Homocysteine-induced内质网应激导致失调的胆固醇和甘油三酯生物合成途径

Introduction Hyperhomocysteinemia is a common, independent risk factor for cardiovascular disease (1–9). Up to 40% of patients diagnosed with premature coronary artery, cerebrovascular, or peripheral vascular disease are reported to have hyperhomocysteinemia. Patients with severe hyperhomocysteinemia due to cystathio- nine β-synthase (CBS) deficiency also exhibit a wide range of clinical manifestations, including athero- sclerosis, thrombosis, mental retardation, ectopia lentis, osteoporosis, and skeletal abnormalities. These patients also develop hepatic steatosis or “fatty liver,” which is characterized by enlarged, multinucleated hepatocytes containing microvesicular lipid droplets (10, 11). Consistent with these findings, homozygous CBS-deficient mice having severe hyperhomocys- teinemia also develop hepatic steatosis (12). Several mechanisms have been proposed to explain the pathological changes associated with hyperhomo- cysteinemia. Homocysteine has been shown to cause cell injury when administered to animals (13, 14) or when added directly to cultured mammalian cells (15–17). Furthermore, cultured vascular endothelial cells from patients with heterozygous CBS deficiency are more sensitive to homocysteine-induced damage than are wild-type cells (17). Because homocysteine is generated intracellularly and can accumulate in cells (18, 19), it has been suggested that homocysteine could act intracellularly to directly modulate the activ- ity of both large (enzymes, receptors) and small mole- cules (nitric oxide, glutathione) (20). We have reported that homocysteine causes protein misfolding in the endoplasmic reticulum (ER) and acti- vates the unfolded protein response (UPR), leading to The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 1263 Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways Geoff H. Werstuck,1 Steven R. Lentz,2 Sanjana Dayal,2 Gazi S. Hossain,1 Sudesh K. Sood,1 Yuan Y. Shi,1 Ji Zhou,1 Nobuyo Maeda,3 Skaidrite K. Krisans,4 M. Rene Malinow,5 and Richard C. Austin1 1Department of Pathology and Molecular Medicine, McMaster University and the Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada 2Department of Internal Medicine, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa, USA 3Department of Pathology and Program for Molecular Biology and Genetics, University of North Carolina, Chapel Hill, North Carolina, USA 4Department of Biology, San Diego State University, San Diego, California, USA 5Oregon Regional Primate Research Center, Beaverton, Oregon, USA Address correspondence to: Richard C. Austin, Hamilton Civic Hospitals Research Centre, 711 Concession Street, Hamilton, Ontario, L8V 1C3, Canada. Phone: (905) 527-2299 ext. 42628; Fax: (905) 575-2646; E-mail: raustin@thrombosis.hhscr.org. Received for publication October 17, 2000, and accepted in revised form April 9, 2001. Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine β-synthase. However, the mechanism by which homocysteine promotes the develop- ment and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regu- latory element–binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intra- cellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by over- expression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the acti- vation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secre- tion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglyc- erides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia. J. Clin. Invest. 107:1263–1273 (2001). See related Commentary on pages 1221–1222. increased expression of the ER stress-response genes, GRP78/BiP and GADD153 (21, 22). These observa- tions suggest that homocysteine acts intracellularly to cause cell dysfunction by perturbing the ER. Consistent with our findings, another group has also demonstrat- ed that homocysteine activates the UPR in cultured vascular endothelial cells, leading to increased expres- sion of GRP78/BiP as well as two novel ER stress- response genes, RTP and Herp (23, 24). However, the mechanism by which the ER stress-inducing effects of homocysteine contribute to the pathophysiology of hyperhomocysteinemia has not been established. An association between UPR activation and lipid biosynthesis has been demonstrated in yeast (25, 26) and human fibroblasts (27). In concordance with these studies, we have demonstrated in cultured human vas- cular endothelial cells that homocysteine increases expression of the sterol regulatory element–binding protein-1 (SREBP-1) (21), an ER membrane-bound transcription factor that functions to activate genes