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Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes
Immunolocalization of a Novel Cholesteryl Ester Hydrolase in the Endoplasmic Reticulum of Murine and Human Hepatocytes ´ ´ ,1 MARı´A TERESA REJAS,2 OLATZ FRESNEDO,1 MIGUEL LOPEZ DE HEREDIA,2 MARı´A JOSE´ MARTı´NEZ,1 SUSANA CRISTOBAL ˜ OCHOA1 JOSE´ M. CUEZVA,2 AND BEGONA
We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissuerestricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism. (HEPATOLOGY 2001;33:662-667.) The mammalian liver plays a central role in the synthesis, redistribution, and regulation of whole body cholesterol and plasma lipoprotein homeostasis. Cholesterol balance in the
Abbreviations: VLDL, very low-density lipoprotein; ACAT, acyl-CoA:cholesterol acyltransferase; ER, endoplasmic reticulum; CEH, cholesteryl ester hydrolase; erCEH, endoplasmic reticulum CEH; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. From the 1Department of Physiology, University of the Basque Country Medical School, Bilbao, Spain; 2Departamento de Biologı´a Molecular, Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, Madrid, Spain. Received August 3, 2000; accepted December 28, 2000. O.F. and M.L.H. contributed equally to this work. S.C.’s present address is: Medical Biochemistry and Biophysics Department, Karolinska Institute, Stockholm, Sweden. Supported by the Spanish Ministry of Education and Culture (Grants PB97/0046 to B.O. and PB97/0018 to J.M.C.), by the Basque Government (Grant PI97/77 to B.O.), and by an Institutional Grant to the CBMSO from the Fundacio´n Ramo´n Areces, Spain. Address reprint requests to: Begon˜a Ochoa, Ph.D., Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, 48080-Bilbao, Spain. E-mail: [email protected]; fax: (34) 94-6015662. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3303-0023$35.00/0 doi:10.1053/jhep.2001.22763
liver involves two input pathways, cholesterol synthesis and lipoprotein uptake; two output pathways, the formation of bile acids and bile and the secretion of very low-density lipoproteins (VLDL); as well as the reversible conversion of cholesterol to cholesteryl esters. Esterification of cholesterol with long-chain fatty acyl-CoA is accomplished by acyl-CoA: cholesterol acyltransferase (ACAT), an integral membrane protein located in the endoplasmic reticulum (ER) that is allosterically regulated by cholesterol (for a recent review, see Chang et al.1). Newly formed cholesteryl esters are secreted as a component of VLDL or are stored in intracellular lipid droplets in the cytoplasm, where cholesteryl esters undergo a constant cycle of hydrolysis and resynthesis. Three major cholesteryl ester hydrolases (CEH) have been identified in the liver that differ in their subcellular localization, function, and enzymatic properties. The lysosomal CEH, or acid lipase, is involved in the hydrolysis of cholesteryl esters and triacylglycerols delivered to the hepatocytes via receptor-mediated endocytosis of lipoproteins.2 The lysosomal CEH has been purified and cloned from several species, including the human liver.3 The major cytosolic CEH from rat liver has also been purified and characterized,4 as well as cloned and expressed.5 This neutral cytosolic CEH is considered to be a key enzyme for releasing free cholesterol from the stores of cholesteryl esters in the cytoplasm and is subjected to complex regulation by hormones and sterols.6,7 Biochemically, a third cholesteryl ester hydrolase activity has been characterized in rat liver microsomes. The microsomal CEH activity of rat liver has a neutral optimal pH and is modulated by changes in cholesterol or bile acid flux (i.e., see Gad and Harrison8 and Martı´nez et al.9), postnatal development,10 circadian rhythm,9 and agonists that trigger activation of cAMPdependent protein kinase,11,12 protein kinase C,12 and AMPactivated protein kinase.13 Recently, we isolated and purified the major microsomal CEH from rat liver.14 The purified CEH from rat liver microsomes is a 106-kd protein that efficiently catalyzes the specific hydrolysis of cholesteryl esters in vitro. Biochemical analysis indicated that the microsomal CEH shares many of the hallmark features of the lipases and carboxylesterases protein family, but differs clearly from other liver cholesteryl ester– splitting enzymes, including the lysosomal and cytosolic CEH isoforms, the hepatic lipase, the retinyl ester hydrolase, the triacylglycerol lipase, and nonspecific carboxylesterases.14 Antibodies raised against the purified CEH from rat liver microsomes neutralized 90% of the CEH activity in rat liver microsomes and 100% of the CEH activity in the preparation of the pure protein.14 In this study we have used the anti-microsomal CEH antibodies to study the subcellular localization and tissue-specific
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expression of microsomal CEH in rodents and human liver samples. Subcellular fractionation of rat hepatocytes, confocal microscopy of human HepG2 cells, and high-resolution immunoelectron microscopy of rat liver samples unambiguosly localized the CEH protein exclusively to the endoplasmic reticulum. The henceforth termed endoplasmic reticulum CEH, erCEH, was not detected in other liver cell types or in other rat tissues with an active cholesterol-cholesteryl ester cycle. The physiologic function and relevance of erCEH in cholesterol homeostasis remains to be established in future studies. MATERIALS AND METHODS Antibodies. Polyclonal antibodies were raised in New Zealand rabbits against the purified CEH from rat liver microsomes.14 Antibodies were purified from the rabbit serum by affinity chromatography on a Hitrap NH-Sepharose column (Amersham Pharmacia Biotech Europe Gmbh, Barcelona, Spain). The coupling of the purified CEH to the column was performed according to the manufacturer’s instructions. Cell and Tissue Processing for Immunoblotting. Microsomes were prepared from adult rat, mouse and human liver, HepG2 cells, and adult rat periportal and perivenous hepatocytes, nonparenchymal liver cells, ovaries, testis, adrenal glands, intestine, aorta, brain, pancreas, spleen, lung, epididymal adipose tissue, mammary glands, and peritoneal macrophages. In brief, cells and small pieces of tissues were homogenized in 4 vol of 20 mmol/L Tris/HCl buffer, pH 7.4, containing 0.25 mol/L sucrose, 2 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 10 mol/L leupeptin, and 1 mmol/L benzamidine, and the homogenates centrifuged as detailed by Ruiz and Ochoa.15 Microsome,15 cytosol,15 lysosome,16 and plasma membrane17 preparations were obtained from hepatocytes isolated from adult female rats fed ad libitum.15 More than 55%, 40%, and 30% of the total activity of marker enzymes for the endoplasmic reticulum (glucose-6-phosphatase17), lysosomes (acid phosphatase16), and plasma membrane (5⬘ nucleotidase17), respectively, was recovered in the preparations. Receptor-rich endosomes were isolated from livers of 17␣-ethinyl estradiol-treated female rats that had been injected low density lipoprotein, as in the study by Belcher et al.18 Periportal and perivenous hepatocytes were prepared by centrifugal elutriation of isolated rat hepatocytes and characterized for biological and biochemical markers, as detailed by Romero et al.19 Three rat nonparenchymal