(Received for publication, June 8, 1995; and in revised form, December 22, 1995)
From the
Transcription of the low density lipoprotein (LDL) receptor gene is regulated by intracellular cholesterol concentration, hormones, and growth factors. We studied the mechanisms by which insulin and estradiol stimulate promoter activity of the LDL receptor gene. Hormonal effects were analyzed in HepG2 cells after transient transfection with promotor reporter gene constructs. Successive 5` deletions of the LDL receptor promoter fragment from -537 to +88 revealed the sterol regulatory element 1 (SRE-1) between -65 and -56 as an insulin- and estradiol-sensitive cis-element. If the SRE-1 is point mutated at position -59 (C to G), which abolishes the binding of the SRE binding proteins (SREBP-1 and SREBP-2), no insulin or estradiol stimulatory effect on reporter gene expression was observed, indicating a role of SRE binding proteins in this regulatory mechanism. The concentration of the 125-kDa membrane-integrated SREBP-1 precursor protein in LDL repressed HepG2 cells is not altered by hormone treatment. Concentrations of SREBP-1 mRNA and precursor protein are reduced significantly by high and stable expression of an SREBP-1 antisense cDNA fragment in HepG2 cells (SREBP1(-) cells). Transfection of SREBP1(-) cells with promoter construct phLDL4 (-105 to +88) reduces induction of reporter gene activity by insulin and insulin-like growth factor-I to 35 and 17%, respectively, compared with HepG2 cells. The stimulatory effect of estradiol remains unchanged, and the inductions by pravastatin are enlarged. We conclude that different regulatory effects converge at SRE-1, but that SREBP-1 is selectively involved in the signal transduction pathway of insulin and insulin-like growth factor-I leading to LDL receptor gene activation.
Expression of the LDL ()receptor is mainly regulated
at the transcriptional level by cellular cholesterol
concentration(1) . The regulatory cellular cholesterol pool is
represented by the concentration of oxysterols, which are regulatory
active substances repressing the transcription rate of the LDL
receptor. The regulatory cis-element in the promoter of these genes is
called SRE-1 (sterol regulatory element 1)(2, 3) .
Recently, two SRE binding proteins (SREBP-1 and -2) have been characterized(4, 5, 6, 7) . They are activated by a novel proteolytic mechanism that controls the concentration of the SRE binding proteins and the transcription rate of the LDL receptor gene. SREBP-1 is synthesized as a 125-kDa precursor protein that is embedded in the membranes of the endoplasmic reticulum and nuclear envelope. The amino-terminal segment of SREBP-1 contains an acidic transcriptional activation domain and a basic helix-loop-helix leucine zipper (bHLH-Zip) region that mediates protein dimerization and DNA binding. In the absence of oxysterols a protease cleaves the precursor protein, and the amino-terminal fragment enters the nucleus and activates transcription. When sterols accumulate within the cells the activity of the protease diminishes, and the active transcription factor is no longer generated.
LDL activity is regulated by different hormones and growth factors in different cells. Insulin (8, 9) and estradiol (10, 11) increase LDL receptor activity at the cell surface. This seems to be a result of enhanced gene transcription(12, 13) . We have focused on the characterization of the still unknown mechanisms by which these hormones stimulate the transcription of the LDL receptor gene.
The following is a list of
generated constructs, subcloned promoter regions, and used primers:
phLDL1 (-537 to +88), subcloned from pHLDLR; phLDL2
(-222 to +88), pr4 (ctcttcaccggagacccaaatacaa)/pr3; phLDL3
(-367 to +88), pr5 (acaatggcattaggctattgga)/pr3; phLDL4
(-105 to +88), pr6 (cgaaactcctcctcttgcag)/pr3; phLDL5
(-69 to +88), pr7 (gaaaatcaccccactgcaaactcctccccctgct)/pr3;
phLDL6 (-35 to +88), pr8 (agaaacctcacattgaaatgct)/pr3;
phLDL7 (-105 to +88, C G at position -59), pr9
(gaaaatcaccGcactgcaaactcctccccctgct)/pr10 (aaatgtcttcacctcactgca);
phLDL10 (-105 to +88, -69 to -57 deleted), pr11
(ctgcaaactcctccccctgct)/pr10.
