Regulation of multidrug resistance 2 P-glycoprotein expression by bile salts in rats and in primary cultures of rat hepatocytes

Regulation of Multidrug Resistance 2 P-Glycoprotein Expression by Bile Salts in Rats and in Primary Cultures of Rat Hepatocytes ¨ SEEMA GUPTA,1 R. TODD STRAVITZ,1 WILLIAM M. PANDAK,1 MICHAEL MULLER ,2 Z. RENO VLAHCEVIC,1 AND PHILLIP B. HYLEMON3

Biliary phospholipid secretion is tightly coupled to the secretion of free cholesterol and bile salts. The secretion of phospholipids across the canalicular membrane of hepatocytes occurs via the multidrug resistance 2 (mdr2) P-glycoprotein (Pgp). The mechanism underlying the coupling of bile salt and phospholipid secretion has not been elucidated. The aims of this study were to determine the effects of bile acid structure on the expression of mdr2 in vitro and in vivo. Under optimal culture conditions, taurine-conjugated bile acids (50 ␮mol/L) increased mdr2 messenger RNA (mRNA) levels in the following order: taurocholate (TCA) (288 ⴞ 36%, P < .005) ⴝ taurodeoxycholate (TDCA) (276 ⴞ 36%, P < .025) > taurochenodeoxycholate (TCDCA) (216 ⴞ 34%, P < .025) > tauroursodeoxycholate (TUDCA) (175 ⴞ 28%, P < .05) of control levels. The increase in mdr2 mRNA levels by TCA was both time and concentration dependent. Cholate feeding to rats with intact enterohepatic circulation increased mdr2 transcriptional activity by 4-fold and protein mass by 1.9-fold. Chronic biliary diversion (CBD) decreased mdr2 mRNA levels to 66 ⴞ 9% (P < .025) of shamoperated controls. Intraduodenal infusion of TCA for 48 hours in CBD rats caused a significant increase in mdr2 mRNA levels (224%) as compared with CBD controls. A diet high in cholesterol (4%) decreased mdr2 mRNA levels to 57% ⴞ 2 (P < .001) of pair-fed controls. Squalestatin (1 ␮mol/L), an inhibitor of cholesterol biosynthesis, increased mdr2 mRNA levels by 8.8-fold (P < .005) in hepatocyte cultures after 24 hours. In conclusion, in the rat, bile acids up-regulated mdr2 transcriptional activity whereas cholesterol decreased mdr2 mRNA both in vitro and in vivo. (HEPATOLOGY 2000;32:341-347.) The coupling of biliary secretion of bile salts, phosphatidylcholine (PC) and cholesterol is important in the maintenance of bile flow, biliary lipid composition, and the solubilization

Abbreviations: PC, phosphatidylcholine; mdr2, multidrug resistance 2; Pgp, P-glycoprotein; mRNA, messenger RNA; TCA, taurocholate; TDCA, taurodeoxycholate; TCDCA, taurochenodeoxycholate; TUDCA, tauroursodeoxycholate; SSC, sodium saline citrate; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; CBD, chronic biliary diversion. From the Departments of 1Medicine-Gastroenterology, and 3Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia; and the 2Division of Gastroenterology and Hepatology, University of Groningen, Groningen, The Netherlands. Received December 9, 1999; accepted May 30, 2000. Supported by a grant from the Veterans Administration and by the National Institutes of Health Grant PO1-DK38030. Address reprint requests to: Phillip B. Hylemon, Ph.D., Department of Microbiology and Immunology, Medical College of VA Campus of VA, Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678. E-mail: [email protected]; fax: 804-828-0676. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3202-0023$3.00/0 doi:10.1053/jhep.2000.9605

of cholesterol. The secretion of PC into the bile is tightly linked to that of cholesterol and the flux of both lipids are coordinated with that of bile salts.1-3 The biliary cholesterol: phospholipid ratios vary substantially in different species (human 0.3, rat 0.1).4 However, these ratios are maintained during moderate changes in overall secretion rates, as bile acid output rises and falls.5,6 In certain pathologic conditions where the secretion of cholesterol is excessive, i.e., out of balance with phospholipids and bile salts, the bile becomes supersaturated with cholesterol. This leads to the precipitation of cholesterol crystals in bile and eventually to the formation of cholesterol gallstones.4,7 Several studies have described a hyperbolic relationship between rates of bile salt, phospholipid, and cholesterol secretion.3,8 As bile salt output increases, the rate of biliary lipid secretion also increases until a maximum is reached.9,10 Moreover, the rate of lipid secretion is proportional to the hydrophobicity of the secreted bile salt species.7,11 Because hydrophobic bile salts have a greater detergent activity than the hydrophilic bile salts, more phospholipid is needed to inactivate the membrane lytic action of the hydrophobic bile salts.12-14 It has been shown recently that rat multidrug resistance 2 (mdr2) P-glycoprotein (Pgp) (mdr3 equivalent in humans) is located in the canalicular membrane of the hepatocyte and is essential for phospholipid secretion.15 Mice in which the mdr2 gene was “knocked out” (mdr2 ⫺/⫺ mice) displayed a complete absence of phospholipids and a dramatic decrease in cholesterol in bile. Mice heterozygous (⫹/⫺) for the mdr2 gene had a 40% decreased phospholipid but normal cholesterol output. Further evidence supporting a role for mdr2 as the PC translocator was provided by Ruetz and Gros,16 who showed that mdr2 could transport a fluorescent PC analog through the membranes of yeast secretory vesicles. Moreover, overexpression of the human MDR3 Pgp could fully restore the secretion of PC into the bile in mdr2 (⫺/⫺) mice.17 The peroxisome proliferators, clofibrate and ciprofibrate, which decrease bile acid biosynthesis and enhance biliary phospholipid output, were found to induce mdr2 gene expression and protein levels in mice.18 In rats, continuous exposure to pravastatin or simvastatin (3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors) lead to a coordinated activation of hepatic PC biosynthesis, biliary output, and mdr2 gene transcription.19,20 Bile acid feeding studies have shown that in mice fed a cholate rich diet, there was an induction in mdr2 messenger RNA (mRNA) levels and a concomitant increase in the maximal phospholipid secretion capacity.21,22 Feeding of ursodeoxycholate, a hydrophilic bile acid, did not influence the mdr2 mRNA content or the phospholipid output rate. However, a detailed study of the effects

