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Originally published In Press as doi:10.1074/jbc.M102516200 on June 13, 2001

J. Biol. Chem., Vol. 276, Issue 32, 30118-30126, August 10, 2001
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In Vivo and in Vitro Regulation of Sterol 27-Hydroxylase in the Liver during the Acute Phase Response

POTENTIAL ROLE OF HEPATOCYTE NUCLEAR FACTOR-1*

Riaz A. MemonDagger, Arthur H. Moser, Judy K. Shigenaga, Carl Grunfeld, and Kenneth R. Feingold

From the Departments of Medicine, University of California, San Francisco and the Metabolism Section, Department of Veterans Affairs Medical Center, San Francisco, California 94121

Received for publication, March 20, 2001, and in revised form, May 31, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The host response to infection is associated with several alterations in lipid metabolism that promote lipoprotein production. These changes can be reproduced by lipopolysaccharide (LPS) administration. LPS stimulates hepatic cholesterol synthesis and suppresses the conversion of cholesterol to bile acids. LPS down-regulates hepatic cholesterol 7alpha -hydroxylase, the rate-limiting enzyme in the classic pathway of bile acid synthesis. We now demonstrate that LPS markedly decreases the activity of sterol 27-hydroxylase, the rate-limiting enzyme in the alternate pathway of bile acid synthesis, in the liver of Syrian hamsters. Moreover, LPS progressively decreases hepatic sterol 27-hydroxylase mRNA levels by 75% compared with controls over a 24-h treatment period. LPS also decreases oxysterol 7alpha -hydroxylase mRNA levels in mouse liver. In vitro studies in HepG2 cells demonstrate that tumor necrosis factor and interleukin (IL)-1 decrease sterol 27-hydroxylase mRNA levels by 48 and 80%, respectively, whereas IL-6 has no such effect. The IL-1-induced decrease in sterol 27-hydroxylase mRNA expression occurs early, is sustained for 48 h, and requires very low doses. In vivo IL-1 treatment also lowers hepatic sterol 27-hydroxylase mRNA levels in Syrian hamsters. Studies investigating the molecular mechanisms of LPS-induced decrease in sterol 27-hydroxylase show that LPS markedly decreases mRNA and protein levels of hepatocyte nuclear factor-1 (HNF-1), a transcription factor that regulates sterol 27-hydroxylase, in the liver. Moreover, LPS decreases the binding activity of HNF-1 by 70% in nuclear extracts in hamster liver, suggesting that LPS may down-regulate sterol 27-hydroxylase by decreasing the binding of HNF-1 to its promoter. Coupled with our earlier studies on cholesterol 7alpha -hydroxylase, these data indicate that LPS suppresses both the classic and alternate pathways of bile acid synthesis. A decrease in bile acid synthesis in liver would reduce cholesterol catabolism and thereby contribute to the increase in hepatic lipoprotein production that is induced by LPS and cytokines.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The host response to infection and inflammation is accompanied by alterations in triglyceride and cholesterol metabolism (1-3). These metabolic changes can also be induced by the administration of lipopolysaccharide (LPS),1 which mimics Gram-negative infections (4). Low doses of LPS rapidly increase hepatic triglyceride synthesis and VLDL production, whereas high doses of LPS inhibit triglyceride clearance by decreasing lipoprotein lipase activity in heart, muscle, and adipose tissue and in post-heparin plasma (5).

In rodents, LPS treatment increases serum cholesterol levels; however, this effect is delayed in onset and is primarily accounted for by an increase in LDL cholesterol (6). LPS stimulates hepatic cholesterol synthesis and increases the mRNA expression, protein mass, and activity of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, in the liver (6). The effect of LPS on hepatic HMG-CoA reductase is specific, because mRNA levels of other important enzymes in cholesterol synthesis, such as HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, are not increased (7, 8). The effect of LPS on LDL clearance is not clear because some studies have shown no change (6), whereas others have demonstrated an increase in LDL receptor mRNA and protein levels in the liver (9). An increase in hepatic cholesterol synthesis and VLDL production could contribute to an increase in serum LDL levels in rodents.

Bile acids are formed in the liver from cholesterol, and their synthesis represents the major pathway for the elimination of cholesterol from the body (10). An increase in bile acid synthesis can lead to a decrease in plasma cholesterol levels. Recent studies have shown that there are two distinct pathways of bile acid synthesis in mammalian liver (reviewed in Ref. 11). The classic or neutral pathway is initiated by microsomal cholesterol 7alpha -hydroxylase (also known as CYP7A) that converts cholesterol into 7alpha -hydroxycholesterol, which is subsequently converted into primary bile acids (12). The alternate or acidic pathway is initiated by mitochondrial sterol 27-hydroxylase (also known as CYP27A) that converts cholesterol into 27-hydroxycholesterol (13), which is then converted into 7alpha ,27-dihydroxycholesterol by oxysterol 7alpha -hydroxylase (also known as CYP7B) and subsequently metabolized into primary bile acids.