Labeling of each probe was
performed by random priming (random primers DNA labeling kit,
Boehringer Mannheim) using 50 µCi of
[-
P]dCTP (3000 Ci/mmol) and 20 ng of
fragment.
Specific mRNA levels of the LDL receptor and
glyceraldehyde-3-phosphate dehydrogenase as an internal standard were
determined by a reverse transcription polymerase chain reaction
titration assay(17) . Standard RNA was prepared by in vitro transcription using T7 RNA polymerase
(AmpliScribe, Biozyme Laboratories, Ltd.) of a human
LDL receptor cDNA (18) fragment (base pairs 1055-1424)
containing a HindIII cutting site generated by in vitro mutagenesis (19) of C/A (base pair 1376).
A polyclonal antibody against SREBP-1 (K10, 1:500, Santa Cruz Biotechnology, Inc.) directed against a peptide sequence from amino acids 470 to 479 and an alkaline phosphatase-coupled anti-rabbit IgG (Boehringer Mannheim), or alternatively the BM chemiluminescence Western blotting kit (Boehringer Mannheim), was used for Western blot analysis of SREBP-1 in the nuclear membrane fractions according to the manufacturer's instructions. Protein extracts were separated on a 7.5% SDS-PAGE calibrated with prestained SDS-PAGE standards high range (Bio-Rad). Protein concentrations were determined by the method of Bradford(22) .
Figure 1: Promoter reporter gene constructs and their relative promoter activity. HepG2 cells were transiently transfected with several 5`-restricted and two mutated constructs as described under ``Experimental Procedures.'' The promoter strength is represented by the luciferase (Luc) activity measured in the cellular extract. The relative luciferase units (RLU) were corrected for transfection efficiency and presented in percent (±S.D. of 4-6 separate experiments) relative to the construct with the highest promoter activity (phLDL2). Binding sites for transcription factor SP1 (sp1), sterol regulatory element binding proteins (sre-1), and RNA polymerase (tata) are indicated, as is the transcription start point (TS) of the LDL receptor gene.
To localize the cis-elements in the promoter of the LDL receptor gene responsible for the insulin and estradiol action, HepG2 cells were transfected with the different promoter reporter gene constructs. One day after transfection the normal culture medium was changed to medium containing 0.5% LPDS for 24 h, leading to a high basal expression of the reporter gene. 5 h before harvesting the hormones were added to the medium. Fig. 2shows the inducibility of luciferase activity for the different constructs by insulin (100 nM) and estradiol (10 µg/ml) relative to the basal reporter gene expression determined in medium containing 0.5% LPDS alone. Insulin increases luciferase activity about 2-fold in cells transfected with construct phLDL4 or a longer one. In all contructs with a deleted or mutated SRE-1 (phLDL6, phLDL7, and phLDL10) luciferase activity is not stimulated by insulin. Deletion of the distal SP1 recognition site reduces the inducibility only slightly, from 2.0- to 1.6-fold. The results are similar for induction with estradiol, but there is already a stepwise reduction of the inducibility from about 2-fold with construct phLDL2 to 1.6-fold with phLDL4 and to 1.35-fold with phLDL5. A complete and functional SRE-1 is necessary for the stimulation of the LDL receptor gene promoter by insulin and estradiol.
Figure 2: Identification of SRE-1 as the insulin- and estradiol-sensitive element. HepG2 cells were transfected with different promoter constructs and then incubated with insulin (100 nM) (black bar) or estradiol (10 µg/ml) (gray bar) as described under ``Experimental Procedures.'' Luciferase activity is presented as x-fold induction relative to the activity measured in unstimulated cells cultured in 0.5% LPDS (open bar), which is set at 1. Bars represent means ± S.D. of 4-6 separate experiments, each performed in triplicate. RLU, relative luciferase units.