341

342 GUPTA ET AL.

of various bile acids on the regulation of mdr2 expression in a defined system of primary hepatocytes has not been previously performed. In this study, we hypothesized that bile salts may up-regulate mdr2 levels in hepatocytes to maintain a constant ratio between bile acids and phospholipids in bile. Indeed, our data show that bile salts up-regulate mdr2 mRNA levels in primary cultures of rat hepatocytes and in vivo. In addition, we report that mdr2 mRNA levels are also directly affected by changes in cholesterol flux in primary hepatocytes and in intact rats. MATERIALS AND METHODS Materials. Taurocholate (TCA), taurodeoxycholate (TDCA), taurochenodeoxycholate (TCDCA), tauroursodeoxycholate (TUDCA), cholesterol, dexamethasone, and thyroxine were obtained from Sigma Chemical Co. (St. Louis, MO). William’s Medium E and the Random Primers DNA Labeling System were from Gibco-BRL (Gaithersburg, MD). Squalestatin, tripotassium salt was kindly provided by Glaxo Research Group (Greenford, Middlesex, UK). Guanidine thiocyanate and cesium chloride was purchased from Fisher Scientific (Springfield, NJ). C219 antibody was from Signet Laboratories Inc. (Dedham, MA). Nitrocellulose membrane and the secondary antibodies were obtained from Bio-Rad (Hercules, CA). All radioisotopes were purchased from ICN Biomedicals Inc. (Costa Mesa, CA). All other reagents were of the highest quality commercially available. Experimental Design. Male Sprague-Dawley rats (Charles River, Cambridge, MA) weighing between 150 to 200 g were housed under controlled lighting conditions on a 12-hour light-dark cycle for 2 weeks. Cholestyramine (5% of diet), cholesterol (4% of diet), and individual bile acids (cholic 1%; chenodeoxycholic 1%; ursodeoxycholic 1%; deoxycholic acid 0.25%) were added to powdered laboratory chow (Ralston Purina, St. Louis, MO). After 14 days of feeding, the rats were killed by decapitation and the livers were harvested for further analysis. For nuclear “run-on” assays, rats were sacrificed after 5 days of cholic acid (1%) feeding. For chronic biliary diversion studies, rats were placed under brief methoxyflurane anesthesia, and biliary fistulas and intraduodenal canulas were placed as described previously.23 After surgery, animals were placed in individual metabolic cages with free access to water and laboratory chow. All animals received continuous intraduodenal infusion of glucose-electrolyte replacement solution. After 72 hours of biliary diversion, taurocholic acid was added to the intraduodenal infusate at a rate at 36 ␮mol/100 g of rat/h for 48 hours. Squalestatin was infused at a rate of 15 ␮g/h for 48 hours. At the conclusion of the experiment, livers were harvested for total RNA extraction. An animal protocol was approved by the institutional Animal Care and Use Committee of the Medical College of Virginia-Virginia Commonwealth University and in compliance with the institution’s guidelines on humane care for laboratory animals, as set forth in the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (publication no. 86-23, revised 1985). Isolation and Culture of Primary Rat Hepatocytes. Primary rat hepatocyte monolayer cultures were prepared using the collagenase-perfusion technique of Bissell and Guzelian.24 Parenchymal cells (2.4 ⫻ 107) were plated onto 150-mm plastic culture dishes coated with rat tail collagen. Before plating, cells were judged to be greater than 90% viable using trypan blue exclusion. Cells were routinely incubated for 48 hours after plating in 20 mL of William’s E media supplemented with insulin (0.25 units/mL medium), 0.1 ␮mol/L dexamethasone and penicillin (100 units/mL medium) in a 5% CO2 atmosphere at 37°C, unless otherwise indicated. Culture medium was changed every 24 hours. The cells were harvested at 72 hours of culture. The taurine conjugate of bile acids (cholic, deoxycholic, chenodeoxycholic, and ursodeoxycholic) were added to the cells at 50 ␮mol/L final concentration unless otherwise indicated. Squale-