Although the regulation of microsomal cholesterol 7alpha -hydroxylase has been extensively studied under physiological and pathological conditions (11, 14), very little is known about the regulation of mitochondrial sterol 27-hydroxylase. Diets supplemented with 5% cholestyramine increase and hydrophobic bile acids decrease the activity, mRNA levels, and transcription of sterol 27-hydroxylase (15). Recent in vivo and in vitro studies suggest that alternate pathway may contribute as much as 50% to the total bile acid synthesis (16, 17). Because bile acid synthesis is the major pathway for elimination of cholesterol from the body, a decrease in bile acid synthesis would increase the availability of cholesterol for lipoprotein production. We have previously shown that LPS and cytokines decrease the activity and mRNA expression of cholesterol 7alpha -hydroxylase in the liver of Syrian hamsters (18). This study was designed to determine the effect of LPS and cytokine treatment on the activity and mRNA expression of sterol 27-hydroxylase in the liver of Syrian hamsters and in HepG2 cells, a human hepatoma cell line. We also studied the effect of LPS on oxysterol 7alpha -hydroxylase mRNA levels in the liver. Finally, we have examined the effect of LPS on the mRNA expression, protein levels, and binding activity of HNF-1, a transcription factor that has been shown to regulate sterol 27-hydroxylase, in the liver of Syrian hamsters.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [alpha -32P]dCTP (3,000 Ci/mmol) and [4-14C]cholesterol (51 mCi/mol) were purchased from PerkinElmer Life Sciences. LPS (Escherichia coli 55:B5) was purchased from Difco Laboratories (Detroit, MI). Recombinant human interleukin (IL)-1beta (specific activity, 1 × 109 units/mg) was kindly provided by Immunex (Seattle, WA). Human tumor necrosis factor-alpha (TNF-alpha ) (specific activity, 1.1 × 105 units/µg) and human IL-6 (specific activity, 1.1 × 105 units/µg) were purchased from R&D Systems (Minneapolis, MN). The endotoxin concentration in the TNF-alpha , IL-1beta , and IL-6 preparations was below the levels detected by the Limulus assay. Multiprime DNA labeling system and Sephadex G-25 columns were purchased from Amersham Pharmacia Biotech; minispin G-50 columns were from Worthington Biochemical Corporation (Freehold, NJ); oligo(dT) cellulose type 77F was from Amersham Pharmacia Biotech; and nitrocellulose and Nytran were from Scleicher & Schuell (Keene, OH). The oligonucleotide sequence for hepatocyte nuclear factor-1 (HNF-1) response element was purchased from Operon Technologies (Alameda, CA). Goat polyclonal antibodies raised against human HNF-1alpha and -beta were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The cDNAs for sterol 27-hydroxylase (13) and oxysterol 7alpha -hydroxylase (19) were kindly provided by Dr. David Russell (University of Texas Southwestern Medical Center, Dallas, TX). The cDNA for HNF-1 (20) was kindly provided by Dr. Gerald Crabtree (Stanford University, Palo Alto, CA).

Animal Procedures-- Male Syrian hamsters (~140-160 g) were purchased from Charles River Laboratories (Wilmington, MA). The animals were maintained in a reverse light cycle room (3 a.m. to 3 p.m. dark, 3 p.m. to 3 a.m. light) and were provided with rodent chow and water ad libitum. Anesthesia was induced with halothane, and the animals were injected intraperitoneally with either LPS (at the indicated doses in 0.5 ml of 0.9% saline) or saline alone. Subsequently food was withdrawn from both control and treated animals because LPS induces anorexia (21). The animals were killed between 4 and 24 h after LPS administration as indicated in the text. The doses of LPS used (1-100 µg/100 g of body weight (BW)) have significant effects on triglyceride and cholesterol metabolism in Syrian hamsters (5-8) but are far below the doses required to cause death in rodents (LD50 = ~5 mg/100 g bw). In a separate experiment, animals were injected with IL-1 (1 µg/100 g of BW) or saline, and 16 h later the livers were obtained for RNA isolation.

Female C57Bl/6 mice (20 g) were purchased from Jackson Laboratories (Bar Harbor, ME). The animals were maintained on a normal 12-h light cycle and were fed mouse chow (Ralston-Purina, St. Louis, MO) and water ad libitum. The animals were injected with LPS (0.1-100 µg/kg of BW, intraperitoneally) or saline, and the food was withdrawn from both groups. Sixteen hours after the treatment, the animals were sacrificed, and the livers were obtained for measuring sterol 27-hydroxylase and oxysterol 7alpha -hydroxylase mRNA levels.