encoding enzymes in the cholesterol/triglyceride biosynthesis and uptake pathways (28, 29). However, it is unknown whether homocysteine alters the expression of SREBP-dependent genes that are involved in choles- terol/triglyceride biosynthesis or whether this effect of homocysteine influences lipid metabolism in vivo. In this study, we used cultured human cells and murine models of hyperhomocysteinemia to test the hypothesis that homocysteine-induced ER stress alters cholesterol/triglyceride metabolism. Our results show that activation of the UPR by homocysteine induces SREBP-dependent genes that are essential for biosyn- thesis and uptake of cholesterol and triglycerides and leads to the accumulation of cholesterol in cultured cells. Furthermore, a significant increase in the levels of cholesterol and triglycerides were observed in the livers, but not plasmas, of hyperhomocysteinemic mice. These results indicate that homocysteine-induced ER stress leads to the transcriptional activation of genes responsible for lipogenesis that likely contribute to hepatic steatosis in hyperhomocysteinemia. Methods Cell culture and treatment conditions. Primary human umbilical vein endothelial cells (HUVECs) were isolat- ed by collagenase treatment of human umbilical veins (30) and cultured in M199 medium (Life Technologies Inc., Burlington, Ontario, Canada) containing 20 µg/ml endothelial cell growth factor, 90 µg/ml porcine intestinal heparin, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 20% FBS (HyClone Laboratories, Logan, Utah, USA). Cells from passages two to four were used in these studies. Human aortic smooth mus- cle cells (HASMCs) were purchased from Cascade Bio- logics (Portland, Oregon, USA) and cultured in M231 media (Cascade Biologics) containing smooth muscle cell growth supplement (Cascade Biologics). The human hepatocarcinoma cell line, HepG2, was obtained from the American Type Culture Collection (ATCC; Rockville, Maryland, USA) and cultured in α- DMEM (Life Technologies Inc.) containing 10% FBS. The human transitional bladder carcinoma cell line, T24/83, was obtained from ATCC and cultured in M199 medium containing 10% FBS. All cells were maintained in a humidified incubator at 37°C with 5% CO2. DL-Homocysteine, L-methionine, DL-cysteine, glycine, DTT, tunicamycin, A23187, and β-mercap- toethanol were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). These compounds were pre- pared fresh in culture medium, sterilized by filtration, and added to the cell cultures. Determination of intracellular levels of homocysteine. HepG2 cells exposed to 1 or 5 mM homocysteine for 0 to 24 hours were washed three times in α-DMEM and three times in PBS. Cells were lysed in water by three freeze/thaw cycles and cellular debris removed by cen- trifugation. Total homocysteine, defined as the total concentration of homocysteine after quantitative reductive cleavage of all disulfide bonds (31), was deter- mined in cellular lysates using the IMx System (Abbott Laboratories, Mississauga, Ontario, Canada) and nor- malized to total protein concentration. Animals and diets. Wild-type C57BL/6 mice (CBS+/+) were obtained from Charles River (Montreal, Québec, Canada) or The Jackson Laboratory (Bar Harbor, Maine, USA). Heterozygous CBS-deficient mice (CBS+/–) (12) were crossbred to CBS+/+ mice. Genotyping for the targeted allele was performed by PCR (12). At the time of weaning, offspring were fed one of four diets: (a) a control diet that contained 4.1 g L-methionine/kg and 7.5 mg folate/kg (LM-485; Harlan Teklad Laboratory, Madison, Wisconsin, USA); (b) a high-methionine diet that was identical to the control diet except that the drinking water was supplemented with 0.5% L-methio- nine; (c) a high-methionine/low-folate diet that con- tained 1.5 mg folate/kg and drinking water that was supplemented with 0.5% L-methionine; or (d) a very high-methionine (24.6 g/kg), low-folate (1.5 g/kg) diet (TD98272; Harlan Teklad Laboratory) (32). Succinyl- sulfathiazole (1.0 mg/kg) was added to the low-folate diets. After 2 to 20 weeks on an experimental diet, mice were sacrificed with an intraperitoneal injection of 75 mg sodium pentobarbital, plasma was collected in EDTA (final concentration 5–10 mM) for measure- ment of total homocysteine, and their tissues removed and snap-frozen in liquid N2 before storage at –70°C. Plasma total homocysteine was measured by HPLC and electrochemical detection as described previously (33). The experimental protocols were approved by the Uni- versity of Iowa and Veterans Affairs Animal Care and Use Committees or the McMaster University Animal Research Ethics Board. Hepatic VLDL-triglyceride secretion rate. CBS+/+ mice fed control or hyperhomocysteinemic diets for 2 weeks were fasted overnight and hepatic VLDL-triglyceride secretion rates quantified by the intravenous adminis- tration of Triton WR1339 (500 mg/kg body weight; Sigma Chemical Co.) as described previously (34). 