liver cell preparations provided by Dr. F. Vidal-Vanaclocha from the University of the Basque Country, Bilbao, Spain, were studied. Each one of the preparations contained a slightly different percentage of endothelial cells (⬃70%), lymphocytes (⬃25%), and Kupffer cells (⬃5%). Human liver biopsies were provided by Dr. F. Dı´az-Aguirregoitia (Cruces Hospital, Baracaldo, Spain). The HepG2 cell line (American Type Culture Collection, Rockville, MD) was grown in Dulbecco’s modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 0.1% (wt/vol) streptomycin (all from Gibco BRL Life Technologies, Paisley, UK). When the cell culture reached approximately 80% confluence, cells were harvested and washed repeatedly in phosphate-buffered saline (PBS; 0.01 mol/L phosphate buffer, 0.15 mol/L NaCl, pH 7.2). Rat peritoneal macrophages were harvested as described previously.20 The protein concentration was determined with the Bradford reagent21 using bovine serum albumin (BSA) as the standard. Immunoblotting. Proteins were fractionated on sodium dodecyl sulfate (SDS)/8% polyacrylamide gel electrophoresis (PAGE).22 After fractionation, proteins were transferred to polyvinylidene difluoride membranes using a Mini Trans-Blot electrophoretic transfer cell (Biorad, Hercules, CA) with 50% (vol/vol) electrophoresis buffer (0.025 mol/L Tris, 0.192 mol/L glycine, 0.1% SDS) and 10% (vol/vol) methanol at 20 V for 60 minutes. Membranes were incubated with gentle agitation in Tris-buffered saline (0.01 mol/L Tris buffer, 0.15 mol/L NaCl, pH 8.0) as follows: (1) blocking for 1 hour in 3% (wt/ vol) BSA; (2) incubation with a 1:1,000 dilution of primary anti-CEH
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antibody in 0.5 % (wt/vol) BSA overnight, followed by 3 10-minute washes with buffer; (3) incubation with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO) in 3% (wt/vol) BSA for 1 hour, followed by 3 10-minute washes with buffer. The blots were developed with freshly prepared peroxidase substrates, hydrogen peroxide, and 4-chloro-1naftol, as described in Cristo´bal et al.,14 or with the ECL reagent from Amersham following the supplier’s instructions. Purified CEH was loaded in the gels as a positive control. Confocal Microscopy. For immunocytochemistry: briefly, HepG2 cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and plated on coverslips the day before the experiment. Coverslips were fixed in methanol for 3 minutes at ⫺20°C and then incubated with a 1:100 dilution of anti-CEH for 1 hour at 37°C. Coverslips were then washed with PBS and further incubated for 1 hour at 37°C with a 1:200 dilution of a goat anti-rabbit IgG fluorescein-conjugate (TAGO Immunochemicals, Burlingame, CA). Next, they were washed with PBS and incubated for 1 hour at 37°C with a 1:200 dilution of the IgG fraction of mouse anti-calnexin (Transduction Laboratories, Lexington, KY). Afterwards, coverslips were washed with PBS and further incubated for 1 hour at 37°C with a 1:100 dilution of a rabbit anti-mouse IgG Texas red-conjugate (Cappel, Turnhout, Belgium). After various PBS washes, the coverslips were mounted on glass slides using Gelvatol (Montsanto, USA). The fluorescence of fluorescein and Texas red were detected by laser confocal microscopy using excitation wavelengths of 488 or 543 nm, and detection wavelengths of 522 or 615, respectively. A Biorad Radiance 2000 Zeiss Axiovert S100 TV confocal microscope was used. Digital images were acquired using the software Lasersharp 2.0 provided by the dealer (Biorad, Hercules, CA). Tissue Processing for Electron Microscopy. Small pieces of liver obtained from 10-week-old overnight fasted female rats were fixed by immersion in freshly prepared 4% paraformaldehyde in 0.1 mol/L So¨rensen phosphate buffer, pH 7.2, for 2 hours at 4°C. Samples were rinsed in buffer and the free-aldehyde groups were quenched with 50 mmol/L ammonium chloride in PBS for 60 minutes at 4°C. Afterwards, the samples were rinsed in PBS, dehydrated in acetone, and finally processed for embedding in Lowicryl K4M (Polysciences Europe, Eppelheim, Germany) according to the manufacturer’s instructions. Gold interferential color ultrathin sections were collected in collodion/carbon-coated nickel grids. The grids were observed in a Jeol 1010 electron microscope under 80 kV accelerating voltage. Immunocytochemical Localization of CEH by Electron Microscopy. Grids were incubated for 5 minutes with PBS containing 1% BSA and then incubated with 1/2, 1/10, or 1/50 dilution of anti-CEH in the same buffer. After 3 washes with PBS, grids were incubated for 45 minutes with goat anti-rabbit IgGs conjugated with 10 nm colloidal gold (British BioCell, Cardiff, UK). The grids were washed twice in PBS and distilled water and air-dried. Counterstaining was performed with 2% aqueous uranyl acetate (6 minutes) and Reynolds lead citrate (45 seconds). Standard controls for immunocytochemical techniques23-25 were conducted in parallel to assess the specificity of the immunoreactive signals: (1) the omission of the primary antibody and (2) the incubation with the pre-immune rabbit serum, in both cases followed by incubation with the gold labeled secondary antibody. Both controls provided no gold decoration of hepatocyte structures. Carbonate Extraction of erCEH From Microsomal Membranes. Rat hepatocyte microsomes (2 mg of protein) were incubated at 0°C for 30 minutes with 0.5 mL of either 100 mmol/L sodium carbonate, pH 11.5, or 150 mmol/L Tris-HCl buffer, pH 8, containing 0.5 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 10 mol/L leupeptin, and 1 mmol/L benzamidine.26 After centrifugation at 230,000g for 1 hour at 4°C, the extract was aspirated and the membrane pellet was resuspended in 0.5 mL of 20 mmol/L Tris-HCl buffer, pH 7.2. Aliquots of the extract and pellet fractions were further subjected to SDS-8% PAGE, and either stained with Coomassie Blue or probed with antibodies against CEH or against calnexin, as indicated previously.
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RESULTS
In this work we have studied the subcellular localization and the expression pattern in rat tissues of a mammalian CEH using antibodies raised against the purified CEH from rat liver microsomes.14 Figure 1A illustrates the specificity of the antibodies developed.14 When the antibodies are used in Western blot analysis of mixtures of proteins, derived either from isolated rat liver microsomes (Fig. 1A, right panel) or from total rat liver homogenates (Fig. 1A, left panel), only one specific immunoreactive band is detected. The immunoreactive protein, of approximately 106 kd, coincides in its electrophoretic migration with that of the purified CEH from rat liver microsomes (Fig. 1A, and see Cristo´bal et al.14). In the more complex protein mixture of total liver homogenates, especially in those tracks where there is protein overloading, several faint immunoreactive signals appear (Fig. 1A, left panel). These signals never represented more than 1% of the total immunoreactive signal of the blot. The nature of these faint immunoreactive proteins, present only in mixtures of liver homogenates, is unknown. In any case, these results unambiguously show that the anti-CEH developed14 is an excellent tool to be used with the purpose of studying the subcellular localization of the protein.
