From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046
Received for publication, August 10, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Sterol regulatory element-binding proteins
(SREBPs) are membrane-bound transcription factors that increase the
synthesis of fatty acids as well as cholesterol in animal cells. All
three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from
membranes. Previous data indicate that the SREBPs are also negatively
regulated by unsaturated fatty acids, but the mechanism is uncertain.
In the current experiments, unsaturated fatty acids decreased the
nuclear content of SREBP-1, but not SREBP-2, in cultured human
embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty
acids increased with increasing chain length and degree of
unsaturation. Oleate, linoleate, and arachidonate were all effective,
but the saturated fatty acids palmitate and stearate were not
effective. Down-regulation occurred at two levels. The mRNAs
encoding SREBP-1a and SREBP-1c were markedly reduced, and the
proteolytic processing of these SREBPs was inhibited. When SREBP-1a was
produced by a cDNA expressed from an independent promoter,
unsaturated fatty acids reduced nuclear SREBP-1a without affecting the
mRNA level. There was no effect when the cDNA encoded a
truncated version that was not membrane-bound. When administered together, sterols and unsaturated fatty acids potentiated each other in
reducing nuclear SREBP-1. In the absence of fatty acids, sterols did
not cause a sustained reduction of nuclear SREBP-1, but they did reduce
nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as
sterols, can down-regulate nuclear SREBPs and that unsaturated fatty
acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c,
whereas sterols have their greatest inhibitory effects on
SREBP-2.
Ingestion of n-3 and n-6 polyunsaturated
fatty acids in place of saturated fatty acids shifts the pattern of fat
metabolism in liver from storage to oxidation (1). Genes involved in
fatty acid oxidation are induced, and genes involved in fatty acid
synthesis or lipogenesis are repressed (2, 3). The increase in genes responsible for oxidation is achieved in part by activation of peroxisome proliferator-activated receptors (4-6) and in part by
conversion of polyunsaturated fatty acids to eicosanoids (1). Neither
of these two pathways is required for the decreased expression of
lipogenic genes elicited by unsaturated fatty acids (6).
New candidates for fatty acid-mediated regulation of lipogenic genes
have emerged in the form of sterol regulatory element-binding proteins
(SREBPs).1 SREBPs are a
unique family of membrane-bound transcription factors that activate
genes involved in the synthesis of cholesterol and fatty acids and
their uptake from plasma lipoproteins (7, 8). The regulation of SREBPs
has been studied most intensively in fibroblast-like cultured cells
such as Chinese hamster ovary cells and human embryonic kidney
(HEK)-293 cells. These studies showed that the SREBPs are synthesized
as membrane-bound precursors averaging 1150 amino acids in length. The
NH2-terminal domain of ~480 amino acids is a
transcription factor of the basic helix-loop-helix-leucine zipper
family. This is followed by a 90-amino acid membrane attachment domain
consisting of two membrane-spanning helices separated by a short
hydrophilic loop. The third domain is a COOH-terminal regulatory domain
of ~590 amino acids. The SREBPs are oriented in a hairpin fashion
with their NH2-terminal and COOH-terminal domains facing
the cytosol and the short hydrophilic loop projecting into the lumen of
the endoplasmic reticulum.
Immediately after synthesis in the endoplasmic reticulum, the SREBPs
form complexes with SREBP cleavage-activating protein (SCAP), a
polytopic membrane protein that escorts them to the Golgi complex
(9-11). Here the SREBPs are cleaved sequentially by two proteases that
release the NH2-terminal domain into the cytosol. The
released fragment travels to the nucleus, where it binds to sterol
regulatory elements located in the 5'-flanking regions of more than 20 genes involved in lipid synthesis and uptake. Target genes include the
low density lipoprotein receptor and the cholesterologenic enzymes
3-hydroxy-3-methylglutaryl CoA synthase and reductase as well as the
lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthetase, and
stearoyl CoA desaturase (8, 12-16).
The proteolytic processing of SREBPs is under feedback control by
cholesterol. Thus, when sterols accumulate in cells, the SCAP·SREBP complex fails to move to the Golgi, and SREBPs are not processed (17). The nuclear SREBPs are rapidly degraded by a
proteasomal process, and the synthesis of sterols and fatty acids
(primarily 18:1 unsaturates) declines.
Three isoforms of SREBP have been identified (8). Two of these
proteins, designated SREBP-1a and SREBP-1c, are derived from a single
gene through use of alternate transcriptional start sites producing
different forms of exon 1 that are spliced to a common exon 2. The
SREBP-1a isoform has a relatively long NH2-terminal acidic
activation domain, and it is a potent transcriptional activator. The
SREBP-1c isoform has a short acidic activation domain, and it may
require post-translational modification to activate transcription (18).
The third isoform, SREBP-2, derived from a separate gene, is ~50%
identical to the SREBP-1 isoforms, and it contains a long activation
domain. Although the target genes for the SREBPs overlap, the two
SREBP-1 isoforms are relatively more potent in stimulating fatty acid
synthesis, whereas SREBP-2 acts primarily on the cholesterol biosynthetic pathway (8, 16, 18).