1264 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 Blood samples (50 µl) were taken at 0, 2, and 4 hours after Triton WR1339 injection, and plasma cholesterol and triglycerides were measured enzymatically as described below. Hepatic cholesterol and triglyceride secretion rates were calculated from the slope of the curve and expressed as micromole per hour per kilo- gram of body weight. Histological analysis. Liver tissue from mice fed control or hyperhomocysteinemic diets for 16 to 20 weeks was fixed in 4% formalin, embedded in paraffin, and tissue sections were stained with hematoxylin and eosin, as described previously (35). Preparation of total RNA. Total RNA was isolated from cultured cells or tissues using the RNeasy total RNA kit (QIAGEN Inc., Mississauga, Ontario, Canada) and resuspended in diethyl pyrocarbonate–treated water. Quantification and purity of the RNA was assessed by A260/A280 absorption, and RNA samples with ratios above 1.6 were stored at –70°C for further analysis. Northern blot analysis. Total RNA (10 µg/lane) was size fractionated on 2.2 M formaldehyde/1.2% agarose gels, transferred to Zeta-Probe GT nylon membranes (Bio- Rad Laboratories Inc., Mississauga, Ontario, Canada), and hybridized using radiolabeled cDNA probes as described previously (21, 22). Signal intensities were quantified by densitometric scanning of the autoradi- ograms using the ImageMaster VDS and Analysis Soft- ware (Amersham Pharmacia Biotech, Baie d’Urfé, Québec, Canada). To correct for differences in gel load- ing, integrated optical densities were normalized to human GAPDH. The human isopentenyl diphos- phate:dimethylallyl diphosphate (IPP) isomerase cDNA encodes an 837-bp DNA fragment from the 3′-untrans- lated region of the IPP isomerase gene. The cDNA probes encoding human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and farnesyl diphosphate (FPP) synthase were kindly provided by Skaidrite Krisans (San Diego State University, San Diego, California, USA). Human SREBP-1 cDNA (number AA568572) was purchased from Genome Sys- tems (St. Louis, Missouri, USA), and LDL receptor cDNA was purchased from ATCC. The cDNA probes encoding GRP78/BiP or GADD153 have been described previously (21, 22). Construction of mammalian expression plasmid encoding human GRP78/BiP. The cDNA encoding the open read- ing frame of human GRP78/BiP (approximately 1.95 kb) was amplified by RT-PCR using total RNA from primary HUVECs. Primers used for RT-PCR were syn- thesized at the Institute for Molecular Biology, McMaster University. GRP78/BiP cDNA was generat- ed using SuperScript RNase H– reverse transcriptase (Life Technologies Inc.) and a primer complimentary to a sequence in the 3′-untranslated region of the human GRP78/BiP mRNA transcript (AB10230: 5′- TAT TAC AGC ACT AGC AGA TCA GTG-3′). For PCR amplification, the forward primer AB10231 (5′-CTT AAG CTT GCC ACC ATG AAG CTC TCC CTG GTG GCC GCG-3′) contained a Kozak consensus sequence (boldface) prior to the initiating ATG and a terminal HindIII restriction site (underlined). The reverse primer AB10232 (5′-AGG CCT CGAG CT ACA ACT CAT CTT TTT CTG CTG T-3′) contained a terminal XhoI restriction site (underlined) adjacent to the authentic termination codon of the GRP78/BiP cDNA. After PCR was performed, the amplified GRP78/BiP cDNA was gel-purified using the QIAEX gel extraction kit (QIAGEN Inc.) and ligated into T- ended pBluescript KS (Stratagene, La Jolla, California, USA). The ligation mixture was then used to trans- form competent DH5α cells (Life Technologies Inc.). Plasmid DNA was isolated from transformed cells using the QIAEX miniprep kit (QIAGEN Inc.), digest- ed with HindIII and XhoI, and the GRP78/BiP cDNA purified from agarose, as described above. The GRP78/BiP cDNA was ligated into the HindIII/XhoI site of the mammalian expression vector pcDNA3.1(+) (Invitrogen Corp., Carlsbad, California, USA) to pro- duce the recombinant plasmid, pcDNA3.1(+)- GRP78/BiP. Authenticity of the human GRP78/BiP cDNA sequence was confirmed by fluorescence-based double-stranded DNA sequencing. Establishment of stable T24/83 cell lines overexpressing GRP78/BiP. T24/83 cells grown to 30% confluence were transfected with 5 µg of the pcDNA3.1(+)- GRP78/BiP expression plasmid using 30 µl of Super- Fect transfection