAAGCTGTCGG-3' and
5'-TTGTTGATGAGCTGGAGCATGT-3'. Mouse SREBP-2-pGEM-T was a generous
gift from Jay Horton (University of Texas, Southwestern). CMX-hTR- and
CMX-hTR- were from Dr. Bruce Blumberg, University of California, Irvine,
and CMX-hRXR- and pCMX-hLXR- were from Dr. Peter Tontonoz
(UCLA).
Electrophoretic Mobility Shift Assays—hTR-,
hTR-, hLXR-, and hRXR- proteins were produced using the
TNT-coupled transcription/translation system (Promega). The
sequences of one strand of the complementary oligonucleotide probes were as
follows: SREBP-2,
5'-CGGACCTGATGCCCTTTTCTGACCTTGGGCACTGGGCATATG-3'; DR-4,
5'-CTAGAGCTTCAGGTCACAGGAGGTCAGAGAGCT-3'; mutant TRE,
5'-TCCCGCTATGCAAATCTGAGCTGCTG-3'. The complementary
oligonucleotides were annealed, and, where indicated, they were labeled with
[
-32P]ATP by T4 polynucleotide kinase. Binding reactions
were carried out at 4 °C for 20 min in 20 µl of binding buffer
containing 12.5 mM Hepes (pH 7.6), 50 mM KCl, 1
mM MgCl2, 5% glycerol, 4 µg of poly(dI·dC), 5
µg of nonfat dry milk, and 5% (w/v) Ficoll. T3 or cold DNA competitors were
added to the binding buffer at the concentrations indicated in the legend to
Fig. 6. The protein-DNA
complexes were analyzed on a 5% polyacrylamide gel with 0.5x TBE buffer
(89 mM Tris borate, 2 mM EDTA). The gels were dried and
analyzed by autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several genes involved in de novo lipogenesis including FAS are
regulated in this manner (18).
As a control, we showed that FAS displayed this pattern of regulation
(Fig. 1A). Genes of
cholesterol metabolism exhibit a similar pattern of expression, and in
agreement with these previous observations
(3) mRNA levels for HMG CoA
reductase and LDL receptor follow this classic T3 regulated pattern
(Fig. 1, B and
C). In contrast, expression of other genes, including
those encoding SREBP-1c and the PPAR- coactivator (PGC-1)
(19), is not T3-responsive
(Fig. 1, E and
F).
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Importantly, the level of SREBP-2 mRNA followed the pattern of a typical T3-regulated gene; however, the magnitude was somewhat lower than for the other T3 target genes (Fig. 1D). Because SREBP-2 activates key genes of cholesterol metabolism, it is possible that the response to variations in TH is secondary to the changes in SREBP-2. This would provide a mechanism for linking cholesterol metabolism to thyroid hormone levels and would explain why genes of cholesterol metabolism follow a classic TR-regulation pattern.
If a decrease in active nuclear SREBP-2 protein was primarily responsible for the repression of LDL receptor mRNA levels in the experiments above, then an increase in nuclear SREBP-2 protein while animals were maintained in a TH-deprived state should result in reactivation of LDL receptor mRNA expression. Therefore, we repeated the experiments of Fig. 1 but we included groups of animals fed a diet containing lovastatin, an HMG CoA reductase inhibitor, and cholestid, a bile acid sequestrant. This feeding protocol is designed to simulate a low cholesterol environment in the liver, and it results in activation of genes of cholesterol metabolism (20). This regimen also increases the nuclear accumulation of SREBP-2 protein (13).