HEPATOLOGY August 2000

statin (1 ␮mol/L) was added to the cells between 48 to 72 hours after plating. Total RNA Extraction. Total RNA was prepared from cultured hepatocytes using the guanidium thiocyanate-CsCl centrifugation method as described previously.25 Northern Blot Hybridization. Total RNA was resolved in a 1% agarose gel containing 7% formaldehyde and then transferred to nitrocellulose membranes by overnight capillary blotting as described by Thomas.26 The membranes were baked at 80°C and prehybridized in 4⫻ sodium saline citrate (SSC), 5⫻ Denhardt’s solution, 1% sodium dodecyl sulfate (SDS), 50% formamide and 100 ␮g/mL salmonsperm DNA at 42°C for 2 to 3 hours. The rat mdr2 complementary DNA (cDNA) was released as a XbaI fragment from the plasmid PGEM7Z-f, kindly provided by Dr. J. A. Silverman (AvMax, Inc., Berkeley, CA), and radiolabeled with [␣-32P] dCTP using a commercially available random primer labeling kit. About 1.5 ⫻ 106 cpm/mL probe was added to the hybridization solution and the membranes were hybridized overnight at 42°C with shaking. The membranes were washed twice at room temperature in 2⫻ SSC for 5 minutes, in 2⫻ SSC and 0.1% SDS for 30 minutes, in 0.1⫻ SSC and 0.1% SDS for 30 minutes, and in 0.1⫻ SSC for 15 minutes. The membranes were exposed to X-ray film for 1 to 3 days at ⫺70°C. The film was developed and radioactivity levels were quantitated using a Molecular Dynamics densitometer. To standardize liver mRNA, the membranes were reprobed with a cyclophilin cDNA insert of plasmid p1B1527 using the same hybridization conditions as above. Nuclear “Run-on” Studies. Nuclei isolation, RNA labeling, and hybridization were performed as described previously.28 In brief, nascent mRNAs of mdr2 and cyclophilin were elongated in vitro in the presence of [␣-32P] GTP. The labeled mRNAs were hybridized to nitrocellulose membranes containing spots of varying dilutions of mdr2 and cyclophilin cDNAs. After washing, the membranes were exposed to X-ray film for 1 to 3 days at ⫺70°C and RNA transcripts were quantitated by means of laser densitometry. Isolation of Canalicular Membranes. Canalicular membrane-enriched fractions from whole rat livers were isolated by the sucrose gradient ultra-centrifugation technique as described by Meier and Boyer.29 The canalicular membrane fractions were suspended in 10 mmol/L Tris-HCl (pH 7.4) with protease inhibitor cocktail (Sigma) by repeated suctioning through a 25-gauge needle and stored at ⫺70°C. Western Blot Analysis. Canalicular membrane-enriched proteins (10 ␮g) were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose using a tankblotting system according to the manufacturer’s instructions (Bio-Rad). Ponceau S staining was performed to check for equal protein transfer. The membranes were blocked for 1 hour in phosphate-buffered saline with 0.05% Tween-20 and 4% nonfat dried milk powder, pH 7.4. The blots were incubated overnight with primary mdr2 antibody (k111) in a 1:500 dilution in blocking buffer; washed in phosphate-buffered saline/ 0.05% Tween-20; incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000 dilution); and the immune complexes were detected using the Western blot chemiluminescence reagent plus (NEN, Boston, MA). The membranes were then stripped as per the manufacturer’s instructions and reprobed with the C219 antibody at a 1:300 dilution in phosphate-buffered saline/0.05% Tween-20 and 1% bovine serum albumin. The blots were developed as described above, except the secondary antibody in this case was goat anti-mouse IgG. Statistical Analyses. Data were analyzed by Student’s t test. Level of significance was set at P ⬍ .05. RESULTS Effect of Hormones on Mdr2 Steady-State mRNA Levels in Primary Hepatocyte Cultures. Previous studies in primary rat hepato-

cyte cultures maintained in the absence of added serum, growth factors, or hormones have shown that the expression of mdr1a and mdr1b mRNA is dramatically increased in cul-

HEPATOLOGY Vol. 32, No. 2, 2000

GUPTA ET AL.

TABLE 1. Effect of Dexamethasone and Insulin on Mdr2 Pgp mRNA Levels in Primary Rat Hepatocyte Cultures Additions

N

% Control

0 h—cells 72 h—no additions 72 h—dexamethasone 72 h—dexamethasone ⫹ insulin

3 3 3 3

100% 158 ⫾ 60 685 ⫾ 78 398 ⫾ 92

NOTE. Dexamethasone (0.1 ␮mol/L) alone or dexamethasone (0.1 ␮mol/L) plus insulin (0.25 units/mL) was added at 0 hour and the medium was changed every 24 hours. Total RNA was isolated at 72 hours and levels of mdr2 Pgp mRNA were determined using cyclophilin as the loading control.

