Impaired Regulation of Sterol Regulatory Element Binding Protein 2 in Cholesterol Gallstone-Susceptible Mice

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Impaired regulation of sterol regulatory element binding protein 2 in cholesterol gallstone-susceptible mice

Guoqiang Xua,1, Oliver Mu¨llera,2, Eduard F. Stangea,2, Michael Fuchsa,b,*

a Division of Gastroenterology, Medical University of Schleswig-Holstein Campus Lu¨beck, D-23538 Lu¨beck, Germany b Department of Medicine I, University of Ulm, Robert-Koch-Strasse 8, Ulm D-89081, Germany Received 14 October 2003; received in revised form 5 January 2004; accepted 6 January 2004

Abstract

Lipid synthesis is under tight transcriptional control involving sterol regulatory element binding proteins (SREBP1, SREBP2). Rising cellular cholesterol levels prevent SREBP2 from entering the nucleus to directly activate the expression of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (Hmgcr), the key enzyme of cholesterol synthesis. The failure to down-regulate cholesterol synthesis in gallstone- susceptible C57L/J but not AKR/J mice prompted us to study the processing of SREBP2 in these mice. Male mice of each strain received a control or lithogenic diet for 28 days. Membrane and nuclear extracts of pooled livers were prepared for immunoblot analysis of SREBP. Steady-state mRNA levels of Hmgcr, Srebp1, Srebp2 and the SREBP cleavage activating protein (Scap) were measured from individual livers. There was no marked difference between the two strains with regard to processing of SREBP1 as well as steady-state mRNA levels of Srebp1, Srebp2 and Scap. However, a near-complete suppression of nuclear SREBP2 related to low Hmgcr mRNA levels was noticed only for gallstone-resistant AKR mice. Abnormal regulation of SREBP2 appears to be responsible for the failure to suppress cholesterol synthesis in genetically cholesterol gallstone-susceptible mice. This defect may contribute to cholesterol hypersecretion and gallstone formation. D 2004 Elsevier B.V. All rights reserved.

Keywords: Gallstone; HMGCoA reductase; Biliary cholesterol; SREBP; SCAP; Lith gene

1. Introduction (SREBPs). While SREBP2 is predominantly involved in cholesterol homeostasis, SREBP1 is also linked to fatty acid The intracellular lipid balance is maintained by coordi- metabolism [1]. SREBPs are synthesized as inactive integral nated transcriptional control. For cholesterol, sterol regula- membrane proteins of the endoplasmic reticulum where they tory elements are the target of transcription factors bind to the cholesterol sensor SCAP or SREBP cleavage- designated sterol regulatory element binding proteins activating protein [1]. In sterol-depleted cells, the SCAP/ SREBP complex moves from the endoplasmic reticulum to the Golgi apparatus via sorting into coat protein II-coated Abbreviations: ACAT2, acyl-CoA:cholesterol acyltransferase 2; CYP7A1, cholesterol 7a-hydroxylase; HMGCR, 3-hydroxy-3-methylglu- vesicles [2]. The sequential action of Site-1 and Site-2 taryl coenzyme A reductase; INSIG, insulin-induced gene; LDLR, low protease, both residing in the Golgi apparatus, cleaves the density lipoprotein receptor; SCAP, sterol regulatory element binding membrane-bound SREBP [1] and allows transport of now protein cleavage-activating protein; SREBP1, sterol regulatory element active SREBP into the nucleus for activation of target genes binding protein 1; SREBP2, sterol regulatory element binding protein 2 that aim at increasing intracellular cholesterol availability. * Corresponding author. Department of Medicine I, University of Ulm, Robert-Koch-Strasse 8, Ulm, D-89081, Germany. Tel.: +49-731-5000; fax: Conversely, when SCAP senses excess cholesterol, the +49-731-500-24302. SCAP/SREBP complex is bound to INSIG, an intrinsic E-mail address: [email protected] (M. Fuchs). membrane protein of the endoplasmic reticulum [3].Asa 1 Present address: Department of Gastroenterology, First Affiliated result, SREBP remains trapped in the endoplasmic reticulum Hospital, Medical College of ZheJiang University, Qing Chun Road 79, and loses access to the proteases of the Golgi apparatus so HangZhou 310003, China. 2 Present address: Department of Gastroenterology, Hepatology and that the transcription of target genes declines. Endocrinology, Robert Bosch Hospital, Auerbachstr. 110, D-70376 Biliary cholesterol hypersecretion appears to be a key Stuttgart, Germany. defect that facilitates gallstone formation in C57L/J but not