We prepared both RNA and nuclear protein from the livers of these animals. First, to evaluate whether nuclear SREBP-2 levels were altered by the PTU/low iodine diet and increased upon feeding cholestid and lovastatin, we measured the nuclear SREBP-2 protein levels by Western blotting (Fig. 2). In agreement with previous studies performed in the hamster (13), nuclear SREBP-2 levels are low when animals are fed a chow diet and they are induced by the addition of cholestid and lovastatin (Fig. 2, lanes 1 and 2). Consistent with lower levels of SREBP-2 mRNA, the PTU/low iodine diet resulted in a decrease in nuclear SREBP-2 protein (Fig. 2, compare lanes 1 and 3). Importantly, treatment of the TH-deprived animals with cholestid and lovastatin resulted in an increase in nuclear SREBP-2 protein (Fig. 2, compare lanes 3 and 4). T3 supplementation resulted in an increase above the PTU/low iodine diet (Fig. 2, lanes 5 and 6).
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RNA blotting with samples from these animals demonstrated that LDL receptor mRNA was down-regulated normally by TH depletion (Fig. 3A, compare lanes 1 and 3). Interestingly, the level of LDL receptor mRNA was increased by the lovastatin/cholestid combination under TH-deprived conditions (Fig. 3A, compare lanes 3 and 4). The SREBP-2 gene is auto-regulated (21), and its expression pattern exhibited a similar regulation profile (Fig. 3B). For specificity, we analyzed hepatic type I 5'-deiodinase, which is a sensitive target of thyroid hormone and TR (22). Its mRNA followed a classic T3 response pattern in our experiments (Fig. 3C). Importantly, it was unaffected by the addition of cholestid and lovastatin (Fig. 3C) indicating that cholestid and lovastatin did not have a secondary affect on thyroid hormone regulation. Taken together, these data are consistent with the hypothesis that the drop in LDL receptor mRNA that occurs under TH deprivation is secondary to the decrease in SREBP-2.
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The LDL receptor protein is responsible for high affinity uptake of LDL-cholesterol in the liver, and a TH-deprived state results in hypercholesterolemia because of the down-regulation in LDL receptor expression (23). To evaluate this in our studies, we measured whether the increase in LDL receptor mRNA resulted in a decrease in serum cholesterol (Fig. 4). As expected, serum cholesterol was down-regulated by cholestid and lovastatin in the normal chow-fed animals (Fig. 4, compare lanes 1 and 2). In the TH-deprived animals, the cholesterol level was dramatically increased as expected (Fig. 4, compare lanes 1 and 3). Importantly, inclusion of lovastatin and cholestid resulted in a significant drop in serum cholesterol levels of these otherwise TH-deprived animals. This indicates that the increase in LDL receptor mRNA resulted in functional expression of LDL receptor protein.
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The studies presented so far indicate that the SREBP-2 gene might be
directly activated by TH. To evaluate this, we fused 4316 bases of the
5'-flanking region of the mouse SREBP-2 gene to the luciferase reporter
and used it in a HepG2 cell-based transfection assay that we established to
evaluate thyroid hormone and TR regulation. When the transfected cells were
cultured in a serum-free defined medium, the addition of expression vectors
for TR and RXR coupled with T3 selectively induced luciferase activity
6-fold (Fig. 5A).
This stimulation required expression vectors and T3 hormone. A similar pattern
of activation was observed for the control murine leukemia virus luciferase
(MLVLuc) reporter, which has been used as a model T3-responsive promoter
(24). The pGL3 vector was
expressed at a very low level and showed a minimal response to TR/RXR that was
not further stimulated by the addition of T3
(Fig. 5A)
Deletion of the 5'-flanking region of the SREBP-2 promoter from –4316 to –2053 resulted in a modest drop in T3 stimulation, and further truncation down to –1019 showed that a moderate level of stimulation by TR/RXR and T3 was retained (Fig. 5B). These results indicate that the SREBP-2 gene is directly activated by TR, and it is likely that there are more than one TR-binding site spread throughout the 5'-flanking region.
