Specific Gene Expression of ATP-binding Cassette Transporters and Nuclear Hormone Receptors in Rat Liver Parenchymal, Endothelial, and Kupffer Cells

Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI (∼3-fold), peroxisome proliferator-activated receptor (PPAR)α and PPARγ (8–20-fold), cholesterol 7α-hydroxylase A1 (>100-fold), and ABCG5/G8 (∼5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)β, PPARδ, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRα, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level. Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI (∼3-fold), peroxisome proliferator-activated receptor (PPAR)α and PPARγ (8–20-fold), cholesterol 7α-hydroxylase A1 (>100-fold), and ABCG5/G8 (∼5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)β, PPARδ, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRα, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level. Transport of cholesterol through the body is an important process in the maintenance of total body cholesterol homeostasis. One specific component is the transport of excessive cholesterol from the periphery to the liver by the high density lipoprotein (HDL). 1The abbreviations used are: HDL, high density lipoprotein; ABC, ATP-binding cassette; C t, threshold cycle number; CYP7A1, cholesterol 7α-hydroxylase; CYP27, cholesterol 27-hydroxylase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; SR-BI, scavenger receptor class BI. This process is called reverse cholesterol transport and consists of three different stages: the efflux of peripheral cellular cholesterol to HDL, the transport of cholesterol esters through the blood to the liver, and the uptake of cholesterol esters by the liver (1Glomset J.A. Adv. Intern. Med. 1980; 25: 91-116PubMed Google Scholar). Cholesterol esters are taken up into the liver through selective uptake by the HDL receptor, scavenger receptor class BI (SR-BI) (2Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2010) Google Scholar), where they are primarily (∼50%) catabolized to bile acids, through conversion by cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27) (3Chiang J.Y.L. Front. Biosci. 1998; 3: D176-D193Crossref PubMed Scopus (262) Google Scholar) for biliary excretion via the bile salt export protein (4Akita H. Suzuki H. Ito K. Kinoshita S. Sato N. Takikawa H. Sugiyama Y. Biochim. Biophys. Acta. 2001; 1511: 7-16Crossref PubMed Scopus (173) Google Scholar). A second major catabolic route (∼40%) is the direct efflux of cholesterol from the liver into the bile via the ABC half-transporters ABCG5 and ABCG8, which together function as a biliary sterol efflux regulator (5Lu K. Lee M.H. Hazard S. Brooks-Wilson A. Hidaka H. Kojima H. Ose L. Stalenhoef A.F. Mietinnen T. Bjorkhem I. Bruckert E. Pandya A. Brewer Jr., H.B. Salen G. Dean M. Srivastava A. Patel S.B. Am. J. Hum. Genet. 2001; 69: 278-290Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Additionally, cholesterol also effluxes from the liver to produce very low density lipoproteins, which are converted to remnants that function as precursors for low density lipoproteins. Native HDL might also be formed by the ABC transporter A1 (ABCA1)-mediated cholesterol efflux (6Tall A.R. Wang N. Mucksavage P. J. Clin. Invest. 2001; 108: 1273-1275Crossref PubMed Scopus (69) Google Scholar). Recently, another member of the ABC transporter family, ABCG1, has been proposed to play a role in the intracellular trafficking and efflux of cholesterol (7Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar). However, the exact role of ABCG1 in the liver, especially in relation to ABCG5 and ABCG8, remains to be clarified. The identification of SR-BI and novel members of the ABC transporter family, including ABCA1, ABCG1, ABCG5, and ABCG8, has allowed the molecular characterization of the individual transporters responsible for the intracellular trafficking and excretion of cholesterol (derivatives). In the liver, it has been shown that SR-BI is responsible primarily for the selective uptake of cholesterol esters from HDL (8Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (470) Google Scholar), whereas ABCG5/G8 and ABCG1, and ABCA1, are proposed mediators of efflux to the bile and to HDL, respectively (6Tall A.R. Wang N. Mucksavage P. J. Clin. Invest. 