ture over a 48-hour period. In contrast, mdr2 mRNA, which is the most abundant of these transporters in the intact liver, exhibited a gradual decline. To establish the culture conditions that could maintain mdr2 RNA levels to nearly in vivo levels, primary rat hepatocytes were cultured in a chemically defined medium without serum. Various hormones were then added to the culture medium (at time 0) individually and in combination. The addition of dexamethasone (0.1 ␮mol/L, 72 hours) alone increased mdr2 mRNA levels about 7-fold as compared with freshly isolated hepatocytes (0 hours) (Table 1). In the presence of dexamethasone (0.1 ␮mol/L) and insulin (0.25 units/mL medium) there was approximately a 4-fold increase in mdr2 mRNA levels between 0 30-32

FIG. 1. Effect of different bile acids on mdr2 mRNA levels in primary rat hepatocytes. Bile acids (50 ␮mol/L) were added to the culture medium 48 hours after plating, and total RNA was isolated at 72 hours. Mdr2 mRNA levels were determined by densitometric analysis of Northern blots. (A) Values are means ⫾ SE; n ⫽ 4. All values were statistically significant when compared with controls. Data are expressed as percent value for no addition hepatocyte controls. (B) representative blot probed for mdr2 and cyclophilin. Lane 1, no addition control; lanes 2-5, 50 ␮mol/L of TCA, TDCA, TCDCA, and TUDCA, respectively.

343

and 72 hours in the absence of added bile salts (Table 1). There was approximately a 2-fold increase in mdr2 mRNA between 48 and 72 hours. The concentration of mdr2 mRNA observed following the addition of hormones for 72 hours was similar to that observed in livers from animals fed a diet containing sodium cholate (data not shown). Therefore, for further studies, hepatocytes were cultured for 48 hours in the presence of dexamethasone (0.1 ␮mol/L) and insulin (0.25 units/mL medium) before making other additions. The combination of insulin and dexamethasone was used as cells were more viable with both hormones. Regulation of Mdr2 mRNA and Transcriptional Activity by Bile Acids. The rate of biliary phospholipid secretion depends on

the phosphatidyl translocating activity of mdr2 and on the structure of the secreted bile salts. Because bile salts are a major factor regulating the secretion of biliary phospholipid, the influence of bile salt structure on mdr2 mRNA levels was determined in both primary rat hepatocyte cultures and in whole animals. As shown in Fig. 1, taurine-conjugated bile acids increased mdr2 mRNA levels in the following order: TCA (288 ⫾ 36%, P ⬍ .005) ⫽ TDCA (276 ⫾ 53%, P ⬍ .025) ⬎ TCDCA (216 ⫾ 34%, P ⬍ .025) ⬎ TUDCA (175 ⫾ 28%, P ⬍ .05) of control levels. Rates of increase in mdr2 mRNA levels due to culture age (48-72 hours) were substracted from each bile salt sample. The increase in mdr2 mRNA levels by TCA in primary hepatocyte cultures was both time (Fig. 2) and concentration dependent (Fig. 3A and B). The time course of the effect of TCA (50 ␮mol/L) on mdr2 mRNA showed an increase in mdr2 mRNA levels beginning within 3 hours of the addition and reaching a maximum at 18 hours. The data in Fig. 3 show that mdr2 mRNA levels increase to 165 ⫾ 21% (P ⬍ .05) of control at TCA concentrations as low as 12.5 ␮mol/L (physiologic concentration range 12.5 to 50 ␮mol/L). Maximal increase of mdr2 mRNA levels to 263 ⫾ 3% (P ⬍ .001) of controls was achieved by addition of 100 ␮mol/L TCA. The TCA-induced increase in mdr2 steady-state mRNA levels could be caused by either a decrease in mdr2 mRNA turn-

FIG. 2. Time course of TCA-induced increase in mdr2 mRNA levels in primary rat hepatocytes. TCA (50 ␮mol/L) was added (open circles) to hepatocyte cultures at 48 hours, total RNA was isolated at time points indicated, and mdr2 mRNA was quantitated as described in the Materials and Methods. Total RNA was also isolated from hepatocyte cultures not receiving TCA and mdr2 mRNA levels determined over the same time course (closed circles). Values are means of 3 independent experiments.

344 GUPTA ET AL.

HEPATOLOGY August 2000

FIG. 4. Transcriptional activities of mdr2 (lanes A) and cyclophilin (lanes B) genes in nuclei isolated from rats fed a control diet or a control diet plus 1% cholic acid (CA) for 5 days. 32P-labeled mRNA transcripts were hybridized to varying amounts (5.0 to 0.625 ␮g) of either mdr2 or cyclophilin cDNA. The relative amounts of RNA hybridizing to probe were quantitated by means of laser densitometry. The ratio of the intensity of mdr2 to cyclophilin was then calculated for each membrane.