FIG. 1. Subcellular localization and cell-specific expression of CEH. (A) Specificity of the anti-CEH antibody in Western blots of mixtures of proteins from rat liver homogenates and microsomes. Proteins were fractionated on SDS-8% PAGE and either stained with Coomassie Blue (C) or probed with anti-CEH antibody (W). In the lane of purified CEH, approximately 0.2 g of protein was loaded. Blots were developed with the ECL reagent. (B) Subcellular fractions were obtained from isolated rat hepatocytes, and microsomes were prepared from the sources indicated. Proteins were fractionated on SDS-8% PAGE and probed with anti-CEH antibody; blots were developed with 4-chloro-1-naftol. Lanes 1-5: microsomes (10 g), lysosomes (15 g), cytosol (50 g), endosomes (50 g), and plasma membrane (50 g) from isolated rat hepatocytes; lanes 6-12: microsomes (20-30 g) from rat liver, rat periportal hepatocytes, rat perivenous hepatocytes, rat nonparenchymal liver cells, mouse liver, human liver, and HepG2 cells. Purified CEH (2 g) was loaded in the gels as a positive control. Molecular mass markers (kd) are shown on the left. The blots shown are representative of several independent experiments.
The localization of CEH was studied in subcellular fractions obtained from isolated rat hepatocytes. Western blots of different membrane fractions revealed that the anti-CEH antibody reacted with a 106-kd protein only in microsomes (Fig. 1B). No immunoreactive signal was detected in other compartments along the secretory pathway, such as the lysosomes, the endosomes and the plasma membrane (Fig. 1B). In agreement with previous findings,14 no immunoreactivity was observed with proteins from the cytosol. The expression of CEH in the microsomal fraction of different cell types of the rat liver was further studied. The results obtained illustrated the presence of the 106-kd protein in microsomes of both periportal and perivenous hepatocytes (Fig. 1B). Remarkably, no immunoreactive signal was detected in the microsomes of non-parenchymal liver cells (Fig. 1B). In addition, the antibody recognized only one protein band in microsomes of mouse and human liver as well as in microsomes of the human hepatoma HepG2 (Fig. 1B). The CEH band in human and mouse samples corresponded to an apparent molecular mass of 106 kd, the molecular mass of purified CEH from rat liver (Fig. 1B). The human microsomal CEH is immunologically related to the rat microsomal CEH (Fig. 1B), which is consistent with the finding of a high specific activity of the neutral CEH in preparations of human liver microsomes (576 ⫾ 24 pmol cholesteryl oleate hydrolyzed ⫻ h⫺1 ⫻ mg of microsomal protein⫺1, mean ⫾ SD of 3 samples analyzed in duplicate). To our knowledge, this is first description of a cholesteryl ester hydrolase in microsomes of human liver. Confocal immunofluorescence analysis of HepG2 cells using antibodies against CEH (Fig. 2, left panel) and calnexin (Fig. 2, middle panel) revealed essentially the same distribution of immunofluorescence throughout the cytoplasm of the cells for the 2 proteins. Calnexin is an integral membrane protein of the ER that functions as a chaperone.27 The clear colocalization of CEH with a marker of the endoplasmic reticulum (see yellow signals in the merge picture on Fig. 2) strongly suggest the localization of CEH in the ER of the cell. High resolution immunoelectron microscopy was further used to localize CEH in the adult rat liver. Figure 3 illustrates that gold particles specifically decorated the rough ER of the hepatocyte. In agreement with previous findings obtained by subcellular fractionation and confocal microscopy, no significant gold-labeling was observed in the cytoplasm (Fig. 3), the nuclei (Fig. 3A), the mitochondria (Fig. 3B and C), and the peroxisomes (Fig. 3B and C) of the hepatocyte at any of the 3 dilutions of the primary antibody used (see Fig. 3). The specificity of the ER labeling could be best assessed by the large differences (1 to 2 orders of magnitude) found in the number of gold particles decorating the rough ER when compared with the number of gold particles decorating nonimmunoreactive organelles in the same field (see Fig. 3). In addition, in all immunolabeling experiments the number of gold particles localized in the ER was more than 10-fold above the number of gold particles found in control sections (grids processed in parallel incubated with a nonimmune sera or without the primary antibody). Taken together, these results show that in rat and human liver parenchymal cells, the 106-kd cholesteryl ester hydrolase exclusively localizes to the ER of the cell and is henceforth designated erCEH.
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FIG. 2. Localization of erCEH in HepG2 cells by confocal microscopy. HepG2 cells were costained for CEH (green) and calnexin (red). Confocal images were taken using a ⫻63 oil objective. The majority of CEH colocalizes with calnexin, a marker of the ER as seen by overlying the green and red images (yellow). The bars represent 6 m.
Finally, the tissue-specific expression of erCEH was examined in microsomes derived from a range of rat tissues including steroid hormone-producing glands: adrenal glands, ovaries, and testis; tissues playing important roles in lipid metabolism: pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, and brain; and coronary heart disease–involved tissues/cells: aorta and macrophages. Interestingly, the anti-CEH antibody did not react with proteins of 106 kd, or of any other molecular mass,
from the tissues studied, indicating the absence of immunologically related proteins in such tissues (data not shown). DISCUSSION
In this report we present the localization of a novel cholesteryl ester hydrolase, erCEH, associated with the ER of the mammalian hepatocyte. The molecular mass of the protein does not differ among human, rat, and mouse. The erCEH
FIG. 3. High-resolution immunolocalization of erCEH in rat liver. Rat liver thin sections were processed for immunolocalization of CEH using 1/50 (A), 1/10 (B), and 1/2 (C) dilution of the primary CEH antibody. The results illustrate that gold particles localize in the endoplasmic reticulum (er) of the hepatocyte (A, B, C). No gold labeling was detected in the nuclei (n, in A), mitochondria (m, in B and C), peroxisomes (p, in B), and other subcellular structures of the liver. Bars, 200 nm.