Sterol 27-Hydroxylase Activity-- The activity of mitochondrial sterol 27-hydroxylase in the liver was measured as described by Souidi et al. (22). Briefly, the livers were homogenized, and the mitochondria were isolated by differential centrifugation. The reaction mixture for the sterol 27-hydroxylase assay contained 75 mM KH2PO4 buffer (pH 7.4), 1 mM EDTA, 0.5 mM dithiothreitol, 5 mM MgCl2, 1 mM NADPH, [14C]cholesterol (1.1 × 106 dpm, 26,000 pmol, 52 µM), 4.5 mg of hydroxypropyl-beta -cyclodextrin, 5 mM sodium isocitrate, 0.2 unit of isocitrate dehydrogenase, and 250-300 µg of mitochondrial protein. The reaction was started with the addition of NADPH and terminated 6 min later by adding 40 µl of 5 N NaOH. After neutralization, the sterols were extracted with 4.3 ml of dichloromethane:ethanol (5:1 v/v) plus 1.2 ml of H2O. Unlabeled 27-hydroxycholesterol (75 µg) was added and separated from cholesterol by TLC on Silica gel G plates using ethyl acetate:hexane (1:1, v/v) as a solvent. The bands were identified and counted by liquid scintillation spectrometry. Control reactions containing no protein were run in parallel to correct for the background. Sterol 27-hydroxylase activity is expressed as picomoles of 27-hydroxycholesterol formed per mg protein per min. The protein was assayed by the method of Bradford (Bio-Rad).

Isolation of RNA and Northern Blotting-- Total RNA was isolated by a variation of the guanidinium thiocyanate method (23) as described earlier (6). Poly(A)+ RNA from liver was isolated using oligo(dT) cellulose and quantified by measuring absorption at 260 nm. Gel electrophoresis, transfer, and Northern blotting were performed as described earlier (6). The uniformity of sample applications was checked by ultraviolet visualization of the acridine orange-stained gels before transfer to Nytran membranes. The cDNA probe hybridization was performed as described previously (6). The blots were exposed to x-ray films for various durations to ensure that measurements were done on the linear portion of the curve, and the bands were quantified by densitometry. The blots were also probed with cyclophilin as a housekeeping gene, and the densitometric values were normalized relative to cyclophilin.

Preparation of Nuclear Extracts-- Nuclear extracts for Western blots and electrophoretic gel mobility shift assays were prepared as described previously (24). Briefly, fresh livers (1.5-2 g), obtained after LPS or saline treatment, were homogenized in 10 mM HEPES (pH 7.9), 25 mM KCl, 0.15 mM spermine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 50 mM NaF, 2 mM sodium metavanadate, 0.5 mM dithiothreitol, and 1% protease inhibitor mixture (Sigma). Immediately following homogenization, the nuclear proteins were extracted as described by Neish et al. (25), except that 1 mM NaF, 0.1 mM sodium metavanadate, and 1% protease inhibitor mixture (Sigma) were added to all buffers. The nuclear protein content was determined by the Bradford assay (Bio-Rad).

Western Blot Analysis-- Denatured nuclear protein (20 µg) was loaded onto 10% polyacrylamide precast gels (Bio-Rad) and subjected to electrophoresis. After electrotransfer onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), the blots were blocked with phosphate-buffered saline containing 0.10% Tween and 5% dry milk for 1 h at room temperature and incubated for 1 h at room temperature with a polyclonal anti-HNF-1alpha antibody (Santa Cruz Biotechnology) at a dilution of 1:10,000. Immune complexes were detected using horseradish peroxidase-linked donkey anti-goat IgG (dilution, 1:10,000) according to the ECL Plus Western blotting kit (Amersham Pharmacia Biotech). Immunoreactive bands obtained by autoradiography were quantified by densitometry.

Electrophoretic Gel Mobility Shift Assays-- Electrophoretic gel mobility shift assays were performed as described earlier (24). Briefly, 10 µg of crude nuclear extract were incubated on ice for 30 min with 6 × 104 cpm of 32P-labeled oligonucleotides in 15 µl of binding buffer and 6 µg of poly(dI-dC). Double-stranded oligonucleotide probes were end-labeled with T4-polynucleotide kinase in the presence of 10 µCi of [gamma -32P]dATP and purified on a Sephadex G-25 column (Amersham Pharmacia Biotech). DNA-protein complexes were separated by electrophoresis, and the gel was dried, exposed to x-ray film, and quantified by densitometry (24). In the competition assay, a 100-fold molar excess of specific or mutated unlabeled oligonucleotide was incubated on ice for 1 h with 10 µg of crude nuclear extract from a control hamster in the binding buffer before adding the labeled oligonucleotide probe. The oligonucleotide sequence used in this study was derived from the rat albumin promoter (26) and was as follows: HNF-1 response element, 5'-GGTAAGTATGGTTAATGATCTACAGTTA-3'; mutant HNF-1 response element, 5'-GGTAAGTATGAGCGATGATCTACAGTTA-3'. In supershift studies, control nuclear extracts were preincubated with 1 µl of one of the following antibodies for 1 h at room temperature prior to the addition of labeled probe: anti-HNF-1alpha , anti-HNF-1beta , and control goat IgG.