AKR/J mice [4]. Although HDL-cholesterol contributes recommendations of the American Veterinary Medical substantially to biliary cholesterol [5,6], the role of newly Association. synthesized cholesterol for biliary secretion has not been explored in these mice. Interestingly, the activity of 2.3. Immunoblot analysis hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme in cholesterol syn- Twelve mice of each strain, either fed a standard or thesis, is not down-regulated in C57L mice fed a lithoge- lithogenic diet, were anesthetized with pentobarbital intra- nic diet which contains large amounts of cholesterol [7,8]. peritoneally (35 mg/kg body weight). Membranes and In the current study, we explored the hypothesis that the nuclear extracts were prepared from pooled livers of six failure of C57L mice to suppress cholesterol synthesis in mice as previously described by others [9]. Equal quantities response to a lithogenic diet is a result of impaired SREBP (30 mg) of membrane fractions and nuclear extracts were processing. A clear-cut difference between the two mouse subjected to 8% SDS-PAGE and proteins were electropho- strains was observed for the processing of SREBP2. Upon retically transferred onto nitrocellulose. The membranes feeding the lithogenic diet, nuclear SREBP2 decreased were exposed to antibodies against SREBP1 (1:2000 dilu- almost completely in AKR mice. This is in line with the tion) and SREBP2 (1:4000 dilution) for at least 2 h. suppression of Hmgcr mRNA and in agreement with an Detection of immunoreactive protein was accomplished intact negative feedback control of cholesterol synthesis. using a chemiluminescence kit (NEN Life Science) follow- Contrary, processing of SREBP2 continued in C57L mice ing incubation with horseradish peroxidase-labeled second- and this was paralleled by a failure to suppress Hmgcr ary antibodies. Protein gels were calibrated with prestained mRNA. These results suggest that impaired reglation of Rainbow markers (Amersham Pharmacia, Freiburg, Ger- SREBP2 may be responsible, at least in part, for the failure many). The protein content of the samples was measured to inhibit cholesterol synthesis in gallstone-susceptible as described previously [5]. C57L mice. 2.4. RNA analysis

2. Materials and methods Relative quantitation of transcript abundance employed reverse transcription-polymerase chain reaction. Due to its 2.1. Materials invariant expression across treatment conditions, 18S ribosomal RNA was used as internal standard [5]. Total mouse Taq DNA polymerase was purchased from Boehringer liver RNA from individual mice (n = 6) was isolated using Mannheim (Germany) and primers were obtained from TRIzol reagent (Life Technology, Munich, Germany). Re- MWG Biotech (Ebersberg, Germany). Polyclonal rabbit verse transcription was performed with the SuperScript II anti-human SREBP1 and goat anti-human SREBP2 anti- preamplification system (Life Technology). An aliquot of the bodies were purchased from Santa Cruz Biotechnology reaction was subjected to PCR amplification using a ‘‘com- (Santa Cruz, USA). It has not been reported that the petimer’’/primer mix specific for 18S ribosomal SREBP1 antibody used is specific for either of the two RNA (QuantumRNA 18S Internal Standards, Ambion, Aus- isoforms (SREBP1a, SREBP1c). Horseradish peroxidase- tin, TX) together with the following gene specific labeled secondary antibodies were from Dako Chemicals primers: Hmgcr (882 bp): 5V-ATCATCTTGGAGAGA- (Hamburg, Germany). Unless otherwise indicated, materials TAAAACTGCCA-3V (sense) and 5V-GGGACGGTGA- of highest purity grade commercially available were ob- CACTTACCATCTGTATGATG-3V (antisense); Ldlr (210 tained from Sigma Chemicals (Deisenhofen, Germany) or bp): 5V-CTC CTC ATT CCC TCT GCC AGC CAT-3V Biometra (Go¨ttingen, Germany). (sense), 5V-GAA GTC GAC ACT GTA CTG ACC ACC-3V (antisense); Acat2 (423 bp): 5V-CAT CTC GCC GAA GGC 2.2. Animals GTT GAG-3V(sense), 5V-CGC TGC GTG CTG GTC TTT GAG-3V(antisense); Cyp7a1 (721 bp): 5V-AAG TCA AAG Male AKR/J and C57L/J mice were obtained from The GGT CTG GGT AG-3V(sense), 5V-GTC ACG GAA GGG Jackson Laboratory (Bar Harbor, ME) and maintained ATG TAT GC-3V(antisense); Srebp1 (1046 bp): 5V-TCAA- under constant light–dark cycles with free access to water CAACCAAGACAGTGACTTCCCTGGCC-3V (sense) and and standard Altromin 1314 chow ( < 0.02% cholesterol; 5V-GTTCTCCTGCTTGAGCTTCTGGTTGCTGTG-3V (an- Altromin GmbH, Lage, Germany). Twelve-week-old male tisense). The primer set is not specific for the two isoforms animals received either a chow or a lithogenic diet con- Srebp1a or Srebp1c, respectively; Srebp2 (1342 bp): 5V- sisting of 15% fat, 1.25% cholesterol and 0.5% cholic acid CATGAG(GC)ACCCTCACGGAGCTGGGCGACGA-3V [5] for 28 days. All animals received human care accord- (sense) and 5V-TGCATCATCCA(AG)(CT)AGAGGAG- ing to the criteria for the care and use of laboratory animal. (CT)TCCTGGCTC-3V(antisense); Scap (585 bp): 5V-ACT- Protocols were approved by the Institutional Animal Care GGGCATCATCCTCATTG-3V(sense) and 5V-GGCACTGT- and Use Committee and euthanasia was consistent with CTGGTTCTCTGG-3V (antisense). ‘‘Competimers’’ are 276 G. Xu et al. / Biochimica et Biophysica Acta 1688 (2004) 274–279 primers that cannot be extended. The ratio of 18S ‘‘competimers’’ to primers was adjusted such that the amount of 18S ribosomal RNA product was within the same range as that of the mRNAs of interest. Amplified samples were subjected to densitometry following agarose gel electropho- resis with ethidium bromide staining using a GS-700 Imag- ing Densitometer and the Molecular Analyst Software (BioRad). Compared with state-of-the-art quantitative real- time PCR, this method of mRNA quantification may represent a weakness. However, control experiments demonstrat- ed that the PCR protocol employed allowed quantitation within the linear range of amplification of both transcripts and yielded similar results when compared with Northern blot analysis [5].