The ratio of SREBP-1a to -1c isoforms varies markedly among different
cells. In most adult tissues, including the liver, SREBP-1c predominates (19), but the reverse is true in cultured cells (19). The
amount of SREBP-2 is generally approximately equal to the combined
total of SREBP-1a and -1c (8).
Inasmuch as unsaturated fatty acids are end-products of SREBP action,
several laboratories have sought to determine whether fatty acids exert
feedback effects on SREBP activity. In general, these studies have
shown regulatory effects, but the details have been contradictory. In
the first of these studies, Thewke et al. (20) showed that
oleate synergized with sterols in reducing the amounts of nuclear
SREBP-1 (nSREBP-1) and nSREBP-2 in Chinese hamster ovary cells.
However, oleate without sterols had no effect. Worgall et
al. (21) found that unsaturated fatty acids (but not saturated
fatty acids) without sterols decreased the amount of nSREBP-1 in
several types of cultured cells and lowered the mRNA levels for
3-hydroxy-3-methylglutaryl-CoA synthase, an SREBP target gene. Xu
et al. (22) fed oils rich in n-6 or
n-3 polyunsaturated fatty acids to rats and observed a
decrease in nSREBP-1 in liver cells. Both n-6 and
n-3 polyunsaturated fatty acids decreased the amount of
mRNA for SREBP-1, but they did not appear to affect transcription
of the SREBP-1 gene as determined by nuclear run-on assays. In
contrast, Xu et al. (22) found no inhibitory effect on
SREBP-1 expression with dietary monounsaturated triolein. Kim et
al. (23) found that long-term feeding (5 months) of a fish oil
diet enriched in n-3 polyunsaturated fatty acids decreased nSREBP-1 and nSREBP-2 levels in the livers of mice. The mRNA for SREBP-1c was reduced dramatically and that of SREBP-2 was reduced partially, but there was no effect on SREBP-1a. In agreement with the
earlier results of Xu et al. (22), Kim et al.
(23) found that oleic acid-enriched safflower oil had little effect in
their animals.
The current studies were undertaken to provide a comprehensive analysis
of the effects of various fatty acids on the levels of mRNA and
protein for all three isoforms of SREBP in nonhepatic human cells
(HEK-293 cells) maintained in tissue culture. The results show that
unsaturated fatty acids lowered the levels of nSREBP-1, even in the
absence of exogenous sterols. In general, the inhibitory effect of the
fatty acids increased with increasing chain length and degree of
unsaturation. The decrease in nSREBP-1 was accompanied by a decrease in
the mRNAs encoding both SREBP-1a and SREBP-1c. However, even when
the level of mRNA for SREBP-1a was held constant through
transfection, arachidonic acid still reduced nSREBP-1a, indicating that
fatty acids have actions at the level of SREBP-1a protein as well as at
the level of mRNA. Arachidonic acid did not have an effect on cells
that were engineered to express a truncated form of SREBP-1a that
enters the nucleus without a requirement for proteolysis, suggesting
that the post-transcriptional effect is exercised at the level of
proteolytic processing of the SREBP-1a precursor. In the absence of
sterols, unsaturated fatty acids had very little effect on the levels
of SREBP-2 mRNA or the nSREBP-2 isoform.
Materials--
We obtained affinity-purified donkey anti-mouse
IgG from Jackson Immunoresearch Laboratories; anti-rabbit IgG from
Amersham Pharmacia Biotech; monoclonal antibodies IgG-HSV-Tag and
IgG-T7-Tag from Novagen; free fatty acids from Sigma-Aldrich, Inc. and
Nu-Chek-Prep, Inc. (Elysian, MN);
N-acetyl-Leu-Leu-norleucinal (ALLN) from Calbiochem; defatted bovine serum albumin (BSA, catalog no. 100069) from Roche Molecular Biochemicals; and triacsin C from BioMol Research
Laboratories, Inc. (Plymouth Meeting, PA); [ Preparation of Delipidated Fetal Calf Serum--
Fetal calf
serum (FCS, Life Technologies, Inc.) was delipidated by a modification
of the method of Cham and Knowles (27). Briefly, 500 ml of serum was
mixed with 400 ml of n-butanol and 600 ml of isopropyl ether
at room temperature for 20 min, followed by a 20-min incubation on ice.