FIG. 3. Effect of TCA concentration on mdr2 mRNA levels in primary rat hepatocytes. Hepatocytes were cultured for 48 hours in serum-free medium containing dexamethasone (0.1 ␮mol/L) and insulin (0.25 units/mL). At 48 hours, varying concentrations (12.5, 25, 50, 100, and 200 ␮mol/L) of TCA was added to the culture medium and total RNA was isolated 24 hours later. Concentration of mdr2 mRNA was determined by Northern blot hybridization. (A) Values are means of 3 independent experiments. Control, mRNA level of hepatocytes with no TCA added. (B) Representative blot probed for mdr2 and cyclophilin after the addition of different concentrations of TCA.

over or an increase in mdr2 gene transcription, or both. The latter possibility was tested by performing nuclear “run-on” assays using nuclei isolated from control rats or rats fed a 1% sodium cholate diet for 5 days. As shown in Fig. 4, cholate feeding increased mdr2 transcriptional activity by approximately 4-fold above controls. Western Immunoblot of Mdr2 Protein. To assess whether the taurocholate-induced increase in mdr2 transcriptional activity and mRNA levels also resulted in changes in the expression levels of mdr2 protein, Western immunoblot analysis was performed on canalicular membrane-enriched fractions using antibody specific for mdr2. As shown in Fig. 5, there was a 1.9-fold increase in the C219 signal (detecting all Pgp) and a 1.9-fold increase in the mdr2-specific signal in sodium cholate fed rats as compared with controls. Steady-State Mdr2 mRNA Levels in Chronic Biliary Diverted Rats and Effect of TCA Infusion. Chronic biliary diversion (CBD),

which depletes the endogenous bile salt pool, decreased mdr2 mRNA levels to 66 ⫾ 9% (P ⬍ .025) of sham-operated controls (Fig. 6). In addition, cholestyramine, a drug that enhances excretion of bile acids, also led to a 63 ⫾ 6.6% (P ⬍ .001) decrease in mdr2 mRNA levels. After infusion of TCA

(48 hours), steady-state mdr2 mRNA levels increased by 224% as compared with chronic biliary diverted control animals (P ⬍ .025). Regulation of Mdr2 mRNA by Changes in Cholesterol Flux. Previous studies in rats under sustained administration of 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors showed an increase in biliary phospholipid and cholesterol secretion and a concomitant up-regulation of mdr2 mRNA and protein levels.19,20 In the current study, to determine the possible role of cholesterol in the regulation of mdr2 mRNA, rats were fed a 4% cholesterol diet for 14 days. As shown in Fig. 7, cholesterol feeding significantly decreased mdr2 mRNA levels (57 ⫾ 2%, P ⬍ .001) compared with the pair-fed controls. We also tested the effect of the squalene synthase inhibitor, squalestatin, on mdr2 mRNA levels in primary cul-

FIG. 5. Effect of cholic acid feeding on mdr2 protein expression in the rat liver. Canalicular membrane-enriched fractions (10 ␮g) isolated from livers of control (C) and cholic acid (CA) fed rats were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane as described under the Materials and Methods. Immunoblot analysis was performed using the primary antibodies C219, detecting all Pgps, and K111, detecting mdr2 specifically. The apparent molecular weight (kd) of the Pgps is indicated to the left of each immunoblot.

HEPATOLOGY Vol. 32, No. 2, 2000

FIG. 6. Effect of cholestyramine feeding and intraduodenal infusion of TCA on mdr2 mRNA levels. Total RNA was isolated from livers of shamoperated rats (n ⫽ 3), rats with CBD (n ⫽ 4), from CBD rats intraduodenally infused with 36 ␮mol/100 g of rat/h of TCA for 48 hours (n ⫽ 3), and from rats fed a 5% cholestyramine (CT; n ⫽ 4) diet for 14 days. Pair-fed and sham-operated rats were used as controls for feeding and infusion studies, respectively. Mdr2 mRNA was quantitated by densitometric analysis of Northern blots. (A) Values are mean ⫾ SE. *P ⬍ .001, **P ⬍ . 025. (B) Representative blot probed for mdr2 and cyclophilin. Lane 1, shamoperated; lane 2, CBD; lane 3, TCA infused.

tures of rat hepatocytes. The hepatocytes used in these studies were cultured for 72 hours in serum-free medium and the media were changed every 24 hours. Under these conditions, the hepatocytes depend solely on newly synthesized cholesterol, because exogenous sources of lipoprotein cholesterol are absent. Squalestatin was added to the culture medium between 48 and 72 hours. As shown in Fig. 8, mdr2 mRNA levels rapidly increased on addition of squalestatin (1 ␮mol/ L). After 24 hours of incubation, mdr2 mRNA levels had increased by 8.8-fold as compared with no addition controls. Similar results were observed in CBD rats infused (15 ␮g/h) with squalestatin where there was approximately a 3.3-fold increase in mdr2 mRNA levels (Fig. 9).

FIG. 7. Effect of cholesterol feeding on mdr2 mRNA levels. Total RNA was isolated from livers of rats fed 4% cholesterol diet (XOL) for 14 days. Mdr2 mRNA was quantitated by densitometric analysis of Northern blots. Values shown are means ⫾ SE; n ⫽ 7 and 5, for control (pair-fed) and XOL fed rats, respectively. *Significant (P ⬍ .001) change.

GUPTA ET AL.

345

FIG. 8. Time course of squalestatin induction of mdr2 mRNA levels in primary hepatocytes. Squalestatin (1 ␮mol/L) was added to hepatocyte cultures before RNA isolations (2 to 24 hours). Northern blots were normalized to 28S rRNA levels. There was an approximately 8.8-fold increase in mdr2 mRNA between 0 and 24 hours.