Because there was a modest drop in T3 regulation upon deletion of the DNA
from –4316 to –2053, we scanned this region for the presence of
putative DR-4-like response elements, which are known to be high affinity TR
sites (25). We noticed there
was a recognizable match to this consensus in this sequence interval (noted in
Fig. 5A), and we
designed complementary oligonucleotides encompassing this putative TR site for
use in a gel mobility shift assay with TR and RXR
(Fig. 6). Heterodimers of each
TR isoform with RXR bound efficiently to this SREBP-2 DR-4 site
(Fig. 6A, lanes
1–11). Efficient binding by both TR- and TR- required
RXR, and binding was unaffected by the presence of thyroid hormone. A similar
DNA binding pattern was observed for a synthetic consensus DR-4 site
(Fig. 6A, lanes
12–20). The formation of the TR/RXR-specific complex was sensitive
to competition by the unlabeled SREBP-2 probe and to the consensus DR-4 site
but not to DNA containing a mutated DR-4 element
(Fig. 6B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results form the basis for a model to account for hypothyroid-linked hypercholesterolemia. The model predicts that as thyroid hormone levels fall, SREBP-2 levels decline followed closely by a drop in LDL receptor mRNA. This results in a decline in high affinity LDL cholesterol uptake in the liver resulting in hypercholesterolemia, which is fundamentally linked to thyroid hormone status. TH depletion in animals not only lowers liver LDL receptor mRNA and protein (26), but hypothyroid humans have increased serum LDL cholesterol (27) and reduced LDL uptake that can be reversed by adding T3 (2). Therefore, our model would account for the association between low serum TH levels and hypercholesterolemia in humans as well.
Because hypercholesterolemia associated with low TH levels is effectively
reversed by T3 supplementation
(2), the therapeutic
significance of our findings is not immediately obvious. However, our data
provide a mechanistic basis for the clinical association and may be helpful in
designing therapeutic strategies to help treat patients with more complicated
diseases such as resistance to thyroid hormone (RTH) where patients have
mutations that directly inactivate the TR gene
(28).
A previous study provided evidence that the LDL receptor promoter was activated in HepG2 cells when an expression construct for TR was added along with T3 (5). However, TR binding was not directly evaluated in this earlier study and the results observed are consistent with TR activating endogenous SREBP-2, which could then activate the LDL receptor gene. Our results indicate that TR directly activates SREBP-2, and it is well established that SREBP-2 activates the LDL receptor gene (13). Thus, our studies clearly establish an indirect mode of regulation for the LDL receptor by T3. However, further studies are required to establish whether there is also a direct effect of TR.
Our studies demonstrate that TR directly activates expression of the SREBP-2 gene, and we have identified at least one binding site for TR in the 5'-flanking sequence (Fig. 6). Based on the gradual loss in T3 responsiveness in the analysis of the promoter deletions, there are likely other functional binding sites for TR and further studies are needed to characterize these additional elements.
SREBP-2 is also autoregulated by SREBPs (21), and SREBP-2 mRNA was also up-regulated by the cholestid and lovastatin under the TH-depleted conditions shown in Fig. 3. SREBPs and TR functionally cooperate to activate both the rodent S14 and chicken acetyl-CoA carboxylase promoters (29–31). Thus, it will be interesting to determine whether SREBPs and TR functionally interact to stimulate expression of the SREBP-2 promoter as well.
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FOOTNOTES |
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To whom correspondence should be addressed. Fax: 949-824-8551; E-mail:
tfosborn{at}uci.edu.
1 The abbreviations used are: TH, thyroid hormone; LDL, low density
lipoprotein; SREBP, sterol regulatory element-binding protein; mSREBP, mouse
SREBP; TR, thyroid hormone receptor; T3, 3,3'
,5-triiodo-L-thyronine; RXR, retinoid X receptor; LXR, liver X
receptor; PTU, propylthiouracil; HMG, 3-hydroxy-3-methylglutaryl; PGC,
peroxisome proliferator-activated receptor--coactivator; FAS, fatty
acid synthase; CMX, cytomegalovirus X.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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