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appears to be specifically expressed in the hepatocyte. We have now demonstrated the presence of a cholesteryl ester hydrolase in the ER of mammals. The protein erCEH seems to be mainly associated to the rough ER of the hepatocyte (Fig. 3). Previous biochemical analysis of the distribution of CEH revealed that RNA-rich microsomes were responsible for about 75% of the CEH activity elicited by rat liver microsomes.28 Because of the very low presence of smooth ER in the liver preparations herein analyzed, it is very difficult to assess whether erCEH is also resident in the smooth ER. Therefore, the association or not of erCEH with the smooth ER will require the development of additional work designed for this purpose. erCEH is a bile salt–independent lipase that specifically catalyzes the hydrolysis in vitro of cholesteryl esters,14 the same esters hydrolyzed by the cytosolic4 and lysosomal3 CEH. Some lipases are intimately linked to the vascular endothelium. One case is the heparin-releasable hepatic lipase that hydrolyzes phospholipids and triglycerides of plasma lipoproteins. Hepatic lipase is synthesized and secreted by the hepatocytes. Some hepatic lipase remains bound to the hepatocyte surface and some other translocates to the endothelial surfaces of liver sinusoids.29 In this work we show the lack of erCEH in liver endothelial cells (Fig. 1) and in hepatocyte cell structures other than the ER (Figs. 1-3). These results indicate that erCEH is confined to the ER of the hepatocyte and that it does not transit through the secretory pathway or translocate to the plasma membrane to a significant extent. It appears that most of the gold signals that reveal erCEH decorate the cytoplasmic side of the ER of the hepatocyte (Fig. 3). Although with this approach it is not possible to provide an indication of the precise location and/or way of association of erCEH with the ER membrane, the fact that erCEH can be solubilized from the microsomal membranes without using detergents,14 or at detergent concentrations that are too low to solubilize integral membrane proteins (Cristo´bal et al., unpublished observation), suggests that erCEH is peripherally associated. In fact, carbonate extraction of rat liver microsomes, a treatment used to ascribe the peripheral or integral nature of a protein in membranes,26 promoted the complete extraction of erCEH but not that of calnexin, an integral membrane protein of the ER (Fig. 4). These findings strongly support the contention that erCEH is a peripheral membrane protein of the ER facing the cytoplasm of the hepatocyte. Cholesteryl ester hydrolases are found ubiquitously. It is generally assumed that their functional activity and regulation depends on the tissue and location of the enzyme. Relatively high activity and expression levels of ACAT and cytosolic CEH have been reported in most tissues analyzed. In contrast, we show that the hepatic parenchyma is likely to be the major, if not the only, tissue in which erCEH is expressed. Of the two ACAT isoforms identified so far, the ACAT2 seems to be preferentially expressed in the liver and intestine and is involved in the particular function of providing cholesteryl esters for lipoprotein secretion.30-33 ACAT1 has a nearly ubiquitous expression pattern and seems to serve more general cellular functions, such as modulation of potentially toxic effects of cholesterol in cell membranes.1,32 The ER of liver parenchymal cells is critical for cholesterol output. The finding that erCEH is present in the ER of both periportal and perivenous hepatocytes suggests that the hydrolysis of cholesteryl esters by erCEH could take place in a site in which cholesterol is
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FIG. 4. erCEH is peripherally associated with ER membranes. To provide an indication of the type of association of erCEH with ER membranes, microsomes were extracted with sodium carbonate and the distribution of erCEH and calnexin studied in the resulting soluble and membrane fractions. Ten to 30 g of microsomal proteins were treated with 100 mmol/L sodium carbonate or 150 mmol/L Tris-HCl buffer. The latter treatment was used as a negative control of the extraction procedure. Proteins from the soluble extract and membrane pellet along with native microsomes were subjected to SDS-8% PAGE and either stained with Coomassie Blue (20 g protein, upper panel) or probed with antibodies against CEH (10 g protein, intermediate panel) or against calnexin (30 g protein, lower panel). Blots were developed with the ECL reagent. Molecular mass markers (kd) are shown on the left. The results obtained indicate a complete extraction of erCEH in the soluble carbonate supernatant whereas calnexin, an integral membrane protein of the ER, remains in the membrane nonextracted fraction. This finding strongly suggests a peripheral association of erCEH to the ER membrane. The blots shown are representative of 2 independent experiments.
either converted to bile acids and exported to the gut or esterified by ACAT and exported to the blood stream. Thus, erCEH might complement ACAT2 in controlling the amount of cholesterol that, in its free and esterified forms, is transferred across the ER membrane by the microsomal triglyceride transport protein34 to be incorporated into VLDL particles. It is tempting to suggest that erCEH might play a role as a local regulator of these hepatocyte-restricted functions in cholesterol metabolism. Alternatively, a regulatory role of erCEH for the maintenance of a cholesterol:cholesteryl ester ratio in ER membranes cannot be excluded. Finally, it is remarkable that essential regulatory molecules, including SREBP (sterol regulatory element-binding protein)35 and SCAP (SREBP cleavage-activating protein),36 and key-limiting enzymes, such as 3-hydroxy-3-methylglutarylCoA reductase,37 ACAT,38 and cholesterol 7␣-hydroxylase,39 involved in cholesterol metabolism are located in the ER. The finding of a CEH in the ER of the hepatocyte strongly suggests its relevant cellular function in cholesterol homeostasis. Understanding the functional relevance of erCEH in the liver will require, as a first step, the yet elusive availability of a specific cDNA probe.
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Acknowledgment: The authors thank Dr. F. Vidal-Vanaclocha and Dr. F. Dı´az-Aguirregoitia for providing the rat nonparenchymal liver cells and human liver biopsies, respectively. J. R. Romero is acknowledged for his help in the preparation and characterization of rat periportal and perivenous hepatocytes. The authors thank C. Sa´nchez and M. Guerra for assisting in the confocal and electron microscopy work, respectively.