Figure 3: Effect of insulin and IGF-I on mRNA levels of the LDL receptor. HepG2 cells were incubated in 0.5% lipid-depleted serum without (LPDS) or with 250 µg of LDL cholesterol/ml (LDL). Then 100 nM insulin or 10 nM IGF-I were added to the LDL-containing LPDS for various periods of time, as indicated. Total RNA was isolated and the relative abundance of LDL receptor (LDLR) mRNA levels were determined by reverse transcription PCR titration assay as described under ``Experimental Procedures.''
Figure 4: Hormonal effects on the proteolytic mechanism cleaving SREBP-1 precursor protein. Nuclei of HepG2 cells were extracted with detergent and salt as described under ``Experimental Procedures,'' and proteins were separated on a 7.5% SDS-PAGE. The concentration of SREBP-1 precursor protein was determined in a Western blot. HepG2 cells were cultured in medium containing 0.5% LPDS alone (lane 1), with LDL (250 µg/ml) for 12 h (lane 2), with insulin for 12 h (100 nM) (lane 3), and with estradiol (10 µg/ml) for 12 h (lane 4). The marker lane is indicated by M.
Figure 5: Reduction of SREBP-1 mRNA concentration by antisense mRNA in SREBP1(-) cells. Two µg of poly(A) mRNA from SREBP1(-) cells and from HepG2 cells were hybridized successively with specific cDNA probes against SREBP-1 (4 kilobases) and SREBP-2 (4.2 and 5.2 kilobases). Hybridization with a cDNA probe against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed for normalization. The marker lane is indicated by M. Autoradiographs were quantified densitometrically.
To compare the amount of SREBP-1 precursor protein in HepG2 cells and SREBP1(-) cells before and after repression with LDL cholesterol, nuclear protein extracts were analyzed by Western blot (Fig. 6). Cells were harvested after culturing in medium containing 0.5% LPDS for 36 h. The SREBP-1-liberating proteolytic mechanism was repressed by the addition of LDL (250 µg/ml) and 25-hydroxycholesterol (10 µg/ml) 12 and 24 h before harvesting. The accumulation of SREBP-1 precursor protein is severalfold stronger in HepG2 cells than in SREBP1(-) cells after 12 and 24 h of incubation (Fig. 6).
Figure 6: Estimation of SREBP-1 precursor protein concentration in SREBP1(-) and HepG2 cells by Western blot analysis. Proteins (50 µg/lane) of nuclei extracts from SREBP1(-) and HepG2 cells, prepared as described under ``Experimental Procedures,'' were separated on a 7.5% SDS-PAGE and analyzed by Western blotting. Cells were cultured in medium containing 0.5% LPDS for 40 h (lanes 1 and 4). LDL (250 µg/ml) and 25-hydroxycholesterol (10 µg/ml) were added 12 h (lanes 2 and 5) and 24 h (lanes 3 and 6) before harvesting, respectively. SREBP-1 (arrow) was detected with antibody K-10.
Figure 7:
Differential role of SREBP-1 in the
induction of LDL receptor promoter activity. SREBP1(-) (gray
bar) and HepG2 cells (black bar) were transiently
transfected with construct phLDL4 and incubated with LDL (250
µg/ml), pravastatin (10M), insulin
(10
M), IGF-I (10
M), estradiol (10 µg/ml), and forskolin
(10
M) before harvesting as described under
``Experimental Procedures.'' Bars represent the
induction of luciferase activity relative to the values measured in
0.5% LPDS-containing medium alone (No). Values are means of
4-8 separate experiments ± S.D., each done in triplicate.
Statistical significances (Student's t test) were
calculated for the difference between the effects observed in HepG2 versus SREBP(-) cells for insulin and IGF-I (p < 0.0001) and pravastatin (p <
0.002).