2.5. Lipid analysis

To determine hepatic total cholesterol and triglycerides contents, separate groups of four mice were used. Lipids were extracted as described [10], dissolved in isopropanol, and aliquots were used for determinations of total cholesterol and triglycerides employing commercially available Fig. 1. Immunoblot analysis of SREBP1 and SREBP2 in AKR and C57L colorimetric kits. mice fed a control (C) or lithogenic (L) diet. For each group and preparation (n = 2), livers of six mice were pooled. Aliquots of membranes and nuclear 2.6. Statistics extracts were subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose. SREBP1 and SREBP2 were visualized with enhanced chemiluminescence. M, mature or nuclear form of SREBP; P, precursor or Data were analyzed by the unpaired, two-tailed Stu- membrane-bound form of SREBP. dent’s t test. Results are expressed as the arithmetic mean F 1 S.D. and P values < 0.05 were considered statis- tically significant. AKR mice (180% vs. 37%). At the same time, nuclear forms of SREBP1 decreased by 70% in the C57L and by 20% in the AKR strain, respectively. As reported previously 3. Results by others, SREBP1 bands may appear as closely spaced doublets [9]. Interestingly, the lower SREBP1 band clearly As reported recently by our group [5], body weights of present in C57L mice fed the control diet consistently AKR and C57L mice fed the control diet were similar. In disappeared upon feeding the lithogenic diet. Because the response to the lithogenic diet, the mean body weight of polyclonal antibody against SREBP1 does not discriminate C57L remained constant but increased by 30% in AKR between SREBP1a and SREBP1c isoforms, it is possible mice. Initial mean liver weights were only slightly higher in that the lower band may represent one of the two isoforms. AKR mice. When challenged with the lithogenic diet and in Employing isoform-specific antibodies may provide an contrast to the C57L strain, livers of AKR mice appeared answer. pale and were enlarged, reflected by a 50% increase in the Precursor forms of SREBP2 increased slightly in both mean liver weight. The hepatic total cholesterol content was strains in the lithogenic state. Interestingly and in contrast to comparable in chow-fed mice of both strains as well as in C57L mice, the AKR strain exhibited a nearly complete response to the lithogenic diet [5]. The hepatic triglyceride absence of nuclear SREBP2. This implicates that impaired content on chow diet was slightly higher in the AKR strain processing of SREBP2 in C57L but not AKR mice may be and a significant ( P < 0.01) increase occurred in both strains responsible for the failure to down-regulate cholesterol in response to the lithogenic diet (AKR: 89.0 F 4.3; C57L: synthesis that has been reported previously [7,8]. To con- 42.6 F 2.7 mg/g). firm impaired feedback regulation of cholesterol synthesis Immunoblot analysis was employed to investigate the in C57L mice under our present experimental conditions, effect of the lithogenic diet on the processing of SREBP1 we measured steady-state Hmgcr mRNA expression (Fig. 2). and SREBP2 in livers of AKR and C57L mice (Fig. 1). On control diet, steady-state Hmgcr mRNA levels were Under control conditions, both strains expressed precursor threefold ( P < 0.001) lower in AKR mice and a 40% and nuclear forms of SREBP1 and SREBP2 at a detectable decrease ( P < 0.01) occurred in the lithogenic state. In level. In response to the lithogenic diet, the precursor form contrast, C57L mice fed the lithogenic diet were unable to of SREBP1 increased more pronounced in C57L than in suppress cholesterol synthesis reflected by unchanged G. Xu et al. / Biochimica et Biophysica Acta 1688 (2004) 274–279 277