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from the liver of estradiol-treated rats. Proc Natl Acad Sci U S A 1987;84: 6785-6789. Romero JR, Fresnedo O, Isusi E, Barrionuevo J, Ochoa B. Hepatic zonation of the formation and hydrolysis of cholesteryl esters in periportal and perivenous parenchymal cells. Lipids 1999;34:907-913. Musanti R, Chiari A, Ghiselli G. Peritoneal macrophage cholesteryl ester content as a function of plasma cholesterol in rats. Arterioscler Thromb 1991;11:1111-1119. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976:72:248-254. Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970;227:680-685. San Martı´n C, Flores AI, Cuezva JM. Cpn60 is exclusively localized into mitochondria of rat liver and embryonic Drosophila cells. J Cell Biochem 1995;59:235-245. Egea G, Izquierdo JM, Ricart J, San Martin C, Cuezva JM. mRNA encoding the -subunit of the mitochondrial F1-ATPase complex is a localized mRNA in rat hepatocytes. Biochem J 1997;322: 557-565. Ricart J, Egea G, Izquierdo JM, San Martin C, Cuezva JM. Subcellular structure containing mRNA for -subunit of mitochondrial H⫹-ATP synthase in rat hepatocytes is translationally active. Biochem J 1997;324: 635-643. Fujiki Y, Hubbard AL, Fowler S, Lazarow PW. Isolation of intracellular membranes by means of sodium carbonate treatment: application to the endoplasmic reticulum. J Cell Biol 1982;93:103-110. High S, Lecomte FJ, Russell SJ, Abell BM, Oliver JD. Glycoprotein folding in the endoplasmic reticulum: a tale of three chaperones? FEBS Lett 2000;476:38-41. Ferna´ndez CJ, Lacort M, Gandarias JM, Ochoa B. Topological studies on rat liver microsomal cholesterol ester hydrolase. Biochem Biophys Res Comm 1987;146:1212-1217. Breedveld B, Schoonderwoerd K, Verhoeven AJM, Willemsen R, Jansen H. Hepatic lipase is localized at the parenchymal cell microvilli in rat liver. Biochem J 1997;321:425-430. Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Shelness GS, Rudel LL. Identification of a form of a acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J Biol Chem 1998; 273:26747-26754. Cases S, Novak S, Zheng YW, Myers HM, Lear SR, Sande E, Welch CB, et al. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. J Biol Chem 1998;273:26755-26764. Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl-CoA:cholesterol acyltransferaserelated enzymes. J Biol Chem 1998;273:26765-26771. Joyce C, Skinner K, Anderson RA, Rudel LL. Acyl-CoA:cholesterol acyltransferase 2. Curr Opin Lipidol 1999;10:89-95. White DA, Bennett AJ, Billet MA, Salter AM. The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triglyceride transfer protein. Br J Nutr 1998;80:219-229. Brown MS, Goldstein JL. The SREBP pathway. Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331-340. Hua X, Nohturfft A, Goldstein JL, Brown MS. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Cell 1996;87:415-426. Reinhart MP, Billheimer JT, Faust JR, Gaylor JL. Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver. J Biol Chem 1987;262:9649-9655. Khelef N, Buton X, Beatini N, Wang H, Meiner V, Chang TY, Farese RV, et al. Immunolocalization of acyl-CoA:cholesterol acyltransferase in macrophages. J Biol Chem 1998;273:11218-11224. Bjo¨rkhem I. Mechanism of bile acid synthesis in mammalian liver. In: Danielsson H, Sjo¨vall J, eds. Sterols and Bile Acids. Amsterdam: Elsevier Scientific Publishers 1985;231-278.
We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissuerestricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism. (HEPATOLOGY 2001;33:662-667.) The mammalian liver plays a central role in the synthesis, redistribution, and regulation of whole body cholesterol and plasma lipoprotein homeostasis. Cholesterol balance in the
Abbreviations: VLDL, very low-density lipoprotein; ACAT, acyl-CoA:cholesterol acyltransferase; ER, endoplasmic reticulum; CEH, cholesteryl ester hydrolase; erCEH, endoplasmic reticulum CEH; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. From the 1Department of Physiology, University of the Basque Country Medical School, Bilbao, Spain; 2Departamento de Biologı´a Molecular, Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, Madrid, Spain. Received August 3, 2000; accepted December 28, 2000. O.F. and M.L.H. contributed equally to this work. S.C.’s present address is: Medical Biochemistry and Biophysics Department, Karolinska Institute, Stockholm, Sweden. Supported by the Spanish Ministry of Education and Culture (Grants PB97/0046 to B.O. and PB97/0018 to J.M.C.), by the Basque Government (Grant PI97/77 to B.O.), and by an Institutional Grant to the CBMSO from the Fundacio´n Ramo´n Areces, Spain. Address reprint requests to: Begon˜a Ochoa, Ph.D., Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, 48080-Bilbao, Spain. E-mail: [email protected]; fax: (34) 94-6015662. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3303-0023$35.00/0 doi:10.1053/jhep.2001.22763
liver involves two input pathways, cholesterol synthesis and lipoprotein uptake; two output pathways, the formation of bile acids and bile and the secretion of very low-density lipoproteins (VLDL); as well as the reversible conversion of cholesterol to cholesteryl esters. Esterification of cholesterol with long-chain fatty acyl-CoA is accomplished by acyl-CoA: cholesterol acyltransferase (ACAT), an integral membrane protein located in the endoplasmic reticulum (ER) that is allosterically regulated by cholesterol (for a recent review, see Chang et al.1). Newly formed cholesteryl esters are secreted as a component of VLDL or are stored in intracellular lipid droplets in the cytoplasm, where cholesteryl esters undergo a constant cycle of hydrolysis and resynthesis. Three major cholesteryl ester hydrolases (CEH) have been identified in the liver that differ in their subcellular localization, function, and enzymatic properties. The lysosomal CEH, or acid lipase, is involved in the hydrolysis of cholesteryl esters and triacylglycerols delivered to the hepatocytes via receptor-mediated endocytosis of lipoproteins.2 The lysosomal CEH has been purified and cloned from several species, including the human liver.3 The major cytosolic CEH from rat liver has also been purified and characterized,4 as well as cloned and expressed.5 This neutral cytosolic CEH is considered to be a key enzyme for releasing free cholesterol from the stores of cholesteryl esters in the cytoplasm and is subjected to complex regulation by hormones and sterols.6,7 Biochemically, a third cholesteryl ester hydrolase activity has been characterized in rat liver microsomes. The microsomal CEH activity of rat liver has a neutral optimal pH and is modulated by changes in cholesterol or bile acid flux (i.e., see Gad and Harrison8 and Martı´nez et al.9), postnatal development,10 circadian rhythm,9 and agonists that trigger activation of cAMPdependent protein kinase,11,12 protein kinase C,12 and AMPactivated protein kinase.13 Recently, we isolated and purified the major microsomal CEH from rat liver.14 The purified CEH from rat liver microsomes is a 106-kd protein that efficiently catalyzes the specific hydrolysis of cholesteryl esters in vitro. Biochemical analysis indicated that the microsomal CEH shares many of the hallmark features of the lipases and carboxylesterases protein family, but differs clearly from other liver cholesteryl ester– splitting enzymes, including the lysosomal and cytosolic CEH isoforms, the hepatic lipase, the retinyl ester hydrolase, the triacylglycerol lipase, and nonspecific carboxylesterases.14 Antibodies raised against the purified CEH from rat liver microsomes neutralized 90% of the CEH activity in rat liver microsomes and 100% of the CEH activity in the preparation of the pure protein.14 In this study we have used the anti-microsomal CEH antibodies to study the subcellular localization and tissue-specific