After centrifugation, the aqueous phase was re-extracted with 200 ml of
isopropyl ether, recentrifuged, subjected to evaporation under a stream
of nitrogen gas, lyophilized, reconstituted with 200 ml of distilled
water, and dialyzed against phosphate-buffered saline (PBS). Multiple
aliquots were stored at Preparation of Albumin-bound Fatty Acids--
A 5 or 10 mM stock solution of each fatty acid was prepared by
diluting the free fatty acid in ethanol and precipitating it with the
addition of NaOH (final concentration of 0.25 M). The precipitated sodium salt was then evaporated under nitrogen gas, reconstituted with 0.9% (w/v) NaCl, and stirred at room temperature for 10 min with defatted BSA (final concentration at 10% (w/v) in 0.15 M NaCl). Each solution was adjusted to pH 7.4 with NaOH and
stored in multiple aliquots at Expression Plasmids--
pTK-HSV-BP1a is an expression vector
that produces epitope-tagged human SREBP-1a under control of the HSV TK
promoter (30).
pTK-HSV-BP1a-T7 encodes human SREBP-1a flanked at the NH2
terminus by two tandem copies of the HSV epitope tag and at the COOH
terminus by three tandem copies of the T7 epitope tag sequence. This
TK-driven plasmid was constructed as follows: pTK-HSV-BP2-Ras-T7 (26)
was digested with BspDI and NdeI to isolate a
6-kb fragment that contained the HSV and T7 epitope repeats but not the
sequence of the SREBP-2/Ras fusion protein. A pair of primers,
5'-CAGCCTGAACTCGCTCCAGAGG-3', corresponding to amino acids 1-7
of the second HSV tag, and 5'-GCTAGCCATATGGCTGGAAGTGACAGTGGTCCCAC-3', corresponding to amino acid residues 1140-1147 of human
SREBP-1a, was used to amplify the coding region of SREBP-1a. The
resulting polymerase chain reaction product was digested with
BspDI and NdeI, and a 3.4-kb fragment encoding
amino acids 1-1147 of SREBP-1a was isolated. The two isolated
fragments were ligated to generate pTK-HSV-BP1a-T7, which was sequenced
in its entirety.
Site-directed Mutagenesis--
A plasmid encoding the nuclear
form of human SREBP-1a (amino acids 1-490), designated
pTK-HSV-BP1a(Stop 490), was constructed by site-directed mutagenesis of
pTK-HSV-BP1a. Mutagenesis to introduce the indicated stop codon was
carried out using the QuikChange site-directed mutagenesis kit
(Stratagene). The resulting expression vector was sequenced in its
entirety to confirm the mutation. Two different clones of the mutant
plasmid were independently transfected.
Cell Culture and Fractionation of HEK-293 Cells--
Unless
otherwise stated, monolayers of human embryonic kidney (HEK)-293 cells
were set up on day 0 (7 × 105 cells/100-mm dish) and
cultured in 8-9% CO2 at 37 °C in medium A (Dulbecco's
modified Eagle's medium (low glucose) containing 100 units/ml
penicillin and 100 µg/ml streptomycin sulfate) supplemented with 10%
(v/v) FCS. Unless otherwise stated, on day 2 the cells were washed once
with PBS and refed with medium A supplemented with 5% (v/v)
delipidated FCS in the absence or presence of BSA-bound fatty acids
(100 µM) and/or sterols (1 µg/ml 25-hydroxycholesterol plus 10 µg/ml cholesterol added in a final concentration of 0.2% (v/v) ethanol) as indicated in the legends. After incubation with fatty
acids and/or sterols at 37 °C for 14 h, ALLN was added directly to the medium at a final concentration of 25 µg/ml unless otherwise stated. 2 h later, the cells were harvested by scraping in the medium, and the cell suspension from triplicate dishes was pooled and
centrifuged at 103 × g for 5 min at 4 °C.
The resulting cell pellet was washed by recentrifugation in PBS at
4 °C, after which the cell pellet was resuspended in buffer A (250 mM sucrose, 10 mM Hepes-KOH at pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, and a mixture
of protease inhibitors that included 2.8 µg/ml aprotinin, 10 µg/ml
leupeptin, 25 µg/ml ALLN, 5 µg/ml pepstatin A, and 0.5 mM Pefabloc). The cell suspension was passed through a
23-gauge needle 20 times and centrifuged at 103 × g at 4 °C for 5 min. The 103 × g
pellet was resuspended in 0.1 ml of buffer B (20 mM
Hepes-KOH at pH 7.6, 0.42 M NaCl, 2.5% (v/v) glycerol, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, and the above mixture of protease inhibitors). This suspension was rotated at 4 °C for 1 h and
centrifuged at 105 × g for 15 min at 4 °C in
a Beckman TLA 100 rotor. The resulting supernatant is designated as the
nuclear extract fraction. The supernatant of the original
103 × g spin was centrifuged at 104 × g for 15 min at 4 °C, after which the pellet was
dissolved in 0.1 ml of SDS lysis buffer (10 mM Tris-HCl at
pH 6.8, 100 mM NaCl, 1% (w/v) SDS, 1 mM sodium
EDTA, 1 mM sodium EGTA, and the above mixture of protease
inhibitors) and designated as the membrane fraction.
Transfection--
HEK-293 cells were transfected for 3 h
with the indicated plasmids using the MBS kit (Stratagene) as described
previously (31).