HepG2 Cell Culture and Cytokine Treatment-- HepG2 cells were obtained from American Type Culture Collection and maintained in minimum essential medium (Mediatech, Inc., Herdon, VA) supplemented with 10% fetal bovine serum under standard culture conditions (5% CO2, 37 °C). The cells were seeded into 100-mm culture dishes and allowed to grow to 80% confluence. Immediately prior to the experiment, the cells were washed with calcium-magnesium-free phosphate-buffered saline, and the experimental medium (Dulbecco's modified Eagle's medium + 0.1% bovine serum albumin) containing TNF, IL-1, or IL-6 (at concentrations indicated in figure legends) was added. The cells were incubated at 37 °C for the indicated times. RNA isolation and Northern blotting were performed according to methods described previously (6).

Statistics-- The results are expressed as the means ± S.E. Statistical significance between two groups was determined by using the Student's t test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies from our laboratory have shown that LPS markedly decreases cholesterol 7alpha -hydroxylase activity and mRNA levels in the liver of Syrian hamsters (18). In this study, we first examined the effect of LPS (100 µg/100 g of BW) on sterol 27-hydroxylase activity in the liver of Syrian hamsters. The data presented in Fig. 1 demonstrate that 24-h LPS treatment produced a 63% decrease in mitochondrial sterol 27-hydroxylase activity in the liver.


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  		Fig. 1.   Effect of LPS on sterol 27-hydroxylase activity in liver. The animals were injected intraperitoneally with either saline or LPS (100 µg/100 g of BW). Twenty-four hours later the animals were killed, the liver mitochondria were isolated, and sterol 27-hydroxylase activity was determined as described under "Experimental Procedures." The value for sterol 27-hydroxylase activity in the control groups was 146 ± 3.47 pmol/min/mg protein. The data are presented as percentages of controls (mean ± S.E.), n = 5 for each group. *, p < 0.001 versus control.

To investigate the mechanism for LPS-induced decrease in sterol 27-hydroxylase activity, we next examined the effect of LPS on sterol 27-hydroxylase mRNA levels in the hamster liver. As shown in Fig. 2A, LPS significantly decreased hepatic sterol 27-hydroxylase mRNA levels. A modest decrease (35%) was first observed at 8 h after LPS administration, and a 75% decrease was seen after 24 h of LPS treatment. A dose-response experiment was therefore performed 24 h after LPS administration. Moderate doses of LPS were required to decrease hepatic sterol 27-hydroxylase mRNA levels, with a half-maximal response seen at less than 10 µg/100 g of BW (Fig. 2B). These data suggest that LPS decreases sterol 27-hydroxylase activity by inhibiting its mRNA expression.


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  		Fig. 2.   Time course (A) and dose response (B) for the effect of LPS on sterol 27-hydroxylase mRNA levels in the liver. Syrian hamsters were injected intraperitoneally with either saline or LPS (100 µg/100 g of BW) (A) or with LPS at doses indicated on the x axis (B). The animals were killed at various time points (A) or 24 h after LPS administration (B), the livers were obtained, and poly(A)+ RNA was isolated. Northern blots were probed with sterol 27-hydroxylase cDNA as described under "Experimental Procedures." The data are presented as the percentages of control values as quantified by densitometry (mean ± S.E.), n = 5 for each group in A and 4 for each group in B. A, *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, *, p < 0.002; **, p < 0.001.

We also examined the effect of LPS treatment on oxysterol 7alpha -hydroxylase, an enzyme that is unique to the alternative pathway of bile acid synthesis (11). Both mouse (19) and human (27) oxysterol 7alpha -hydroxylase have recently been cloned. These studies demonstrate a marked size difference between human and mouse oxysterol 7alpha -hydroxylase mRNA transcripts (9 kilobases in human and 2.5 kilobases in mouse). Our initial studies showed that the murine cDNA for oxysterol 7alpha -hydroxylase did not cross-react with oxysterol 7alpha -hydroxylase mRNA in hamster liver. Hence, we switched species and examined the effect of 16-h LPS treatment on both oxysterol 7alpha -hydroxylase and sterol 27-hydroxylase in mouse liver. The data presented in Fig. 3 show that, as seen in hamsters, LPS markedly decreased sterol 27-hydroxylase mRNA levels in mouse liver. This effect of LPS was very sensitive, and doses as low as 0.1 µg/mouse produced a 58% decrease in sterol 27-hydroxylase mRNA expression in mouse liver; a maximum decrease of 83% was seen at a dose of 1 µg/mouse. On the other hand, higher doses of LPS (10 µg/mouse) were required to produce a 50% decrease in oxysterol 7alpha -hydroxylase mRNA expression in mouse liver (Fig. 3).