DISCUSSION

Bile salts are important in stimulation of bile flow and biliary lipid secretion.1,7 In bile, cholesterol is solubilized in PC vesicles and in mixed micelles of bile salts, PC, and cholesterol. This association serves to attenuate the deleterious detergent action and potential cytotoxicity of hydrophobic bile acids on biliary epithelial cells.14,33 Although the secretion of bile salts, biliary phospholipids, and cholesterol occur via separate processes,34 the ratio between these 3 lipids in bile is tightly maintained in mammalian species.5 An increase in biliary cholesterol concentration, either absolute or relative to bile acid and phospholipid, has been shown both in humans and in animals to result in supersaturation of cholesterol in bile, subsequent precipitation of cholesterol crystals, and an increased risk of cholesterol gallstone formation.4,7 Because biliary lipid secretion is driven by bile salts, the hepatocyte must sense the concentration and structure of bile salts and regulate the uptake, biosynthesis, intracellular transport, and secretion of these lipids in relation to the bile acid pool composition and concentration. The coordinate regulation of these processes at the cellular level is not fully under-

FIG. 9. Effect of squalestatin infusion on induction of mdr2 mRNA in chronic biliary diverted rats. Squalestatin was infused at 15 ␮g/h for 48 hours, RNA was isolated, and Northern blot analysis was performed. Lanes 1, 2, and 3 represent different animals infused with squalestatin for 48 hours. Lanes 4 and 5 represent control solution infusion for 48 hours and sham operated controls, respectively. Cyclophilin (lower lanes) was used as the RNA loading control.

346 GUPTA ET AL.

FIG. 10. Cholesterol and bile acid metabolism in the hepatocyte. The various input and output pathways for cholesterol (C), the de novo biosynthesis of bile acids (BA) and their biliary secretion along with that of phosphatidylcholine (PC) are shown. Many intermediates and enzymes have been omitted for clarity. Primary and secondary bile acids down-regulate C7␣H and S27H activities (arrow with minus sign) and up-regulate mdr2 transcriptional activity (arrow with plus sign). Cholesterol down-regulates mdr2 (arrow with minus sign). Abbreviations: ACAT, acyl CoA:cholesterol acyltransferase; CEH, cholesterol ester hydrolase.

stood. Recently, the genes encoding the PC transporter (mdr2 Pgp)35 and the bile acid transporter (spgp/bsep)36 were cloned and characterized allowing for a more detailed determination of their molecular regulation. A regulatory role for bile salts has been described previously in secretion of phospholipids as well as in mdr2 gene expression7,9-11,21,22 (Fig. 10). Hydrophobic bile salts have been shown to down-regulate the activity, mRNA levels, and protein mass of CYP7a1, CYP8a1, and CYP2737-40 (Fig. 10). The regulation of these enzymes by bile acids was mainly at the level of transcription. Cholesterol, a metabolic precursor of bile acids, has been shown to up-regulate C7␣H specific activity and mRNA levels in the rat.28,41 In this study, we determined the effects of bile acid structure on the regulation of rat mdr2 gene expression. TCA and TDCA increased mdr2 mRNA to a greater extent than TCDCA or TUDCA in primary hepatocyte cultures (Fig. 1A). In vivo, the infusion of hydrophobic bile acids increases phospholipid secretion to a greater extent than hydrophilic bile acids.7,11-14 It is unknown why TCDCA is a less effective inducer of mdr2 mRNA than TCA or TDCA in primary hepatocyte cultures. However, we have previously shown that TCDCA is rapidly metabolized to ␤-muricholate, a hydrophilic bile acid.37 The up-regulation of mdr2 by different bile acids shows a similar pattern as down-regulation of CYP7a1, CYP8a1, and CYP27 by bile acids.37-40 The increase in mdr2 mRNA appears to be caused by an increase in transcriptional activity (Fig. 4). The bile acid–mediated repression of CYP7a1 gene transcription has been reported to be mediated by 2 bile acid responsive elements located in the 5⬘-upstream region of the promoter.42,43 Although the protein factors binding to these sequences have not yet been identified, recent studies indicate that bile acids, such as chenodeoxycholate, activate the farnesoid X receptor, which then heterodimerizes with retinoid X receptor and decreases CYP7a1 transcription.44-46 However, no similar bile acid responsive element sequences were identified in the first 1,380 base pairs of the rat promoter of mdr2. It is plausible that regulation of mdr2 might proceed via an “indirect or cell signaling mechanism” involving protein ki-