SDS-PAGE and Immunoblot Analysis--
Protein concentration of
nuclear extract and membrane fractions was measured with a BCA kit
(Pierce). A given amount of nuclear extract or membrane fraction was
mixed with 5× SDS loading buffer (1× loading buffer contains 30 mM Tris-HCl at pH 7.4, 3% SDS, 5% (v/v) glycerol, 0.004%
(w/v) bromphenol blue, 2.5% (v/v) Northern Blot Hybridization of mRNA--
Total RNA was
prepared from triplicate dishes of monolayers of HEK-293 cells using
the RNA Stat-60 kit (Tel-test, Inc., Friendswood, TX), followed by
phenol/chloroform extraction and ethanol precipitation. For Northern
gel analysis, 30 µg of total RNA was mixed with RNA sample loading
buffer (containing 50 µg/ml ethidium bromide) (Sigma), denatured with
formaldehyde and formamide, subjected to electrophoresis in a 1%
agarose formaldehyde gel, and transferred to Hybond N+
membranes. The cDNA probes for human SREBP-1 and SREBP-2 were prepared by excising a 1.4-kb NotI/XbaI fragment
from pIND-BP1c-FLAG (18) and a 1.2-kb HindIII fragment from
pTK-HSV-BP2 (30). The cDNA probe for human RNase Protection Assay--
The cDNA fragments for human
SREBP-1a and SREBP-1c used as templates for cRNA probe synthesis were
generated by polymerase chain reaction using the plasmids
pIND-BP1a-FLAG and pIND-BP1c-FLAG (18). The primers used to amplify
SREBP-1a were: 5' primer, 5'-CGCTCCCTAGGAAGGGCCGTA-3'; and 3' primer,
5'-ACTGTCTTGGTTGTTGATAAGC-3'. The primers used to amplify SREBP-1c
were: 5' primer, 5'-AGGGGTAGGGCCAACGGCCT-3' and 3' primer,
5'-CAAGCTGCCTGGGGAGCTGGTA-3'. The amplified human SREBP-1a fragment
corresponds to 66 bp of 5'-untranslated region, exon 1a and 36 bp of
exon 2. The amplified human SREBP-1c fragment corresponds to 54 bp of
5'-untranslated region, 15 bp of exon 1c, and 128 bp of exon 2. Note
that exon 2 is common to SREBP-1a and SREBP-1c. The amplified fragments
were subcloned into the pCRII vector (Invitrogen). After linearization
of plasmid DNA with XbaI, antisense RNA was transcribed with
[ In previous studies from this laboratory, the sterol-mediated
regulation of SREBP processing was measured in cultured cells that were
grown in lipoprotein-deficient serum, which is essentially devoid of
low density lipoprotein cholesterol, owing to the removal of low
density lipoprotein by ultracentrifugal flotation (25). Ultracentrifugation does not remove free fatty acids, which are bound
to albumin. To delipidate serum more completely, in the current studies
we used a solvent extraction procedure that employs butanol and
isopropyl ether (see "Experimental Procedures"). The concentrations
of free fatty acids in medium containing 5% lipoprotein-deficient serum and 5% delipidated serum were 18 and 0.39 µM,
respectively (45-fold reduction).
Fig. 1 shows the content of SREBPs in
membrane fractions and nuclear extracts of HEK-293 cells that were
incubated for 16 h in medium containing either
lipoprotein-deficient serum or delipidated serum with or without added
sterols or arachidonate. Inasmuch as our antibody does not
differentiate between SREBP-1a and -1c, we use the general term SREBP-1
to refer to the results of immunoblotting. When the cells were
incubated in lipoprotein-deficient serum, we observed the full-length
precursor form of SREBP-1 in cell membranes and the cleaved mature form
in nuclear extracts (Fig. 1A, lane A). Addition
of sterols (mixture of 25-hydroxycholesterol plus cholesterol) led to
an increase in the 120-kDa precursor form in membranes and a decrease
in the nuclear form (lane B). When arachidonate (100 µM) was added, the nuclear form of SREBP-1 (designated
nSREBP-1) also disappeared, but there was no buildup of the precursor
(lane C). In the presence of sterols plus arachidonate, the
nuclear form remained low, but the precursor again accumulated (lane D). When the experiments were conducted in delipidated
serum, nSREBP-1 (lane E) was increased as compared with that
observed in lipoprotein-deficient serum (lane A). When
sterols were added in delipidated serum, the precursor accumulated, but
there was only a slight decline in nSREBP-1 (lane F). The
addition of arachidonate abolished nSREBP-1, again without a buildup of
the precursor (lane G). When arachidonate and sterols were
present, nSREBP-1 was also undetectable, but the precursor accumulated
(lane H).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP
(800 Ci/mmol); redivue [
-32P]dCTP (3000 Ci/mmol) and Rediprime II Random Primer Labeling kit from Amersham
Pharmacia Biotech; and human
-actin cDNA control probe from
CLONTECH Laboratories, Inc. Rabbit polyclonal
antibodies against human SREBP-1 (amino acids 31-175 of the SREBP-1a
isoform) and human SREBP-2 (amino acids 48-403) were prepared as
previously described (24). Lipoprotein-deficient fetal calf serum
(d > 1.215 g/ml) was prepared by ultracentrifugation
(25). Other reagents were obtained from previously described sources
(9, 26).