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  		Fig. 3.   Dose response for the effect of LPS on sterol 27-hydroxylase (CYP27) and oxysterol 7alpha -hydroxylase (CYP7b) mRNA expression the liver in mice. C57Bl/6 mice were injected intraperitoneally with either saline or LPS at the doses indicated on the x axis. The animals were killed at 16 h after LPS administration, the livers were obtained, and poly(A)+ RNA was isolated. Northern blots were probed with sterol 27-hydroxylase and oxysterol 7alpha -hydroxylase cDNAs as described under "Experimental Procedures." The data are presented as the percentages of control values as quantified by densitometry (mean ± S.E.), n = 4 for each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Because pro-inflammatory cytokines such as TNF, IL-1, and IL-6 mediate many of the metabolic alterations that occur during the host response to infection and inflammation (4), it is possible that these cytokines could directly regulate sterol 27-hydroxylase. To address this question, we examined the effect of TNF-alpha , IL-1beta , and IL-6 in HepG2 cells, a human hepatoma cell line. TNF-alpha and IL-1beta decreased sterol 27-hydroxylase mRNA by 48 and 80%, respectively, in HepG2 cells, whereas IL-6 had no such effect (Fig. 4). We were unable to detect oxysterol 7alpha -hydroxylase mRNA expression in HepG2 cells using the murine oxysterol 7alpha -hydroxylase cDNA probe. Because IL-1beta was most effective in decreasing sterol 27-hydroxylase mRNA levels in HepG2 cells, we performed additional studies on the effect of IL-1beta in HepG2 cells. The data presented in Fig. 5A show that IL-1beta induces a marked decrease in sterol 27-hydroxylase mRNA levels by 8 h, and the effect is sustained for at least 48 h. Furthermore, the dose-response studies demonstrate that the maximal decrease in sterol 27-hydroxylase mRNA levels is observed at 1 ng/ml IL-1beta , and the half-maximal response occurs at less than 0.1 ng/ml (Fig. 5B), suggesting that the decrease in sterol 27-hydroxylase mRNA levels in HepG2 cells is a very sensitive response to IL-1beta .


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  		Fig. 4.   Effect of cytokines on sterol 27-hydroxylase mRNA in HepG2 cells. TNF, IL-1, or IL-6 were added to HepG2 cells at a concentration of 100 ng/ml. Twenty-four hours later, poly(A)+ RNA was isolated. Northern blots were probed with sterol 27-hydroxylase cDNA as described under "Experimental Procedures." The data are presented as the percentages of control values as quantified by densitometry (mean ± S.E.). n = 4 for each group. *, p < 0.05; **, p < 0.001.


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  		Fig. 5.   Time course (A) and dose response (B) for the effect of IL-1 on sterol 27-hydroxylase mRNA in HepG2 cells. HepG2 cells were incubated with 100 ng/ml IL-1 at the indicated times (A) or with the indicated concentrations of IL-1 (B) for 24 h. At the end of the incubations, poly(A)+ RNA was isolated, and Northern blots were probed with sterol 27-hydroxylase cDNA as described under "Experimental Procedures." The data are presented as the percentages of control values as quantified by densitometry (mean ± S.E.), n = 4 for all groups. A, *, p < 0.002; **, p < 0.001. B, *, p < 0.001.

We also examined the in vivo effect of IL-1 on sterol 27-hydroxylase mRNA levels in the liver of Syrian hamsters. The data presented in Fig. 6 show that 16-h IL-1 treatment (1 µg/100 g of BW) produced a 50% decrease in sterol 27-hydroxylase mRNA levels in the liver. Taken together, these data suggest that sterol 27-hydroxylase is down-regulated during the acute phase response, and IL-1 is capable of mediating this metabolic response in vivo.


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  		Fig. 6.   Effect of IL-1 on sterol 27-hydroxylase mRNA in liver. The animals were injected intraperitoneally with saline or IL-1 (1 µg/100 g of BW). Sixteen hours later the animals were killed, the livers were obtained, and poly(A)+ RNA was isolated. Northern blots were probed with sterol 27-hydroxylase cDNA as described under "Experimental Procedures." The data are presented as the percentages of control values as quantified by densitometry (means ± S.E.), n = 5 for each group. *, <0.002.

To investigate the molecular mechanism involved in the down-regulation of sterol 27-hydroxylase mRNA levels, we examined the effect of LPS on HNF-1 in vivo. A recent study reported that bile acids suppress sterol 27-hydroxylase transcription by decreasing the binding of HNF-1 to sterol 27-hydroxylase promoter (28). The effect of 16-h LPS treatment on the binding of nuclear proteins to the HNF-1 response element is presented in Fig. 7A. Densitometric analysis of the data shows that LPS decreased the binding to the HNF-1 response element by 70% in nuclear extracts in the hamster liver. Competition with a 100-fold molar excess of specific oligonucleotide, but not of mutated oligonucleotide, demonstrates the specificity of binding. In addition, the band was supershifted after incubation of control nuclear extracts with anti-HNF-1alpha antibody, but not with anti-HNF-1beta or nonspecific IgG (Fig. 7B), suggesting that the shifted bands in Fig. 7A are due to HNF-1alpha in the nuclear extracts. These results indicate that LPS specifically decreases the binding activity to the HNF-1 response element in the hamster liver. Thus, it is possible that LPS decreases sterol 27-hydroxylase mRNA levels by decreasing the binding of HNF-1 to sterol 27-hydroxylase promoter in the liver.