HEPATOLOGY August 2000

nase C activation by bile acids. Stravitz et al.47,48 have shown that bile acids activate calcium-independent protein kinase C isoforms in proportion to their relative hydrophobicity. Interestingly, our results showed that cholesterol feeding led to a marked decrease in mdr2 mRNA levels. Incubation of primary rat hepatocytes with squalestatin, an inhibitor of squalene synthase, the first committed step in the cholesterol biosynthetic pathway, resulted in a dramatic increase in mdr2 mRNA levels. Similar results were obtained in vivo (Fig. 9). A possible explanation for this observation could be that the mdr2 promoter may have sterol responsive elements.49 A decline in cellular sterol levels, such as during treatment with squalestatin, results in activation of sterol regulatory element binding proteins.50,51 These proteins then bind to sterol responsive elements in the promoter regions of the target genes and activate transcription. Indeed, the 5⬘-upstream region of the mdr2 gene contains 2 putative sterol responsive elements at positions ⫺278 to ⫺269 and ⫺193 to ⫺18820 possibly suggesting an explanation for cholesterol regulation of mdr2. We can now hypothesize that bile acid pool composition may be an important regulator determining the amount of phospholipid in bile probably by regulation of genes involved in lipid biosynthesis and transport. It is unclear why cholesterol down-regulates the mdr2 gene and if cholesterol flux alters phospholipid secretion in vivo. However, recent data do show that inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase increase mdr2 expression and phospholipid secretion in the rat.20 The bile acid pool composition and the size of the free cholesterol pool may be important factors that allow the hepatocyte to balance the secretion of phospholipid and cholesterol going into bile and into plasma lipoproteins. In summary, our data indicate an important role for bile salt structure in regulating mdr2 gene expression and excretion of PC into bile. REFERENCES 1. Cohen DE. Hepatocellular transport and secretion of biliary phospholipids. Semin Liver Dis 1996;16:191-200. 2. Hardison WGM, Apter JT. Micellar theory of biliary cholesterol excretion. Am J Physiol 1972;222:61-67. 3. Oude Elferink RP, Ottenhoff R, van Wijland M, Smit JJ, Schinkel AH, Groen AK. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse. J Clin Invest 1995;95:31-38. 4. Turley SD, Dietschy JM. The metabolism and excretion of cholesterol by the liver. In: Arias IM, Jackoby WB, Popper H, Schacter D, Shafritz, eds. The Liver: Biology and Pathobiology. New York: Raven Press, 1988:617641. 5. Coleman R, Rahman K, Bellringer ME, Kan K, Hamlin S. In: Paumgartner G, Stichl A, Gerok W, eds. Trends in Bile Acid Research. London: Kluwer, 1989:161-176. 6. Coleman R, Rahman K, Bellringer ME, Carella M. Biliary lipids secretion and its control. In: Northfield T, Jazrawi R, Zentler-Munro P, eds. Bile Acids in Health and Disease. New York: Kluwer, 1988:43-60. 7. Coleman R, Rahman K. Lipid flow in bile formation. Biochim Biophys Acta 1992;1125:113-133. 8. Marzolo MP, Rigotti A, Nervi F. Secretion of biliary lipids from the hepatocyte. HEPATOLOGY 1990;12(Suppl):134S-142S. 9. Barnwell SG, Tuchweber B, Yousef IM. Biliary lipid secretion in the rat during infusion of increasing doses of unconjugated bile acids. Biochim Biophys Acta 1987;922:221-233. 10. Yousef IM, Barnwell S, Gratton F, Tuchweber B, Weber A, Roy CC. Liver cell membrane solubilization may control maximum secretory rate of cholic acid in the rat. Am J Physiol 1987;252(1 Pt 1):G84-G91. 11. Cohen DE, Leighton LS, Carey MC. Bile salt hydrophobicity controls vesicle secretion rates and transformations in native bile. Am J Physiol 1992;263(3 Pt 1):G386-G395. 12. Coleman R. Bile salts and biliary lipids. Biochem Soc Trans 1987; 15(Suppl):68S-80S.

HEPATOLOGY Vol. 32, No. 2, 2000 13. Jacyna MR, Ross PE, Bouchier IA. Biliary cholesterol—friend or foe? Q J Med 1986;61:991-995. 14. Sagawa H, Tazuma S, Kajiyama G. Protection against hydrophobic bile salt-induced cell membrane damage by liposomes and hydrophilic bile salts. Am J Physiol 1993;264(5 Pt 1):G835-G839. 15. Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993;75:451-462. 16. Ruetz S, Gros P. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell 1994;77:1071-1081. 17. Smith AJ, de Vree JM, Ottenhoff R, Oude Elferink RP, Schinkel AH, Borst P. Hepatocyte-specific expression of the human MDR3 P-glycoprotein gene restores the biliary phosphatidylcholine excretion absent in Mdr2 (⫺/⫺) mice. HEPATOLOGY 1998;28:530-536. 18. Chianale J, Vollrath V, Wielandt AM, Amigo L, Rigotti A, Nervi F, Gonzalez S, et al. Fibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse. Biochem J 1996;314(Pt 3):781-786. 19. Carrella M, Feldman D, Cogoi S, Csillaghy A, Weinhold PA. Enhancement of mdr2 gene transcription mediates the biliary transfer of phosphatidylcholine supplied by an increased biosynthesis in the pravastatintreated rat. HEPATOLOGY 1999;29:1825-1832. 20. Hooiveld GJ, Vos TA, Scheffer GL, Van Goor H, Koning H, Bloks V, Loot AE, et al. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) induce hepatic expression of the phospholipid translocase mdr2 in rats. Gastroenterology 1999;117:678-687. 21. Frijters CM, Ottenhoff R, Van Wijland MJ, Van Nieuwkerk C, Groen AK, Oude Elferink RP. Influence of bile salts on hepatic mdr2 P-glycoprotein expression. Adv Enzyme Regul 1996;36:351-363. 22. Frijters CM, Ottenhoff R, van Wijland MJ, van Nieuwkerk CM, Groen AK, Oude Elferink RP. Regulation of mdr2 P-glycoprotein expression by bile salts. Biochem J 1997;321(Pt 2):389-395. 23. Heuman DM, Hernandez CR, Hylemon PB, Kubaska WM, Hartman C, Vlahcevic ZR. Regulation of bile acid synthesis. I. Effects of conjugated ursodeoxycholate and cholate on bile acid synthesis in chronic bile fistula rat. HEPATOLOGY 1988;8:358-365. 24. Bissell DM, Guzelian PS. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci 1980;349:85-98. 25. Hylemon PB, Gurley EC, Stravitz RT, Litz JS, Pandak WM, Chiang JY, Vlahcevic ZR. Hormonal regulation of cholesterol 7 alpha-hydroxylase mRNA levels and transcriptional activity in primary rat hepatocyte cultures. J Biol Chem 1992;267:16866-16871. 26. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A 1980;77:52015205. 27. Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 1988;7:261-267. 28. Pandak WM, Li YC, Chiang JY, Studer EJ, Gurley EC, Heuman DM, Vlahcevic ZR, et al. Regulation of cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat. J Biol Chem 1991;266:3416-3421. 29. Meier PJ, Boyer JL. Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. Methods Enzymol 1990;192:534-545. 30. Lee CH, Bradley G, Zhang JT, Ling V. Differential expression of P-glycoprotein genes in primary rat hepatocyte culture. J Cell Physiol 1993;157: 392-402. 31. Fardel O, Ratanasavanh D, Loyer P, Ketterer B, Guillouzo A. Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture. Eur J Biochem 1992;205:847-852. 32. Fardel O, Loyer P, Morel F, Ratanasavanh D, Guillouzo A. Modulation of multidrug resistance gene expression in rat hepatocytes maintained