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expression of microsomal CEH in rodents and human liver samples. Subcellular fractionation of rat hepatocytes, confocal microscopy of human HepG2 cells, and high-resolution immunoelectron microscopy of rat liver samples unambiguosly localized the CEH protein exclusively to the endoplasmic reticulum. The henceforth termed endoplasmic reticulum CEH, erCEH, was not detected in other liver cell types or in other rat tissues with an active cholesterol-cholesteryl ester cycle. The physiologic function and relevance of erCEH in cholesterol homeostasis remains to be established in future studies. MATERIALS AND METHODS Antibodies. Polyclonal antibodies were raised in New Zealand rabbits against the purified CEH from rat liver microsomes.14 Antibodies were purified from the rabbit serum by affinity chromatography on a Hitrap NH-Sepharose column (Amersham Pharmacia Biotech Europe Gmbh, Barcelona, Spain). The coupling of the purified CEH to the column was performed according to the manufacturer’s instructions. Cell and Tissue Processing for Immunoblotting. Microsomes were prepared from adult rat, mouse and human liver, HepG2 cells, and adult rat periportal and perivenous hepatocytes, nonparenchymal liver cells, ovaries, testis, adrenal glands, intestine, aorta, brain, pancreas, spleen, lung, epididymal adipose tissue, mammary glands, and peritoneal macrophages. In brief, cells and small pieces of tissues were homogenized in 4 vol of 20 mmol/L Tris/HCl buffer, pH 7.4, containing 0.25 mol/L sucrose, 2 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 10 mol/L leupeptin, and 1 mmol/L benzamidine, and the homogenates centrifuged as detailed by Ruiz and Ochoa.15 Microsome,15 cytosol,15 lysosome,16 and plasma membrane17 preparations were obtained from hepatocytes isolated from adult female rats fed ad libitum.15 More than 55%, 40%, and 30% of the total activity of marker enzymes for the endoplasmic reticulum (glucose-6-phosphatase17), lysosomes (acid phosphatase16), and plasma membrane (5⬘ nucleotidase17), respectively, was recovered in the preparations. Receptor-rich endosomes were isolated from livers of 17␣-ethinyl estradiol-treated female rats that had been injected low density lipoprotein, as in the study by Belcher et al.18 Periportal and perivenous hepatocytes were prepared by centrifugal elutriation of isolated rat hepatocytes and characterized for biological and biochemical markers, as detailed by Romero et al.19 Three rat nonparenchymal liver cell preparations provided by Dr. F. Vidal-Vanaclocha from the University of the Basque Country, Bilbao, Spain, were studied. Each one of the preparations contained a slightly different percentage of endothelial cells (⬃70%), lymphocytes (⬃25%), and Kupffer cells (⬃5%). Human liver biopsies were provided by Dr. F. Dı´az-Aguirregoitia (Cruces Hospital, Baracaldo, Spain). The HepG2 cell line (American Type Culture Collection, Rockville, MD) was grown in Dulbecco’s modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 0.1% (wt/vol) streptomycin (all from Gibco BRL Life Technologies, Paisley, UK). When the cell culture reached approximately 80% confluence, cells were harvested and washed repeatedly in phosphate-buffered saline (PBS; 0.01 mol/L phosphate buffer, 0.15 mol/L NaCl, pH 7.2). Rat peritoneal macrophages were harvested as described previously.20 The protein concentration was determined with the Bradford reagent21 using bovine serum albumin (BSA) as the standard. Immunoblotting. Proteins were fractionated on sodium dodecyl sulfate (SDS)/8% polyacrylamide gel electrophoresis (PAGE).22 After fractionation, proteins were transferred to polyvinylidene difluoride membranes using a Mini Trans-Blot electrophoretic transfer cell (Biorad, Hercules, CA) with 50% (vol/vol) electrophoresis buffer (0.025 mol/L Tris, 0.192 mol/L glycine, 0.1% SDS) and 10% (vol/vol) methanol at 20 V for 60 minutes. Membranes were incubated with gentle agitation in Tris-buffered saline (0.01 mol/L Tris buffer, 0.15 mol/L NaCl, pH 8.0) as follows: (1) blocking for 1 hour in 3% (wt/ vol) BSA; (2) incubation with a 1:1,000 dilution of primary anti-CEH
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antibody in 0.5 % (wt/vol) BSA overnight, followed by 3 10-minute washes with buffer; (3) incubation with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO) in 3% (wt/vol) BSA for 1 hour, followed by 3 10-minute washes with buffer. The blots were developed with freshly prepared peroxidase substrates, hydrogen peroxide, and 4-chloro-1naftol, as described in Cristo´bal et al.,14 or with the ECL reagent from Amersham following the supplier’s instructions. Purified CEH was loaded in the gels as a positive control. Confocal Microscopy. For immunocytochemistry: briefly, HepG2 cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and plated on coverslips the day before the experiment. Coverslips were fixed in methanol for 3 minutes at ⫺20°C and then incubated with a 1:100 dilution of anti-CEH for 1 hour at 37°C. Coverslips were then washed with PBS and further incubated for 1 hour at 37°C with a 1:200 dilution of a goat anti-rabbit IgG fluorescein-conjugate (TAGO Immunochemicals, Burlingame, CA). Next, they were washed with PBS and incubated for 1 hour at 37°C with a 1:200 dilution of the IgG fraction of mouse anti-calnexin (Transduction Laboratories, Lexington, KY). Afterwards, coverslips were washed with PBS and further incubated for 1 hour at 37°C with a 1:100 dilution of a rabbit anti-mouse IgG Texas red-conjugate (Cappel, Turnhout, Belgium). After various PBS washes, the coverslips were mounted on glass slides using Gelvatol (Montsanto, USA). The fluorescence of fluorescein and Texas red were detected by laser confocal microscopy using excitation wavelengths of 488 or 543 nm, and detection wavelengths of 522 or 615, respectively. A Biorad Radiance 2000 Zeiss Axiovert S100 TV confocal microscope was used. Digital images were acquired using the software Lasersharp 2.0 provided by the dealer (Biorad, Hercules, CA). Tissue Processing for Electron Microscopy. Small pieces of liver obtained from 10-week-old overnight fasted female rats were fixed by immersion in freshly prepared 4% paraformaldehyde in 0.1 mol/L So¨rensen phosphate buffer, pH 7.2, for 2 hours at 4°C. Samples were rinsed in buffer and the free-aldehyde groups were quenched with 50 mmol/L ammonium chloride in PBS for 60 minutes at 4°C. Afterwards, the samples were rinsed in PBS, dehydrated in acetone, and finally processed for embedding in Lowicryl K4M (Polysciences Europe, Eppelheim, Germany) according to the manufacturer’s instructions. Gold interferential color ultrathin sections were collected in collodion/carbon-coated nickel grids. The grids were observed in a Jeol 1010 electron microscope under 80 kV accelerating voltage. Immunocytochemical Localization of CEH by Electron Microscopy. Grids were incubated for 5 minutes with PBS containing 1% BSA and then incubated with 1/2, 1/10, or 1/50 dilution of anti-CEH in the same buffer. After 3 washes with PBS, grids were incubated for 45 minutes with goat anti-rabbit IgGs conjugated with 10 nm colloidal gold (British BioCell, Cardiff, UK). The grids were washed twice in PBS and distilled water and air-dried. Counterstaining was performed with 2% aqueous uranyl acetate (6 minutes) and Reynolds lead citrate (45 seconds). Standard controls for immunocytochemical techniques23-25 were conducted in parallel to assess the specificity of the immunoreactive signals: (1) the omission of the primary antibody and (2) the incubation with the pre-immune rabbit serum, in both cases followed by incubation with the gold labeled secondary antibody. Both controls provided no gold decoration of hepatocyte structures. Carbonate Extraction of erCEH From Microsomal Membranes. Rat hepatocyte microsomes (2 mg of protein) were incubated at 0°C for 30 minutes with 0.5 mL of either 100 mmol/L sodium carbonate, pH 11.5, or 150 mmol/L Tris-HCl buffer, pH 8, containing 0.5 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 10 mol/L leupeptin, and 1 mmol/L benzamidine.26 After centrifugation at 230,000g for 1 hour at 4°C, the extract was aspirated and the membrane pellet was resuspended in 0.5 mL of 20 mmol/L Tris-HCl buffer, pH 7.2. Aliquots of the extract and pellet fractions were further subjected to SDS-8% PAGE, and either stained with Coomassie Blue or probed with antibodies against CEH or against calnexin, as indicated previously.