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  		Fig. 7.   Effect of LPS on HNF-1 binding activity in nuclear extracts in liver. Syrian hamsters were injected intraperitoneally with either saline or LPS (100 µg/100 g of BW). Sixteen hours later hepatic nuclear extracts were prepared and incubated with radiolabeled oligonucleotide representing specific binding site for HNF-1 as described under "Experimental Procedures." A, representative electrophoretic gel mobility shift assay. Unlabeled specific (wild type) and nonspecific (mutant) competing oligonucleotides at 100-fold molar excess were added 1 h prior to the addition of the labeled oligonucleotide probes. Lane 1, free probe (FP); lanes 2-6, control; lanes 7-11, LPS; lane 12, specific unlabeled oligonucleotide (WT); lane 13, mutated unlabeled oligonucleotide (MU). B, electrophoretic gel mobility supershift assay using a nuclear extract from a control hamster and performed in the absence (lane 1) and the presence of antibodies raised against HNF-1alpha (lane 2), HNF-1beta (lane 3), and control goat IgG (lane 4). The arrow shows the complex supershifted by HNF-1alpha antibody.

To identify the mechanism for the LPS-induced decrease in HNF-1 activity in liver, we examined the effect of LPS on hepatic HNF-1 mRNA and protein levels. We examined the HNF-1 mRNA levels in the hamster liver at 4 and 8 h after LPS treatment. The data presented in Fig. 8A show that LPS produced 90-95% decrease in HNF-1 mRNA levels in the liver at both 4 and 8 h after treatment. We also examined the effect of 16-h LPS treatment on HNF-1alpha protein in the nuclei from hamster liver by Western blot (Fig. 8B). Densitometric analysis of the data show that LPS produced a 82% decrease in HNF-1alpha protein level in the liver nuclei. Taken together, these results indicate that LPS acutely decreases the transcription of HNF-1 in the liver, which is followed by a decrease in HNF-1alpha protein mass and its binding activity, suggesting that a LPS-induced decrease in HNF-1 activity may be the potential mechanism for the down-regulation of sterol 27-hydroxylase during the acute phase response.


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  		Fig. 8.   Effect of LPS treatment on HNF-1 mRNA expression (A) and protein mass (B) in the liver. Syrian hamsters were injected IP with either saline or LPS (100 µg/100 g of BW). The animals were killed at 4 and 8 h after LPS administration for poly(A)+ RNA isolation for Northern blots and at 16 h for isolating hepatic nuclear extracts for Western blots. Northern blots were probed with sterol 27-hydroxylase cDNA as described under "Experimental Procedures." A, HNF-1 mRNA expression presented as the percentages of control values as quantified by densitometry (mean ± S.E.), n = 5 for each group. *, p < 0.001. B, a representative Western blot for HNF-1alpha protein.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous studies from our laboratory have reported that in vivo administration of LPS, a component of Gram-negative bacterial cell wall, markedly decreases the activity and mRNA expression of cholesterol 7alpha -hydroxylase in the livers of Syrian hamsters (18). We now demonstrate that LPS decreases the activity and mRNA levels of sterol 27-hydroxylase in the liver of Syrian hamsters. The earliest decrease in sterol 27-hydroxylase mRNA levels was observed after 8 h of LPS treatment, and the maximal decrease occurred after 24 h. LPS also decreased hepatic sterol 27-hydroxylase activity, suggesting that LPS decreases the enzyme activity by suppressing its gene expression. Additionally, LPS produced a marked decrease in sterol 27-hydroxylase mRNA levels and a modest decrease in oxysterol 7alpha -hydroxylase in mice, and these effects were dose-dependent, with sterol 27-hydroxylase being more sensitive to LPS. These results suggest that the effect of LPS on sterol 27-hydroxylase is consistent across different rodent species. Our previous studies demonstrated that the LPS-induced decrease in cholesterol 7alpha -hydroxylase mRNA levels occurred very rapidly (65% decrease by 90 min, with a sustained decrease of 90% for 16 h) after LPS administration (18). Taken together, these data suggest that both the classic and alternate pathways of bile acid synthesis are sequentially down-regulated during the acute phase response.