Fig. 2. Amounts of various mRNAs in livers of AKR and C57L mice a control (C) or lithogenic (L) diet. Total RNA from small parts of livers of the animals (n = 6) used in Fig. 1 was prepared and steady-state mRNA levels were analyzed by RT-PCR. Signals corresponding to specific mRNAs were quantified by densitometry. The lower of the two bands represents 18S ribosomal RNA. The fold change in each mRNA, relative to that of mice fed the control diet, was calculated after correction with 18S ribosomal RNA. These values are shown below a representative gel. Srebp1, sterol regulatory element binding protein 1; Srebp2, sterol regulatory element binding protein 2; Scap, sterol regulatory element binding protein cleavage activating protein; Hmgcr, 3-hydroxy-3- methylglutaryl coenzyme A; Ldlr, low density lipoprotein receptor; Acat2, acyl-CoA:cholesterol acyltransferase 2; Cyp7a1, cholesterol 7a-hydroxylase.

Hmgcr mRNA levels. To confirm that adaptive changes of 4. Discussion lipid regulatory enzymes augment the bioavailability of free cholesterol, we also measured steady-state Ldlr, Acat2 and The C57L gallstone-susceptible mouse reflects metabolic Cyp7a1 mRNA expression (Fig. 2). Basal steady-state Ldlr abnormalities frequently observed in cholesterol gallstone and Acat2 mRNA levels were similar in both strains. When patients [12,13]. Detailed characterization of this specific the animals received the lithogenic diet, Ldlr and Acat2 mouse strain [4,5,8] provided compelling evidence that mRNA levels increased significantly ( P < 0.05). AKR mice HDL-cholesterol contributes substantially to biliary choles- had threefold ( P < 0.01) higher basal Cyp7a1 mRNA ex- terol hypersecretion that is crucial to initiate cholesterol pression levels than C57L mice. In response to the litho- crystallization and gallstone formation. However, employ- genic diet, steady-state Cyp7a1 mRNA levels of C57L and ing other mouse models, the role of HDL cholesterol as AKR mice were suppressed by 50% ( P < 0.01) and 80% substrate for biliary cholesterol during lithogenesis has been ( P < 0.001), respectively. These results are in line with an questioned [14]. C57L mice also fail to suppress mRNA increased bioavailability of free cholesterol in response to levels and activity of HMGCR in the lithogenic state [5,7,8], the lithogenic diet. suggesting that newly synthesized cholesterol further con- Regulation of SREBP does not only occur at the tributes to cholesterol hypersecretion. We hypothesized that posttranscriptional but also at the transcriptional level. the molecular mechanism that prevents suppression of We also analyzed steady-state Srebp1, Srebp2 and Scap HMGCR under these conditions may be related to impaired mRNA levels (Fig. 2). In AKR mice, Srebp1 mRNA proteolytic processing of SREBPs. levels were twofold lower than in C57L mice (0.65 F In the non-lithogenic state (control diet), steady-state 0.04 vs. 1.47 F 0.13; P < 0.001) and increased 1.5-fold Hmgcr mRNA levels were significantly lower in AKR than ( P < 0.01) upon challenge with the lithogenic diet. Basal in C57L mice. This is in good agreement with a previously Srebp2 mRNA expression levels were also lower in AKR reported nearly 50% lower HMGCR activity in AKR mice mice (1.39 F 0.16 vs. 2.12 F 0.58; P>0.05) and decreased [7]. This may suggest that in the face of a dietary challenge by 60–70% ( P < 0.01) when the animals received the with cholesterol, C57L mice have a reduced ability to lithogenic diet. This confirms that SREBP2 activity is down-regulate HMGCR that normally would compensate regulated not only posttranslationally by proteolytic cleav- for the increased dietary supply of cholesterol. Unexpect- age of the precursor form, but also at the transcriptional edly, we found similar Srebp2 mRNA levels in addition to level [11]. Basal steady-state Scap mRNA levels were 1.4- expressed nuclear SREBP2 in both mouse strains. One fold higher in AKR than in C57L mice (1.67 F 0.14 vs. explanation may be the complex transcription of the 1.21 F 0.03; P < 0.05). Feeding the lithogenic diet resulted Hmgcr