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RESULTS
In this work we have studied the subcellular localization and the expression pattern in rat tissues of a mammalian CEH using antibodies raised against the purified CEH from rat liver microsomes.14 Figure 1A illustrates the specificity of the antibodies developed.14 When the antibodies are used in Western blot analysis of mixtures of proteins, derived either from isolated rat liver microsomes (Fig. 1A, right panel) or from total rat liver homogenates (Fig. 1A, left panel), only one specific immunoreactive band is detected. The immunoreactive protein, of approximately 106 kd, coincides in its electrophoretic migration with that of the purified CEH from rat liver microsomes (Fig. 1A, and see Cristo´bal et al.14). In the more complex protein mixture of total liver homogenates, especially in those tracks where there is protein overloading, several faint immunoreactive signals appear (Fig. 1A, left panel). These signals never represented more than 1% of the total immunoreactive signal of the blot. The nature of these faint immunoreactive proteins, present only in mixtures of liver homogenates, is unknown. In any case, these results unambiguously show that the anti-CEH developed14 is an excellent tool to be used with the purpose of studying the subcellular localization of the protein.
FIG. 1. Subcellular localization and cell-specific expression of CEH. (A) Specificity of the anti-CEH antibody in Western blots of mixtures of proteins from rat liver homogenates and microsomes. Proteins were fractionated on SDS-8% PAGE and either stained with Coomassie Blue (C) or probed with anti-CEH antibody (W). In the lane of purified CEH, approximately 0.2 g of protein was loaded. Blots were developed with the ECL reagent. (B) Subcellular fractions were obtained from isolated rat hepatocytes, and microsomes were prepared from the sources indicated. Proteins were fractionated on SDS-8% PAGE and probed with anti-CEH antibody; blots were developed with 4-chloro-1-naftol. Lanes 1-5: microsomes (10 g), lysosomes (15 g), cytosol (50 g), endosomes (50 g), and plasma membrane (50 g) from isolated rat hepatocytes; lanes 6-12: microsomes (20-30 g) from rat liver, rat periportal hepatocytes, rat perivenous hepatocytes, rat nonparenchymal liver cells, mouse liver, human liver, and HepG2 cells. Purified CEH (2 g) was loaded in the gels as a positive control. Molecular mass markers (kd) are shown on the left. The blots shown are representative of several independent experiments.
The localization of CEH was studied in subcellular fractions obtained from isolated rat hepatocytes. Western blots of different membrane fractions revealed that the anti-CEH antibody reacted with a 106-kd protein only in microsomes (Fig. 1B). No immunoreactive signal was detected in other compartments along the secretory pathway, such as the lysosomes, the endosomes and the plasma membrane (Fig. 1B). In agreement with previous findings,14 no immunoreactivity was observed with proteins from the cytosol. The expression of CEH in the microsomal fraction of different cell types of the rat liver was further studied. The results obtained illustrated the presence of the 106-kd protein in microsomes of both periportal and perivenous hepatocytes (Fig. 1B). Remarkably, no immunoreactive signal was detected in the microsomes of non-parenchymal liver cells (Fig. 1B). In addition, the antibody recognized only one protein band in microsomes of mouse and human liver as well as in microsomes of the human hepatoma HepG2 (Fig. 1B). The CEH band in human and mouse samples corresponded to an apparent molecular mass of 106 kd, the molecular mass of purified CEH from rat liver (Fig. 1B). The human microsomal CEH is immunologically related to the rat microsomal CEH (Fig. 1B), which is consistent with the finding of a high specific activity of the neutral CEH in preparations of human liver microsomes (576 ⫾ 24 pmol cholesteryl oleate hydrolyzed ⫻ h⫺1 ⫻ mg of microsomal protein⫺1, mean ⫾ SD of 3 samples analyzed in duplicate). To our knowledge, this is first description of a cholesteryl ester hydrolase in microsomes of human liver. Confocal immunofluorescence analysis of HepG2 cells using antibodies against CEH (Fig. 2, left panel) and calnexin (Fig. 2, middle panel) revealed essentially the same distribution of immunofluorescence throughout the cytoplasm of the cells for the 2 proteins. Calnexin is an integral membrane protein of the ER that functions as a chaperone.27 The clear colocalization of CEH with a marker of the endoplasmic reticulum (see yellow signals in the merge picture on Fig. 2) strongly suggest the localization of CEH in the ER of the cell. High resolution immunoelectron microscopy was further used to localize CEH in the adult rat liver. Figure 3 illustrates that gold particles specifically decorated the rough ER of the hepatocyte. In agreement with previous findings obtained by subcellular fractionation and confocal microscopy, no significant gold-labeling was observed in the cytoplasm (Fig. 3), the nuclei (Fig. 3A), the mitochondria (Fig. 3B and C), and the peroxisomes (Fig. 3B and C) of the hepatocyte at any of the 3 dilutions of the primary antibody used (see Fig. 3). The specificity of the ER labeling could be best assessed by the large differences (1 to 2 orders of magnitude) found in the number of gold particles decorating the rough ER when compared with the number of gold particles decorating nonimmunoreactive organelles in the same field (see Fig. 3). In addition, in all immunolabeling experiments the number of gold particles localized in the ER was more than 10-fold above the number of gold particles found in control sections (grids processed in parallel incubated with a nonimmune sera or without the primary antibody). Taken together, these results show that in rat and human liver parenchymal cells, the 106-kd cholesteryl ester hydrolase exclusively localizes to the ER of the cell and is henceforth designated erCEH.
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FIG. 2. Localization of erCEH in HepG2 cells by confocal microscopy. HepG2 cells were costained for CEH (green) and calnexin (red). Confocal images were taken using a ⫻63 oil objective. The majority of CEH colocalizes with calnexin, a marker of the ER as seen by overlying the green and red images (yellow). The bars represent 6 m.