2001; 108: 1273-1275Crossref PubMed Scopus (69) Google Scholar, 9Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1361) Google Scholar). However, the liver is a complex tissue and contains, in addition to the parenchymal cells, which are localized around the bile canaliculi, endothelial cells, and tissue macrophages (Kupffer cells). To assess the individual function of the ABC transporters and their regulation by nuclear hormone receptors it is therefore essential to establish their cellular localization in the liver. Here we report that key mediators in liver cholesterol homeostasis, in particular PPARα, PPARγ, and ABCG1, are expressed differentially in specific cell types of the rat liver. Our data stress that it is necessary to focus on the regulation of genes involved in cholesterol homeostasis in the different cell types of the liver to get molecular insight in their mechanism of regulation and the consequences for liver cholesterol transport. Animals—In the study, three male Wistar WU rats (200–250 g) were fed a chow diet containing 4.3% (w/w) fat and no cholesterol, and three rats were fed a high cholesterol diet containing 2% (w/w) cholesterol, 5% olive oil (w/w), and 0.5% (w/w) cholic acid for 2 weeks. Rats were anesthetized and the vena porta was cannulated. Subsequently, the liver was perfused for 10 min with oxygenated Hanks' buffer, pH 7.4, containing HEPES (1.6 g/liter). The perfusion was continued for 10 min with Hanks‘/HEPES buffer containing 0.05% (w/v) collagenase (type IV, Sigma) and 1 mm CaCl2. Parenchymal cells were isolated after mincing the liver in Hanks’ buffer containing 0.3% bovine serum albumin, filtering through nylon gauze and centrifugation for three times 10 min at 50 × g. The pellets consisted of pure (>99%) parenchymal cells as judged by light microscopy. The supernatants were centrifuged for 10 min at 500 × g to harvest the non-parenchymal cells. By means of centrifugal elutriation the endothelial cells and Kupffer cells were separated (10Nagelkerke J.F. Barto K.P. van Berkel T.J. J. Biol. Chem. 1983; 258: 12221-12227Abstract Full Text PDF PubMed Google Scholar). The purity of each cell fraction (>95% for both) was checked by light microscopy, after staining for peroxidase activity with 3,3-diaminobenzidine (Sigma). Analysis of Gene Expression by Real Time Quantitative PCR—Total RNA was isolated from rat liver parenchymal, endothelial, and Kupffer cells using TriZol reagent (Invitrogen) according to the manufacturer's instructions. Purified RNA was DNase treated (DNase I, 10 units/μg of total RNA) and reverse transcribed (RevertAid M-MuLV reverse transcriptase) according to the protocols supplied by the manufacturer. Quantitative gene expression analysis was performed on an ABI PRISM 7700 machine (Applied Biosystems, Foster City, CA) using SYBR Green technology. PCR primers (Table I) were designed using Primer Express 1.7 software with the manufacturer's default settings (Applied Biosystems) and validated for identical efficiencies (slope = –3.3 for a plot of the threshold cycle number (C t) versus log ng cDNA). In 96-wells optical plates, 12.5 μl of SYBR Green master mix was added to 12.5 μl of cDNA (corresponding to 50 ng of total RNA input) and 300 nm forward and reverse primers in water. Plates were heated for 2 min at 50 °C and 10 min at 95 °C. Subsequently 40 PCR cycles consisting of 15 s at 95 °C and 60 s at 60 °C were applied. At the end of the run, were heated to 95 °C with a time of min to to that PCR were The of in the RNA was by using total RNA that been to reverse was used as the of gene and expression levels gene expression were calculated by the C of the gene from the C of and 2 to the of C are as the number of PCR cycles at which the the PCR a gene mRNA are expressed to for real time PCR on an of and Primer on an of and in a of in gene expression the different liver cell types, from three different cell by real time PCR was calculated using a on the in C (C than were The in reverse cholesterol transport is the hepatic uptake and cellular processing of cholesterol esters from HDL by SR-BI (1Glomset J.A. Adv. Intern. Med. 1980; 25: 91-116PubMed Google Scholar). The mRNA expression of SR-BI in the different cell types of the liver was with real time PCR to which cell is A higher SR-BI expression was in parenchymal cells compared with endothelial and Kupffer cells In the liver, cholesterol is used for bile acid Therefore, the mRNA expression of key in bile acid and CYP27, in the different hepatic cell types were A high level of expression was in parenchymal cells, which was than higher than the expression levels in endothelial and Kupffer cells expression was in parenchymal cells and also in endothelial cells. Because a second route of cholesterol from the liver is through direct excretion of cholesterol into the bile via the half-transporters ABCG5 and ABCG8, we the expression of is also higher in parenchymal cells than in non-parenchymal cells. that ABCG5 and expression was higher in parenchymal cells compared with endothelial and Kupffer cells. In the liver, was to be involved in the efflux of cholesterol for of HDL (6Tall A.R. Wang N. Mucksavage P. J. Clin. Invest. 