It has been proposed that the alternate pathway of bile acid synthesis may play at least two distinct roles (29, 30). In its first role, it may act as a backup pathway for the classic cholesterol 7alpha -hydroxylase pathways, whereas in its second role it may serve to increase the diversity of bile acids species in the bile to ensure maximum solubilization of different dietary fats and vitamins. Studies in cholesterol 7alpha -hydroxylase knockout mice have shown that the initial bile acid deficiency caused by disruption of classic pathway in the knockout mice is compensated by the induction of enzymes of alternate pathway of bile acid synthesis (29, 30). Conversely, deletion of sterol 27-hydroxylase gene in mice results in a compensatory increase in cholesterol 7alpha -hydroxylase expression in the liver (31). However, our results indicate that during the acute phase response no compensatory response occurs, because both cholesterol 7alpha -hydroxylase (18) and sterol 27-hydroxylase (present study) mRNA levels and activity in the liver are markedly decreased by LPS treatment. These data suggest that during the host response to infection the need of the body to conserve cholesterol is so essential that both pathways of bile acid synthesis are down-regulated to prevent the elimination of cholesterol. Others have shown that LPS administration in rodents reduces basal bile flow, bile salt secretion, and mRNA expression of several transporters that are involved in the hepatocellular uptake and canalicular excretion of bile salts (32, 33).

The LPS-induced down-regulation of sterol 27-hydroxylase (present study) and cholesterol 7alpha -hydroxylase (18) could play an important role in the changes in hepatic production of lipoproteins during the acute phase response. Studies have shown that overexpression of cholesterol 7alpha -hydroxylase gene in hamster liver by the adenovirus-mediated gene transfer technique increases total bile acid synthesis and decreases serum total and LDL levels (34). Similar results were obtained by overexpression of cholesterol 7alpha -hydroxylase gene in LDL receptor knockout mice (35), suggesting that an increase in bile acid synthesis can markedly lower circulating lipoprotein levels. Conversely, a LPS-induced decrease in total bile acid production through inhibition of the regulatory enzymes of the classic as well as alternate pathways of bile acid synthesis as shown here should result in an increase in the pool of cholesterol available for lipoprotein production. Previous studies from our and other laboratories have shown that LPS and cytokines increase serum triglyceride and cholesterol levels by increasing hepatic lipoprotein production and by decreasing triglyceride clearance (4-8). The increase in serum lipid levels during the acute phase response may be beneficial. Studies have shown that lipoproteins bind LPS and that this binding can prevent the deleterious effects of LPS (36, 37). Moreover, an increase in serum lipids may result in an enhanced delivery of lipids to cells that are activated during the host response to infectious and inflammatory stimuli.

The effects of LPS are mediated by its ability to stimulate a variety of immune cells resulting in the synthesis and secretion of a multitude of cytokines including TNF-alpha , IL-1beta , and IL-6 (38, 39). The stimulation of acute phase protein synthesis and the changes in lipid and lipoprotein metabolism during infection and inflammation are now known to be mediated by these cytokines (40). We have previously shown that TNF-alpha and IL-1beta increase serum triglyceride and cholesterol levels, stimulate hepatic lipogenesis, and enhance VLDL production (4). Moreover, both TNF-alpha and IL-1beta decrease cholesterol 7alpha -hydroxylase mRNA levels in the liver (18). In the present study, we demonstrate both TNF-alpha - and IL-1beta -decreased sterol 27-hydroxylase mRNA levels in HepG2 cells, a human hepatoma cell line, suggesting that cytokine mediators of acute phase response can directly affect hepatocyte sterol 27-hydroxylase. Furthermore, we show that IL-1beta mimics the effect of LPS on hepatic sterol 27-hydroxylase mRNA levels in vivo. It is likely that IL-1beta and TNF-alpha are not the only mediators of this effect because LPS treatment induces a number of cytokine and peptide mediators (38), and it is now well recognized that cytokines have redundant actions (40) with multiple cytokines influencing lipid metabolism (4).

Several recent studies have addressed the molecular mechanisms involved in the transcriptional regulation of cholesterol 7alpha -hydroxylase (41, 42). It was shown recently that oxysterol-induced activation of cholesterol 7alpha -hydroxylase involves liver X receptor (LXR) (43, 44), whereas suppression of cholesterol 7alpha -hydroxylase gene by bile acids is mediated by farnesoid X receptor (45). The cholesterol 7alpha -hydroxylase gene promoter also contains binding sites for peroxisome proliferator activated receptor-alpha (PPAR-alpha ) and retinoid X receptor (RXR) (46). It has also been shown that the proximal promoter region of hamster cholesterol 7alpha -hydroxylase gene contains HNF-3 and HNF-4 response elements (47). We have recently shown that LPS acutely decreases the mRNA and protein levels of RXR isoforms (alpha , beta , and gamma ), LXR-alpha , and PPAR-alpha in the liver of Syrian hamsters (24). Moreover, LPS treatment reduces RXR-RXR, RXR-PPAR, and RXR-LXR binding activities in nuclear extracts in the liver (24). The LPS-induced decrease in LXR-alpha and RXR-LXR heterodimerization could be a potential mechanism for the decrease in cholesterol 7alpha -hydroxylase that is observed during the acute phase response. On the other hand, it is possible that the decrease in cholesterol 7alpha -hydroxylase is mediated by a decrease in HNF-3 and/or HNF-4, because their mRNA expression is also decreased during the acute phase response (48, 49).