Finally, the tissue-specific expression of erCEH was examined in microsomes derived from a range of rat tissues including steroid hormone-producing glands: adrenal glands, ovaries, and testis; tissues playing important roles in lipid metabolism: pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, and brain; and coronary heart disease–involved tissues/cells: aorta and macrophages. Interestingly, the anti-CEH antibody did not react with proteins of 106 kd, or of any other molecular mass,
from the tissues studied, indicating the absence of immunologically related proteins in such tissues (data not shown). DISCUSSION
In this report we present the localization of a novel cholesteryl ester hydrolase, erCEH, associated with the ER of the mammalian hepatocyte. The molecular mass of the protein does not differ among human, rat, and mouse. The erCEH
FIG. 3. High-resolution immunolocalization of erCEH in rat liver. Rat liver thin sections were processed for immunolocalization of CEH using 1/50 (A), 1/10 (B), and 1/2 (C) dilution of the primary CEH antibody. The results illustrate that gold particles localize in the endoplasmic reticulum (er) of the hepatocyte (A, B, C). No gold labeling was detected in the nuclei (n, in A), mitochondria (m, in B and C), peroxisomes (p, in B), and other subcellular structures of the liver. Bars, 200 nm.
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appears to be specifically expressed in the hepatocyte. We have now demonstrated the presence of a cholesteryl ester hydrolase in the ER of mammals. The protein erCEH seems to be mainly associated to the rough ER of the hepatocyte (Fig. 3). Previous biochemical analysis of the distribution of CEH revealed that RNA-rich microsomes were responsible for about 75% of the CEH activity elicited by rat liver microsomes.28 Because of the very low presence of smooth ER in the liver preparations herein analyzed, it is very difficult to assess whether erCEH is also resident in the smooth ER. Therefore, the association or not of erCEH with the smooth ER will require the development of additional work designed for this purpose. erCEH is a bile salt–independent lipase that specifically catalyzes the hydrolysis in vitro of cholesteryl esters,14 the same esters hydrolyzed by the cytosolic4 and lysosomal3 CEH. Some lipases are intimately linked to the vascular endothelium. One case is the heparin-releasable hepatic lipase that hydrolyzes phospholipids and triglycerides of plasma lipoproteins. Hepatic lipase is synthesized and secreted by the hepatocytes. Some hepatic lipase remains bound to the hepatocyte surface and some other translocates to the endothelial surfaces of liver sinusoids.29 In this work we show the lack of erCEH in liver endothelial cells (Fig. 1) and in hepatocyte cell structures other than the ER (Figs. 1-3). These results indicate that erCEH is confined to the ER of the hepatocyte and that it does not transit through the secretory pathway or translocate to the plasma membrane to a significant extent. It appears that most of the gold signals that reveal erCEH decorate the cytoplasmic side of the ER of the hepatocyte (Fig. 3). Although with this approach it is not possible to provide an indication of the precise location and/or way of association of erCEH with the ER membrane, the fact that erCEH can be solubilized from the microsomal membranes without using detergents,14 or at detergent concentrations that are too low to solubilize integral membrane proteins (Cristo´bal et al., unpublished observation), suggests that erCEH is peripherally associated. In fact, carbonate extraction of rat liver microsomes, a treatment used to ascribe the peripheral or integral nature of a protein in membranes,26 promoted the complete extraction of erCEH but not that of calnexin, an integral membrane protein of the ER (Fig. 4). These findings strongly support the contention that erCEH is a peripheral membrane protein of the ER facing the cytoplasm of the hepatocyte. Cholesteryl ester hydrolases are found ubiquitously. It is generally assumed that their functional activity and regulation depends on the tissue and location of the enzyme. Relatively high activity and expression levels of ACAT and cytosolic CEH have been reported in most tissues analyzed. In contrast, we show that the hepatic parenchyma is likely to be the major, if not the only, tissue in which erCEH is expressed. Of the two ACAT isoforms identified so far, the ACAT2 seems to be preferentially expressed in the liver and intestine and is involved in the particular function of providing cholesteryl esters for lipoprotein secretion.30-33 ACAT1 has a nearly ubiquitous expression pattern and seems to serve more general cellular functions, such as modulation of potentially toxic effects of cholesterol in cell membranes.1,32 The ER of liver parenchymal cells is critical for cholesterol output. The finding that erCEH is present in the ER of both periportal and perivenous hepatocytes suggests that the hydrolysis of cholesteryl esters by erCEH could take place in a site in which cholesterol is
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FIG. 4. erCEH is peripherally associated with ER membranes. To provide an indication of the type of association of erCEH with ER membranes, microsomes were extracted with sodium carbonate and the distribution of erCEH and calnexin studied in the resulting soluble and membrane fractions. Ten to 30 g of microsomal proteins were treated with 100 mmol/L sodium carbonate or 150 mmol/L Tris-HCl buffer. The latter treatment was used as a negative control of the extraction procedure. Proteins from the soluble extract and membrane pellet along with native microsomes were subjected to SDS-8% PAGE and either stained with Coomassie Blue (20 g protein, upper panel) or probed with antibodies against CEH (10 g protein, intermediate panel) or against calnexin (30 g protein, lower panel). Blots were developed with the ECL reagent. Molecular mass markers (kd) are shown on the left. The results obtained indicate a complete extraction of erCEH in the soluble carbonate supernatant whereas calnexin, an integral membrane protein of the ER, remains in the membrane nonextracted fraction. This finding strongly suggests a peripheral association of erCEH to the ER membrane. The blots shown are representative of 2 independent experiments.
either converted to bile acids and exported to the gut or esterified by ACAT and exported to the blood stream. Thus, erCEH might complement ACAT2 in controlling the amount of cholesterol that, in its free and esterified forms, is transferred across the ER membrane by the microsomal triglyceride transport protein34 to be incorporated into VLDL particles. It is tempting to suggest that erCEH might play a role as a local regulator of these hepatocyte-restricted functions in cholesterol metabolism. Alternatively, a regulatory role of erCEH for the maintenance of a cholesterol:cholesteryl ester ratio in ER membranes cannot be excluded. Finally, it is remarkable that essential regulatory molecules, including SREBP (sterol regulatory element-binding protein)35 and SCAP (SREBP cleavage-activating protein),36 and key-limiting enzymes, such as 3-hydroxy-3-methylglutarylCoA reductase,37 ACAT,38 and cholesterol 7␣-hydroxylase,39 involved in cholesterol metabolism are located in the ER. The finding of a CEH in the ER of the hepatocyte strongly suggests its relevant cellular function in cholesterol homeostasis. Understanding the functional relevance of erCEH in the liver will require, as a first step, the yet elusive availability of a specific cDNA probe.
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Acknowledgment: The authors thank Dr. F. Vidal-Vanaclocha and Dr. F. Dı´az-Aguirregoitia for providing the rat nonparenchymal liver cells and human liver biopsies, respectively. J. R. Romero is acknowledged for his help in the preparation and characterization of rat periportal and perivenous hepatocytes. The authors thank C. Sa´nchez and M. Guerra for assisting in the confocal and electron microscopy work, respectively.
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19. 20. 21.
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