gene with several transcription initiation sites in a 20% ( P>0.05) decrease of Scap mRNA levels in both [15]. It is possible that transcription factors other than strains. SREBPs or co-regulators such as NF-Y/CBF [16] may 278 G. Xu et al. / Biochimica et Biophysica Acta 1688 (2004) 274–279 contribute to the difference in Hmgcr mRNA expression SCAP/SREBP2 complex from the endoplasmic reticulum under these conditions. with the inability to turn off proteolysis may impair choles- In the lithogenic state, the increase of membrane-bound terol sensing. This may be related to a mutation in the SREBP1 and the concomitant reduction of nuclear SREBP1 cholesterol sensing domain of SCAP [3]. The recent iden- in both mouse strains indicate that the proteolytic process tification of INSIG1 and INSIG2 isoforms [25] may provide governing the release of membrane-bound SREBP1 is another explanation. Under conditions of cholesterol excess, intact. Although the SREBP1 antibody used did not allow INSIG2 alone is mediating SREBP regulation. Thus, it is us to distinguish between SREBP1a and SREBP1c, the possible that an INSIG2 defect allows continuous process- latter constitutes more than 90% of the SREBP pool in the ing of SREBP2 but may be insufficient to trap SREBP1 in mouse [1]. Together with the fact that SREBP1c is a potent the endoplasmic reticulum [26]. regulator of lipogenesis [1], the more pronounced decrease In summary, the current study provides first insights how of nuclear SREBP1 in C57L compared with AKR mice may impaired feedback regulation of SREBP2 processing may protect them from developing fatty liver [17]. explain, at least in part, why gallstone-susceptible C57L The decrease of nuclear SREBP2 in AKR but not C57L mice, despite ingestion of large amounts of cholesterol, do mice in the lithogenic state is in line with a decline of not suppress hepatic cholesterol synthesis. We speculate that steady-state Hmgcr mRNA levels and a suppression of in contrast to the non-lithogenic state, the contribution of HMGCR activity [5,7,8]. Although preformed cholesterol newly synthesized cholesterol to biliary cholesterol secre- represents a predominant source for biliary cholesterol tion and gallstone formation is appreciable. Based on [18,19] with a significant contribution of HDL cholesterol published quantitative trait locus analysis data for C57L [5,6], our results suggest that the contribution of newly mice [4], we infer that one or more Lith gene(s) may be synthesized cholesterol to lithogenic bile and gallstone involved in the processing of SREBP2. formation is appreciable. Indeed, this is in good agreement with recent preliminary studies where AKR and C57L mice were fed the squalene epoxidase inhibitor Tu 2208 in Acknowledgements addition to the lithogenic diet [20]. Under these conditions, HMGCR activity was suppressed by 40% associated with a The authors are greatly indebted to Nadine Katzberg for reduction of gallstone prevalence by 50% and a gallstone excellent technical assistance. mass less than assayable. Further support comes from a Dr. Fuchs was supported in part by a grant from the study where the cholesterol saturation index could be Deutsche Forschungsgemeinschaft (Fu 288/2-3), a grant-in- decreased in patients with a history of gallstones by long- aid from AstraZeneca GmbH (Wedel, Germany) and term administration of the HMGCR inhibitor fluvastatin Go¨decke/Parke-Davis GmbH (Freiburg, Germany). Addi- [21]. Clearly, future studies to measure directly the contri- tional support came from the ‘‘Freunde und Fo¨rderer der bution of newly synthesized cholesterol to biliary choles- Medizinischen Universita¨t zu Lu¨beck’’ (to M.F.) and the terol under such conditions are needed. Substantial amounts ‘‘Reinhold Jarchow-Stiftung’’ (to M.F.). Dr. Xu was of dietary fat and cholic acid, both of which can influence supported by an exchange scholarship program of the the hepatic regulation of SREBPs, are included in the Landesregierung Schleswig-Holstein and a grant-in-aid lithogenic diet. 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