reagent (QIAGEN Inc.), as described by the manufacturer. As a vector control, T24/83 cells were transfected with pcDNA3.1(+) under the same conditions. Stable transfectants were selected in com- plete medium containing 1.2 mg/ml G418 (Life Tech- nologies Inc.) for 2 weeks. G418-resistant clones were subsequently isolated and cultured in complete medi- um containing 1.0 mg/ml G418. Overexpression of GRP78/BiP was assessed using immunoblotting and indirect immunofluorescence, as described below. The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 1265 Figure 1 Intracellular homocysteine levels in HepG2 cells. HepG2 cells were cultured in the absence or presence of 1 mM (circles) or 5 mM (tri- angles) homocysteine for the indicated time periods, washed, and lysed by three freeze/thaw cycles. Total intracellular homocysteine was determined and normalized to total protein. Data are present- ed as the mean ± SD of three separate experiments. Immunoblot analysis. The anti-KDEL mAb (SPA- 827), which recognizes both GRP78/BiP and GRP94, was purchased from StressGen Biotech- nologies Corp. (Victoria, British Columbia, Cana- da). The anti-GADD153 polyclonal Ab (sc-575) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). The mAb’s reactive against human SREBP-1 or -2 (clones IgG-2A4 and IgG-1C6, respectively) were purchased from PharMingen (Mississauga, Ontario, Canada). Total protein lysates from mouse tissues or cultured cells were solubilized in SDS-PAGE sample buffer and separated on SDS-polyacrylamide gels under reduc- ing conditions, as described previously (21, 22). After incubation with the appropriate primary and horseradish peroxidase-conjugated (HRP-conjugat- ed) secondary Ab’s (Life Technologies Inc.), the membranes were developed using the SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, Illinois, USA). Immunohistochemistry and image analysis. Immunohis- tochemistry using polyclonal Ab’s against GRP78/BiP (sc-1050; Santa Cruz Biotechnology Inc.) was per- formed as described previously (21, 22). Images were captured with Northern Eclipse image analysis/archival software (Empix Imaging Inc., Mis- sissauga, Ontario, Canada). Uptake of BODIPY FL LDL. Cells treated in the absence or presence of homocysteine were washed with PBS and incubated in media containing 10 µg/ml BODIPY FL LDL (Molecular Probes, Eugene, Oregon, USA). After incubation at 37°C for 2 hours, cells were washed with PBS, fixed in 3% formaldehyde in PBS, and the uptake of LDL was detected by fluo- rescence microscopy. Total cholesterol and triglyceride levels. Cultured cells or tissues were homogenized in lysis buffer containing 0.1% Triton X-100. Lysates were saponified, and lipids were extracted with hexane/isopropanol (3:2) (36). Col- orimetric cholesterol and triglyceride assays were car- ried out using the Sigma Diagnostics Cholesterol and 1266 The Journal of Clinical Investigation | May 2001 | Volume 107 | Number 10 Figure 2 Homocysteine induces the expression of GRP78/BiP and GADD153 in HepG2 cells. (a) Northern blot analysis of the steady-state mRNA levels of GRP78/BiP and GADD153 in HepG2 cells cultured for 4 hours in the absence (control) or presence of either 5 mM homocysteine, 5 mM cysteine, 5 mM methionine, 10 µg/ml tunicamycin, 2.5 mM DTT, 5 mM homoserine, or 5 mM glycine. Total RNA (10 µg/lane) was size fractionated by agarose-gel electrophoresis, trans- ferred to nylon membranes, and subjected to blot hybridization using radiola- beled cDNA probes encoding human GRP78/BiP or GADD153. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe. (b) Immunoblot analysis of GRP78/BiP and GADD153 protein in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. HepG2 cells were also treated with 2.5 mM DTT or 10 µg/ml tunicamycin for 8 hours. Total protein lysates (40 µg/lane) were sepa- rated on 12% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes and immunostained with Ab’s against either GRP78/BiP (anti-KDEL) or GADD153. Figure 3 Homocysteine induces the expression of SREBP-1 in HepG2 cells. (a) Immunoblot analysis of SREBP-1 protein in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. Total protein lysates (40 µg/lane) were separated on 10% SDS-polyacrylamide gels under reducing conditions, trans- ferred to nitrocellulose membranes, and immunostained with an mAb that recog- nizes both the precursor (P) and mature (M) forms of SREBP-1. (b) Northern blot analysis of the steady-state mRNA levels of SREBP-1 in HepG2 cells cultured in the presence of 5 mM homocysteine for the indicated time periods. Total RNA (10 µg/lane) was size fractionated by agarose-gel electropho