In contrast to cholesterol 7alpha -hydroxylase, much less is known about the transcription factors that may regulate sterol 27-hydroxylase. A recent study reported that bile acids suppress sterol 27-hydroxylase transcription by decreasing the binding of HNF-1 to sterol 27-hydroxylase promoter (28). HNF-1 is a transcription factor that is expressed in liver, digestive tract, pancreas, and kidney (50) and is involved in the regulation of large number of hepatic genes including albumin, fibrinogen, alpha 1-antitrypsin. Burke et al. (51) have previously reported that HNF-1 mRNA levels and binding activity are decreased in the liver in a severe burn injury model in mice (48). The data presented in this study show that LPS markedly decreases HNF-1 binding activity in nuclear extracts in the hamster liver as demonstrated by gel shift and supershift assays using the HNF-1 response element. Moreover, LPS suppresses HNF-1 binding activity by decreasing its mRNA expression and protein mass. Thus, it is possible that a LPS-induced decrease in HNF-1 expression could be a potential mechanism for the decrease in sterol 27-hydroxylase that is seen during the acute phase response. It is also interesting to note that several other genes that contain a functional HNF-1 binding sequence such as albumin (52), UDP-glucuronosyltransferase (26), alpha -fetoprotein (53), microsomal triglyceride transfer protein (54), liver fatty acid binding protein (55), phosphoenolpyruvate carboxykinase (56), and insulin-like growth factor-I (57) are also down-regulated during the host response to infection and inflammation (58-62). To definitively demonstrate that the decrease in sterol 27-hydroxylase expression is due to a decrease in HNF-1, one could mutate the HNF-1 response element in HepG2 cells and see whether the inhibitory effect of cytokine treatment on sterol 27-hydroxylase expression is lost. Unfortunately, in HNF-1-responsive genes such as microsomal triglyceride transfer protein and sterol 27-hydroxylase, mutations of the HNF-1 response element result in very low basal expression (28, 61), which makes it difficult to demonstrate that the removal of this response element would prevent suppression of sterol 27-hydroxylase gene expression by cytokines.

It has been shown previously that, in addition to its modulation by nuclear hormone receptors, cholesterol 7alpha -hydroxylase is also regulated by activation of protein kinase C (11). Because protein kinases are involved in the phosphorylation of transcription factors such as PPARs and RXRs (63, 64), it is likely that there is cross-talk between kinase signaling cascades and nuclear hormone receptors in regulating bile acid homeostasis. A very recent study by Gupta et al. (65) reported that bile acids can rapidly down-regulate cholesterol 7alpha -hydroxylase transcription by activation of c-Jun N-terminal kinase pathway in cultured rat hepatocytes. They also showed that small heterodimer partner-1, which is induced by farnesoid X receptor to repress cholesterol 7alpha -hydroxylase transcription, is a direct target of activated c-Jun. Finally, they showed that TNF-alpha rapidly activated c-Jun N-terminal kinase pathway and down-regulated cholesterol 7alpha -hydroxylase mRNA levels. Whether similar additional regulatory mechanism(s) are involved in the modulation of enzymes of alternate bile acid synthesis pathway is currently not known.

In summary, the present study demonstrates that LPS produces a marked decrease in sterol 27-hydroxylase activity and mRNA levels in the liver of Syrian hamsters that may be mediated by a LPS-induced decrease in HNF-1 binding activity. Coupled with our earlier studies on cholesterol 7alpha -hydroxylase, these data indicate that LPS suppresses both the classic and alternate pathways of bile acid synthesis. A decrease in total bile acid synthesis could facilitate the formation and secretion of lipoproteins in the liver that may contribute to the host defense during the acute phase response.

    FOOTNOTES

* This work was supported by grants from the Research Service of the Department of Veterans Affairs.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.

Dagger To whom correspondence should be addressed: Metabolism Section (111F), Dept. of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel: 415-750-2005; Fax: 415-750-6927; E-mail: rmemon@itsa.ucsf.edu.

Published, JBC Papers in Press, June 13, 2001, DOI 10.1074/jbc.M102516200

    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; HMG-CoA, hydroxymethylglutaryl coenzyme A; IL, interleukin; TNF-alpha , tumor necrosis factor-alpha ; BW, body weight; HNF-1, hepatocyte nuclear factor-1; LXR, liver X receptor; PPAR-alpha , peroxisome proliferator activated receptor-alpha ; RXR, retinoid X receptor; LDL, low density lipoprotein; VLDL, very low density lipoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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