2001; 108: 1273-1275Crossref PubMed Scopus (69) Google Scholar). no has been in the liver ABCG1, is proposed to play a role in biliary efflux G. T. 2001; PubMed Scopus Google Scholar). In and ABCG1 expression is in to cholesterol and are involved in cholesterol efflux to Y. G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Because and ABCG1 are in the physiological we genes also a expression the different cell types of the liver. to the was expressed in parenchymal and Kupffer cells whereas ABCG1 was expressed and higher in Kupffer and endothelial cells than in parenchymal cells, respectively In to ABCA1, ABCG1 is expressed in non-parenchymal cells, which a role of ABCG1 in the excretion of cholesterol directly into the bile the In ABCG1 expression was in the different hepatic cells isolated from rats on a high cholesterol hepatic parenchymal cell ABCG1 expression increased in to a high cholesterol diet, whereas no on endothelial and Kupffer cell ABCG1 expression was ABCG1 levels were increased in parenchymal cells in to a high cholesterol diet, ABCG1 expression levels were and higher in endothelial and Kupffer cells compared with parenchymal of a high cholesterol diet on the ABCG1 mRNA expression levels as by real time PCR in liver parenchymal endothelial and Kupffer cells. from rats on a chow diet and on a high cholesterol diet are expressed to expression compared with the ABCG1 expression on a chow diet as Analysis of expression in the different cell types was performed to a relation with expression of the ABC a to that for ABCA1, with a high expression in parenchymal and Kupffer cells compared with endothelial cells A higher expression of was in endothelial liver cells compared with parenchymal and Kupffer cells respectively which that may be a important in endothelial cells. It has been shown that PPARγ are to and mRNA levels A. Lee Y. S.B. L. P. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and that for directly the expression of via an mechanism Jr., M.H. Xu Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). Therefore, we also the gene expression in the different cell that expression is in parenchymal cells, with an and higher expression in in these cells than in endothelial cells and Kupffer cells, The PPARγ is with that for suggesting a major function of these genes in parenchymal cells. mRNA levels were in the different cells, endothelial cell expression was higher as compared with parenchymal and Kupffer cells data indicate that the liver cell types a Hepatic cholesterol uptake from serum coupled with intracellular processing and bile excretion are important features in the of reverse cholesterol transport. It has been shown that of cholesterol homeostasis an essential role in the of as E. M. A. A. H. M. Full Text Full Text PDF PubMed Scopus Google Scholar) and A. M. H. J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the liver, SR-BI a role in the selective uptake of cholesterol esters from HDL (2Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2010) Google Scholar). Additionally, studies on ABC transporters that hepatic is involved in HDL (6Tall A.R. Wang N. Mucksavage P. J. Clin. Invest. 2001; 108: 1273-1275Crossref PubMed Scopus (69) Google Scholar), whereas ABCG5/G8 and ABCG1 were to biliary efflux of cholesterol from the liver (7Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 9Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. 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Additionally, we in the the SR-BI mRNA expression in the parenchymal cells compared with endothelial and Kupffer cells, which is in with the high protein expression data for SR-BI as by K. van van Berkel T.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). that mRNA data for the cell types are for the activity of the particular genes of and their metabolic SR-BI, the mRNA and protein expression data in parenchymal cells are with data that the parenchymal cholesterol uptake is in SR-BI as compared with K. and J. C. van and are the key in and bile acid PubMed Scopus Google Scholar). The high expression of these bile acid in parenchymal cells is in with the data by S. A. van Berkel T.J. Google Scholar), as it was shown that uptake of HDL cholesterol esters by the parenchymal cells is coupled to bile acid an high expression of compared with parenchymal cells was in liver endothelial cells. In J. 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Google Scholar, van Berkel T.J. PubMed Google Scholar). The high uptake and an high efflux of cholesterol from Kupffer cells might be the metabolic mechanism for the high expression level in these cells. M. J. Clin. Invest. 