GUPTA ET AL.

33. 34.

35. 36.

37.

38.

39. 40. 41. 42. 43. 44. 45. 46. 47.

48.

49. 50. 51.

347

under various culture conditions. Biochem Pharmacol 1992;44:22592262. Carey MC, Small DM. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man. J Clin Invest 1978;61:998-1026. Barnwell SG, Lowe PJ, Coleman R. The effects of colchicine on secretion into bile of bile salts, phospholipids, cholesterol and plasma membrane enzymes: bile salts are secreted unaccompanied by phospholipids and cholesterol. Biochem J 1984;220:723-731. Brown PC, Thorgeirsson SS, Silverman JA. Cloning and regulation of the rat mdr2 gene. Nucleic Acids Res 1993;21:3885-3891. Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, Meier PJ, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998; 273:10046-10050. Stravitz RT, Hylemon PB, Heuman DM, Hagey LR, Schteingart CD, Ton-Nu HT, Hofmann AF, et al. Transcriptional regulation of cholesterol 7 alpha-hydroxylase mRNA by conjugated bile acids in primary cultures of rat hepatocytes. J Biol Chem 1993;268:13987-13993. Pandak WM, Vlahcevic ZR, Heuman DM, Redford KS, Chiang JY, Hylemon PB. Effects of different bile salts on steady-state mRNA levels and transcriptional activity of cholesterol 7 alpha-hydroxylase. HEPATOLOGY 1994;19:941-947. Vlahcevic ZR, Jairath SK, Heuman DM, Stravitz RT, Hylemon PB, Avadhani NG, Pandak WM. Transcriptional regulation of hepatic sterol 27-hydroxylase by bile acids. Am J Physiol 1996;270(Pt 1):G646-G652. Einarsson K, Akerlund JE, Reihner E, Bjorkhem I. 12 Alpha-hydroxylase activity in human liver and its relation to cholesterol 7 alpha-hydroxylase activity. J Lipid Res 1992;33:1591-1595. Doerner KC, Gurley EC, Vlahcevic ZR, Hylemon PB. Regulation of cholesterol 7 alpha-hydroxylase expression by sterols in primary rat hepatocyte cultures. J Lipid Res 1995;36:1168-1177. Chiang JY, Stroup D. Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter. J Biol Chem 1994;269:17502-17507. Stroup D, Crestani M, Chiang JY. Identification of a bile acid response element in the cholesterol 7 alpha-hydroxylase gene CYP7A. Am J Physiol 1997;273(Pt 1):G508-G517. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, et al. Identification of a nuclear receptor for bile acids. Science 1999;284: 1362-1365. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365-1368. Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 1999;3:543553. Stravitz RT, Vlahcevic ZR, Gurley EC, Hylemon PB. Repression of cholesterol 7 alpha-hydroxylase transcription by bile acids is mediated through protein kinase C in primary cultures of rat hepatocytes. J Lipid Res 1995;36:1359-1369. Stravitz RT, Rao YP, Vlahcevic ZR, Gurley EC, Jarvis WD, Hylemon PB. Hepatocellular protein kinase C activation by bile acids: implications for regulation of cholesterol 7 alpha-hydroxylase. Am J Physiol 1996;271(Pt 1):G293-G303. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343:425-430. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331-340. Edwards PA, Ericsson J. Sterol and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway. Annu Rev Biochem 1999;68:157-185.