We showed that reporter gene constructs of the LDL receptor promoter transiently transfected into HepG2 cells are stimulated approximately 2-fold by both insulin and estradiol. Although several insulin-responsive elements have been identified recently (23) there seems to be no unique sequence mediating the insulin effect. The molecular mechanisms that show how steroid hormones activate transcriptions are understood in more detail(24) , but the DNA consensus sequence (25) recognized by estrogen receptors is not present in the promoter region of the LDL receptor gene. It is possible that the estrogen receptor does not bind directly to the DNA.
Analysis of the hormonal effects with 5`-deleted constructs revealed the region between -69 and -36, where the SRE-1 and the proximal SP1 site are located, as the hormone-sensitive promoter sequence. The point mutation (C to G at position -59) in construct phLDL7 within the SRE-1 destroys the recognition sequence for SRE binding proteins(3, 4) . Insulin and estradiol have no promoter-stimulating effect on this mutated construct. Similar results were obtained with construct phLDL10 in which the SRE-1 is deleted. This construct has a higher promoter activity than phLDL7, which can be explained by the reduced distance of the two SP1 binding sites. We conclude that SRE-1 might be the hormone sensitive cis-element and that binding of the transcription factors SREBP-1 or -2 is involved in the promoter activation by insulin and estradiol.
Insulin increases the LDL receptor mRNA concentration in the presence of repressing concentrations of LDL (12) (see Fig. 3). One possible explanation for such action is that insulin increases the nuclear concentration of SRE binding proteins by activating the proteolytic mechanism that liberates the native SRE binding proteins from their membrane-bound precursor form. We studied the influence of insulin and estradiol on the liberation mechanism of SREBP-1 in LDL-repressed HepG2 cells and found no hormone-induced changes of the SREBP-1 precursor protein pool (Fig. 4) and no change of the mRNA concentrations of SREBP-1 and -2 under these conditions (data not shown). Therefore, it appears that insulin and estradiol do not affect the liberation mechanism of SREBP-1; it is unlikely that a higher SREBP-1 gene expression compensates for this mechanism.
Reduction of SREBP-1 concentration in HepG2 cells enabled us to differentiate between stimulatory effects on LDL receptor promoter activity. High expression of SREBP-1 antisense mRNA in SREBP1(-) cells resulted in a 60% reduction of SREBP-1 mRNA concentration and an even greater decrease in specific protein concentration in the nuclear membrane fraction of LDL cholesterol-repressed HepG2 cells. In transfected SREBP-1-deficient cells only the stimulatory effects of insulin and IGF-1 were reduced to 35 and 17%, respectively, whereas the stimulatory effect of estradiol was unchanged and the induction by the cholesterol synthesis inhibitor pravastatin was even increased. However, the reduced SREBP-1 mRNA level in SREBP1(-) cells was not compensated by a higher expression of SREBP-2. The role of SREBP-2 was not determined in greater detail in this study, but one possible explanation is that SREBP-2 plays a more dominant role in SREBP-1-deficient HepG2 cells, e.g. mediating the effect of the cholesterol synthesis inhibitor. In accordance with that explanation, the depletion of sterols in hamster liver by treating animals with a cholesterol synthesis inhibitor and a bile acid-binding resin led to an increase in nuclear SREBP-2(26) .
Although expression and processing of SRE binding proteins might not be similar between cultured cells, including HepG2 and intact liver, these data indicate that SREBP-1 and SREBP-2 couple different intracellular signal transduction pathways to the sterol response promotor element. After our paper was submitted, a report by Lloyd and Thompson (27) appeared in which the in vivo pattern of protein-DNA interactions in the LDL receptor promotor element was investigated. Interestingly, they show that incubation of HepG2 cells with insulin is associated with a hypermethylation at position -59 in the SRE of the LDL receptor promotor, indicating insulin-induced alterations in protein binding to the SRE region. We conclude from our results that different regulatory effects converge at SRE-1 but that SREBP-1 is selectively involved in the signal transduction pathway of insulin and IGF-I leading to LDL receptor gene activation.