2001; 108: PubMed Scopus Google Scholar) and M. Kaminski W.E. E. G. J. A. van Berkel Schmitz G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) shown that to the HDL The high expression levels of in parenchymal cells, with the that functions on the of Jr., J.A. C. N. S. Brewer Jr., H.B. Biophys. PubMed Scopus (69) Google Scholar), that the liver to HDL by the efflux of cholesterol from parenchymal cells via A catabolic route for hepatic cholesterol is the direct excretion into the which for of the total liver Recently, members of the ABC ABCG5 and ABCG8, been shown to in the hepatic sterol into bile L. J. K.E. Hobbs H.H. J. Clin. Invest. PubMed Scopus Google Scholar). in ABCG5 are to a that is by levels of S. T. C. R. Dean M. K. Schmitz G. Hum. Scopus Google Scholar). Because parenchymal cells are responsible for bile acid a high expression of the biliary transporters as ABCG5/G8 in these cells compared with endothelial and Kupffer cells is with their The expression of ABCG5 ABCG8, which is in with the that these transporters as to biliary cholesterol efflux K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1361) Google Scholar, G.A. I. A. Hobbs H.H. J. Clin. Invest. PubMed Scopus Google Scholar). a novel member of the ABC transporter family, ABCG1, has also been proposed to a function in the intracellular trafficking and biliary efflux of cholesterol in the liver G. T. 2001; PubMed Scopus Google Scholar). to the ABCG1 expression was in non-parenchymal cells of the rat liver. A and higher ABCG1 expression was in Kupffer and endothelial cells than in parenchymal cell Kupffer and endothelial cells and to the total liver they contain and 24% of total liver ABCG1 a high specific ABCG1 expression in Kupffer cells was ABCG1 has also been proposed to play a role in the cholesterol efflux from peripheral macrophages (7Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar). after rats on a high cholesterol diet for 2 ABCG1 expression increased in parenchymal cells, whereas no in ABCG1 expression in endothelial and Kupffer cells was The of a of ABCG1 in endothelial and Kupffer cells in to diet may be by an activity of ABCG1 in these cells on a chow the in expression and intracellular localization of direct and of ABCG1 might to the in regulation different cell types as by Schmitz and G. T. 2001; PubMed Scopus Google Scholar). endothelial and Kupffer cell ABCG1 were and higher than that in parenchymal cells, the contribution of ABCG1 in the parenchymal cells to total liver increased from 25 to 60%. This that high cholesterol ABCG1 might to the transport of cholesterol in the parenchymal cells. is the regulation of SR-BI and the ABC transporters by nuclear hormone the and respectively J. Med. PubMed Scopus Google Scholar, P. PubMed Scopus Google Scholar). Therefore, we also their cellular localization in the different cell types of the liver. different types of the been and A high expression of was in parenchymal cells. In the liver, an essential role in the regulation of and the of bile R. Gene 2001; PubMed Scopus Google Scholar). was in the parenchymal cells, which with the high expression of in these cells. high expression levels were in Kupffer cells, where expression was In a role in the regulation of efflux via A. S.B. P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). Kupffer cells also contain high expression levels of ABCA1, which is with a role for in the regulation of in these cells. the different cell types of the liver, was with a higher expression in endothelial cells versus parenchymal and Kupffer cells. The expression of that for LXRα, which that in the liver, may a function different from that of In the liver, is to play a role in the of bile it is to a in the sterol leading to increased levels of cholic acid G. M. C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). This might the high expression of in parenchymal cells compared with endothelial and Kupffer cells. The high expression of PPARγ in parenchymal cells that PPARγ, PPARα, also has a major function in these cells. is expressed the different cell types of the liver, suggesting a function for in cell types of the liver. In we data that ABC transporters and nuclear hormone receptors involved in liver cholesterol homeostasis are expressed differentially in the specific cell types of the liver. To their intracellular transport function the liver it appears to be essential to into their cellular as especially for This appears for studies on the regulation of the transporters by nuclear receptors metabolic are coupled directly to the specific (intra)cellular expression level of the cholesterol