20 °C. The concentration of free fatty
acids in the reconstituted delipidated serum was measured with the Free
Fatty Acid, Half-micro Test assay kit (Roche Molecular Biochemicals);
the concentrations of cholesterol and triglycerides were measured as
previously described (28, 29). In 12 preparations of delipidated fetal
calf serum, the mean concentration of free fatty acids was reduced from
840 to 7.7 µM, the mean concentration of cholesterol was
reduced from 280 to 7.5 µg/ml, and the mean concentration of
triglycerides was reduced from 600 to 23 µg/ml.
20 °C protected from light in tubes
evacuated under nitrogen gas.
-mercaptoethanol). After boiling
for 5 min, aliquots of the proteins (50 µg) were subjected to 8%
SDS-PAGE and transferred to Hybond-C extra-nitrocellulose filters
(Amersham Pharmacia Biotech). The filters were incubated with
antibodies as described in the figure legends. Bound antibodies were
visualized with peroxidase-conjugated donkey anti-rabbit IgG or donkey
anti-mouse IgG using the SuperSignal CL-HRP substrate system (Pierce)
according to the manufacturer's instructions. Gels were calibrated
with prestained molecular weight markers (New England BioLabs). Filters
were exposed to Reflection NEF0496 film (PerkinElmer Life Sciences) at
room temperature for the indicated time. The intensity of the nuclear
bands was quantified with the National Institutes of Health IMAGE 1.61 software.
-actin was obtained
from CLONTECH. cDNA probes were labeled with
[
-32P]dCTP using a Rediprime II Random Labeling kit.
The membranes were hybridized with the indicated
32P-labeled probes (2 × 106 cpm/ml) for
15 h at 65 °C using Rapid-hyb buffer (Amersham Pharmacia Biotech); washed twice with 0.1% (w/v) SDS/2× SSC at room temperature for 10 min followed by 0.1% (w/v) SDS/0.1× SSC at 65 °C for 15 min
and exposed at
80 °C to film with intensifying screens for the
indicated time. The amount of radioactivity was quantified using the
NIH IMAGE 1.61 software.
-32P]CTP (20 mCi/ml) using bacteriophage SP6 RNA
polymerase (Ambion, Austin, TX) as described previously (19). Aliquots
of total RNA (50 µg) from each sample were incubated with the above
SREBP-1 cRNA probe plus a cRNA probe for the mRNA of human
-actin (19) plus the reagents contained in the HybSpeed RPA kit
(Ambion). In preparing the probes, we adjusted the specific activity of the [
-32P]CTP to give comparable signals for
-actin
and SREBP-1. After digestion with RNase A/T1, protected fragments were
separated on 8 M urea/5% polyacrylamide gels, and the gels
were dried and subjected to autoradiography by using reflection film
and BioMax intensifying screens (Kodak). To assess endogenous
regulation, an SREBP-1c cRNA probe was used. The protected fragments
corresponding to SREBP-1a (129 bp) and SREBP-1c (198 bp) were
visualized on gels analyzed quantitatively with the NIH IMAGE 1.61 software. For studying transfected cells, a SREBP-1a cRNA probe was
used to distinguish the endogenous SREBP-1a as a 189-bp protected
fragment and transfected SREBP-1a as a 123-bp fragment. The level of
-actin mRNA in each sample was used to normalize signals
obtained for the SREBP mRNAs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunoblot analysis of endogenous SREBP-1
(A) and SREBP-2 (B) in HEK-293 cells
incubated with lipoprotein-deficient serum or delipidated serum:
effects of sterols and fatty acids. On day 0, HEK-293 cells were
set up for experiments as described under "Experimental Procedures"
except that the cells were plated at a density of 4 × 105 cells/60-mm dish. On day 2, the cells were refed with
medium A containing either 5% fetal calf lipoprotein-deficient serum
(lanes A-D and I-L) or 5% delipidated FCS
(lanes E-H and M-P) and one of the following
additions as indicated: 100 µM sodium arachidonate, 1 µg/ml 25-hydroxycholesterol plus 10 µg/ml cholesterol (sterols), or
sodium arachidonate plus sterols. All culture medium was adjusted to
contain 0.1% BSA and 0.2% ethanol. After incubation for 16 h,
the cells were harvested, and nuclear extracts and membrane fractions
were prepared as described under "Experimental Procedures."
Aliquots of the membranes (40 µg) and nuclear extracts (40 µg) were
subjected to SDS-PAGE. Immunoblot analysis was carried out with 5 µg/ml rabbit anti-SREBP-1 IgG (lanes A-H) or anti-SREBP-2
IgG (lanes I-P). The filters were exposed to film for
20 s (membranes) or 10 s (nuclear
extracts). P and N denote the precursor and
cleaved nuclear forms of SREBP-1 or SREBP-2, respectively. The
intensity of the nuclear bands was quantified with the NIH IMAGE 1.61 software as described under "Experimental Procedures." The values
are expressed relative to the signal intensity in lane A
(SREBP-1) or lane I (SREBP-2).
In lipoprotein-deficient serum, sterols regulated SREBP-2 processing in a manner that was similar to that of SREBP-1 (Fig. 1B, lanes I and J). However, in contrast to the findings with SREBP-1, arachidonate did not effect the nuclear form of SREBP-2 (lane K). Sterols plus arachidonate gave a result that was similar to that of sterols alone (lane L). In delipidated serum, sterols alone abolished nSREBP-2 (lanes M and N). Again, arachidonate had virtually no effect (lane O), and sterols plus arachidonate was similar to sterols alone (compare lanes N and P).
The data of Fig. 1 indicate a difference in the ability of sterols and arachidonate to regulate SREBP-1 and SREBP-2. Sterols were unable to abolish nSREBP-1 unless a fatty acid such as arachidonate was present. In contrast, sterols eliminated nSREBP-2 even in the absence of fatty acids. Moreover, arachidonate alone reduced the amount of nSREBP-1, but it did not reduce the content of nSREBP-2. The data further suggest that the previously observed ability of sterols to suppress nSREBP-1 in lipoprotein-deficient serum is dependent on the presence of fatty acids in the lipoprotein-deficient serum.
To study the effects of sterols and fatty acids independently, all
further experiments in this paper were performed with cells that were
incubated in delipidated serum plus and minus added sterols or fatty
acids. Fig. 2A shows that the
ability of various fatty acids to reduce nSREBP-1 increased in
proportion to chain length and degree of unsaturation. Thus, palmitate
(16:0) had no effect, but palmitoleate (16:1) caused a 50% reduction.
Stearate (18:0) reduced nSREBP-1 only slightly, but its monounsaturated derivative, oleate (18:1), had a pronounced effect (40% of control). Polyunsaturated linoleate (18:2) and linolenate (18:3) were even more effective. Arachidonate (20:4) and docosatetraenoate (22:4) were
also potent. None of these fatty acids had a significant effect on
nSREBP-2 (Fig. 2B) with the possible exception of
arachidonate, which produced a slight reduction.
|
To measure the effects of these fatty acids on the mRNA for SREBP-1a and -1c, we used an RNase protection assay (Fig. 2C). We adjusted the exposure times to compensate partially for the fact that the absolute level of SREBP-1a mRNA was 10-fold greater than that of SREBP-1c. Unsaturated fatty acids reduced both the SREBP-1a and -1c mRNAs. The effectiveness of individual fatty acids increased with increasing chain length and degree of unsaturation in parallel with the effects on the nuclear protein. In experiments not shown, we found that saturated and monounsaturated fatty acids up to 14 carbons in length failed to lower the SREBP-1 mRNAs or the levels of the nuclear protein.
To determine the amount of time required for unsaturated fatty acids to
decrease the SREBP-1 mRNA and the nuclear protein, we measured the
time course of the response to arachidonate (Fig. 3). At zero time, the cells were switched
from medium containing 10% FCS to medium containing 5% delipidated
serum, and the cells were harvested 20 h later. Arachidonate (100 µM) was added to the medium at the indicated times before
harvest. A decline in the amount of nSREBP-1 was detectable when
arachidonate was added 4 h before harvest (lane C), and
the level declined by 70% at 6 h (lane D) and 90% at
16 h. There was little fall in nSREBP-2 even after 16 h
(lane K). The precursor form of SREBP-2, but not SREBP-1,
increased with time after addition of arachidonate. We observed a
parallel decrease in the mRNAs for both SREBP-1a and SREBP-1c that
was detectable within 2-4 h after arachidonate addition (Fig.
3C, lanes N and O) and reached 90% in
6 h (lane P). The 90% decline persisted for the
16 h duration of the experiment (lane Q). We observed
no change in the SREBP-2 mRNA, which was measured by Northern
blotting (Fig. 3D).
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As noted in Fig. 1, in the absence of fatty acids, sterols alone cause
only a slight decrease in nSREBP-1 when measured at 16 h. To
determine whether this sterol resistance was constant with time, we
performed a time course experiment (Fig.
4). At zero time, the cells were switched
from medium containing FCS to medium containing delipidated serum plus
sterols in the absence or presence of arachidonate. Cells were
harvested at different intervals, and SREBPs were measured by
immunoblotting (panels A and B). At zero time,
nSREBP-1 and nSREBP-2 were both present (lanes A,
G, H, and N). At the 4- and 8-h time
points after addition of sterols in delipidated serum, both nuclear
proteins were markedly reduced (lanes B, C,
I, and J). The nSREBP-2 remained low throughout the experiment (lanes K and L). However, by
12 h nSREBP-1 had reappeared (lane D), and it remained
high at 16 h (lane F). The reappearance of nSREBP-1 was
associated with an increase in the mRNAs encoding SREBP-1a and -1c
(Fig. 4C). Sterols may suppress nSREBP-1 transiently because
the cells initially have sufficient unsaturated fatty acids as a result
of prior incubation in FCS. The nSREBP-1 may reappear only after the
unsaturated fatty acids become depleted and the SREBP-1 mRNA
increases. This reappearance is prevented by maintaining arachidonate
in the culture medium (lane F).
|
To study the interaction between sterols and fatty acids on levels of
SREBP-1 mRNAs and protein, we incubated cells for 16 h in
delipidated serum with varying amounts of arachidonate in the absence
or presence of sterols. The mRNAs and protein were quantified by
RNase protection and immunoblotting, respectively, and the results are
plotted in Fig. 5. For simplicity, we
show only the mRNA for SREBP-1a, which accounts for 90% of the
total SREBP-1 mRNA in the HEK-293 cells. The results for SREBP-1c
were qualitatively similar (data not shown). The addition of sterols raised the level of SREBP-1a mRNA (panel A) and the
precursor form of the protein (panel B). The addition of
arachidonate reduced the mRNA and the protein precursor under both
conditions. The results with the nuclear form of SREBP-1 (panel
C) did not parallel those observed for the mRNA and the
precursor. In the absence of arachidonate, sterols did not increase
nSREBP-1 (panel C), despite the increase in mRNA and
precursor levels. The addition of arachidonate in the presence of
sterols led to a sharper decline in SREBP-1 than it did in the absence
of sterols (panel C). In the presence of sterols, the
nuclear protein became undetectable at 20 µM
arachidonate. In the absence of sterols, nSREBP-1 remained detectable,
even at 100 µM arachidonate.
|
Considered together, the data of Fig. 5 raise the possibility that arachidonate had two effects on SREBP-1a. It lowered the SREBP-1a mRNA, but it decreased the amount of nuclear protein to an extent that was greater than the reduction in the mRNA. This discrepancy was most apparent in the presence of sterols. With sterols plus 20 µM arachidonate, nSREBP-1 became undetectable even though the mRNA remained relatively high and the amount of precursor had fallen by less than 50%. Even in the absence of sterols, arachidonate changed the ratio of precursor to nuclear protein. Thus, at 20 µM arachidonate the level of precursor had fallen by less than 50%, whereas the level of nuclear protein declined by 80%.
To test the possibility that arachidonate affects nSREBP-1a
independently of changes in the level of mRNA, we studied the behavior of epitope-tagged SREBP-1a that was produced by transfection under the control of the TK promoter, which should be resistant to
transcriptional regulation by arachidonate. Following transfection, cells were incubated in the absence or presence of arachidonate, and
membranes and nuclear extracts were subjected to electrophoresis and
immunoblotted with an antibody against the epitope tag. In the absence
of arachidonate, epitope-tagged SREBP-1a was processed, and the nuclear
form was detected (Fig. 6A, lane
D). Addition of increasing amounts of arachidonate eliminated
nSREBP-1a (lanes E and F) even though it did not
decrease the mRNA of the transfected SREBP-1a (Fig. 6B,
lanes M-O). As a positive control in the same cells, we
found that arachidonate decreased the mRNA of endogenous SREBP-1a
as determined by RNase protection (Fig. 6B, lanes
M-O).
|
If arachidonate reduces nSREBP-1a by blocking proteolytic processing of the precursor, then the fatty acid should have no effect when cells express a truncated SREBP-1 that terminates before the membrane-spanning region. To test this hypothesis, we transfected HEK-293 cells with a cDNA encoding an epitope-tagged truncated version of SREBP-1a with a stop codon at position 490. This protein terminates at Leu-489, which corresponds to the COOH terminus of nSREBP-1a after it has been processed by the Site-1 and Site-2 proteases (26). Nuclear extracts from cells expressing SREBP-1a(Stop490) exhibited the properly sized nuclear form of SREBP-1a as visualized by blotting with an antibody against the epitope tag (Fig. 6A, lane G). The amount of this nuclear protein did not change when increasing amounts of arachidonate were added (lanes H and I). As expected, the TK-driven HSV-SREBP-1a(Stop 490) mRNA was not down-regulated by arachidonate (Fig. 6B, lanes P-R), even though the endogenous SREBP-1a was decreased by arachidonate under the same conditions.
As an additional test of the hypothesis that arachidonate inhibits the
proteolytic processing of SREBP-1, we tested its effect on the amount
of the COOH-terminal fragment that is generated when SREBP-1 is cleaved
by Site-1 protease. For this purpose, we transfected HEK-293 cells with
a cDNA encoding full-length SREBP-1a with a T7 epitope tag at the
COOH terminus and an HSV epitope tag at the NH2 terminus.
Membranes and nuclear extracts were subjected to electrophoresis and
immunoblotted with antibodies against the T7 and HSV epitope tags,
respectively (Fig. 7). When the cells
were incubated in delipidated serum, the membranes contained the
cleaved COOH terminus, which blotted with the anti-T7 antibody (designated C in lane C). The nuclear extracts
contained the NH2-terminal fragment, which blotted with the
anti-HSV antibody (designated N in lane C). The
amounts of the NH2- and COOH-terminal fragments were both
diminished markedly when the cells were incubated with arachidonate
(lane D), whereas the intact precursor remained in the
membranes (designated P). The transfected mRNA was
unaffected by arachidonate addition, as measured by the RNase
protection assay (data not shown).
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DISCUSSION |
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Taken as a whole, the data in this paper indicate that SREBP-1 is regulated in a different fashion than SREBP-2 in HEK-293 cells. In these cells sterols are the primary regulators of SREBP-2, and they do so primarily by affecting proteolytic processing with only minor changes in the level of mRNA. On the other hand, SREBP-1 is regulated by a combination of the action of sterols and unsaturated fatty acids, and this regulation involves major reductions in mRNA levels (by fatty acids) as well as an apparent reduction in proteolytic processing (by both fatty acids and sterols) and perhaps other post-transcriptional events.
The mechanism by which unsaturated fatty acids lower SREBP-1a and -1c mRNA levels roughly in parallel in HEK-293 cells remains to be explored. These two mRNAs use different promoters that are separated by at least 10 kb (19, 32). The two transcripts contain different first exons with different 5'-untranslated regions and translation start sites. These transcripts splice into a common second exon, and thereafter they are identical. The fatty acids might act on a single transcription factor that regulates both promoters. Alternately, fatty acids might affect transcription elongation through an action on a sequence that is common to both transcripts. They might also affect the stability of the two mRNAs, as suggested for liver by Xu et al. (22). Fatty acids did not affect the level of mRNA encoded by a transfected cDNA. This transcript was driven by a different promoter than the endogenous SREBPs. It contained neither of the two 5'-untranslated regions, and it lacked all of the introns. However, it did contain nearly all of the 3'-untranslated region of the SREBP-1a and -1c transcripts (549 bp).
The likely mechanism for the protein level regulation of SREBP-1a and -1c lies in proteolytic processing. This follows from the observation that fatty acids down-regulate nSREBP-1a when it is produced from a transgene encoding the full-length precursor, but not when it encodes the truncated nuclear form of SREBP-1a. This conclusion is supported strongly by the observation that the level of the other product of SREBP cleavage, i.e. the membrane-bound COOH-terminal fragment is also decreased by arachidonic acid (Fig. 7). We do not know whether this apparent regulation of SREBP-1 cleavage by fatty acids is mediated by SCAP, which mediates the suppressive effects of sterols on SREBP cleavage. Under some conditions fatty acids have a synergistic effect with sterols (see Fig. 5), but whether this synergy is exerted by a combined effect on a single regulatory machinery remains to be determined.
The question of whether fatty acids inhibit SREBP-2 processing is not directly addressed by these studies. In sharp contrast to the reduction in mRNA levels for SREBP-1a and SREBP-1c, fatty acids did not reduce the mRNA for SREBP-2. Moreover, the content of nSREBP-2 did not decline. Yet, we consistently observed an increase in the precursor form of SREBP-2 in the cell membranes when arachidonate was added. These findings raise the possibility that unsaturated fatty acids partially inhibit SREBP-2 processing, but the reduction in nSREBP-2 is minor because the SREBP-2 mRNA remains high and the cells continue to produce relatively large amounts of the precursor form.
Whether the results in HEK-293 cells extend to other cells, most
notably hepatocytes, is unknown. As detailed in the introduction, several laboratories have noted a decrease in SREBP-1 mRNA and nSREBP-1 protein when hepatocytes are incubated with various fatty acids, or when animals are fed diets rich in polyunsaturated fatty acids, but there is disagreement over which fatty acids are most effective, and the mechanism has not been explored in detail. Inasmuch
as the liver is the major site of synthesis of fatty acids and inasmuch
as diets rich in polyunsaturated fatty acids reduce plasma levels of
cholesterol and triglycerides (1-3), the feedback regulation of SREBPs
by fatty acids in liver merits further study.
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ACKNOWLEDGEMENTS |
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We thank Clinton Steffey and Debra Morgan for excellent technical assistance. Lisa Beatty and Dana Applewhite provided invaluable help with the cell cultures.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health (NIH) Research Grant HL20948 and a grant from the Perot Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by NIH Medical Scientist Training Grant GM08014.
§ Supported by NIH Division of Cellular and Molecular Biology Training Grant GM08203.
¶ To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Rm. L5.238, Dallas, TX 75390-9046. Tel.: 214-648-2141; Fax: 214-648-8804; E-mail: jgolds@mednet.swmed.edu.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M007273200
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ABBREVIATIONS |
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The abbreviations used are: SREBP, sterol regulatory element-binding protein; nSREBP, cleaved nuclear form of SREBP; ALLN, N-acetyl-Leu-Leu-norleucinal; BSA, bovine serum albumin; FCS, fetal calf serum; HEK-293 cells, human embryonic kidney-293 cells; HSV, herpes simplex virus; PBS, phosphate-buffered saline; SCAP, SREBP cleavage-activating protein; TK, thymidine kinase; PAGE, polyacrylamide gel electrophoresis.
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