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Regulation of oxysterol 7α-hydroxylase (CYP7B1) in primary cultures of rat hepatocytes
Regulation of Oxysterol 7␣-Hydroxylase (CYP7B1) in Primary Cultures of Rat Hepatocytes William M. Pandak,1 Phillip B. Hylemon,2 Shunlin Ren,1 Dalila Marques,1 Gregorio Gil,3 Kaye Redford,1 Darrell Mallonee,2 and Z. Rano Vlahcevic1† Conversion of cholesterol into 7␣-hydroxylated bile acids is a principal pathway of cholesterol disposal. Cholesterol 7␣-hydroxylase (CYP7A1) is the initial and rate-determining enzyme in the “classic” pathway of bile acid synthesis. An “alternative” pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (CYP27) with subsequent 7␣-hydroxylation of 27-hydroxycholesterol by oxysterol 7␣-hydroxylase (CYP7B1). The regulation of CYP7B1, possibly a rate-determining enzyme in the alternative pathway, has not been thoroughly studied. The aims of this study were to (1) study the regulation of liver CYP7B1 by bile acids, cholesterol, adenosine 3ⴕ, 5ⴕ-cyclic monophosphate (cAMP), and phorbol myristate acetate (PMA) in primary rat hepatocytes and (2) determine the effect of CYP7B1 overexpression on rates of bile acid synthesis. The effects of different bile acids (3-150 mol/L), cAMP (50 mol/L), PMA (100 nmol/L; protein kinase C stimulator), cholesterol (200 mol/L), and squalestatin (1 mol/L; cholesterol synthesis inhibitor) on CYP7B1 expression in primary rat hepatocytes were studied. Taurocholic acid and taurodeoxycholic acid decreased CYP7B1 activity by 45% ⴞ 10% and 36% ⴞ 7%, respectively. Tauroursodeoxycholic acid and taurochenodeoxycholic acid did not alter CYP7B1 activity. Inhibition of cholesterol synthesis with squalestatin decreased CYP7B1 activity by 35%, whereas addition of cholesterol increased activity by 39%. Both PMA and cAMP decreased CYP7B1 activity by 60% and 34%, respectively, in a time-dependent fashion. Changes in CYP7B1 messenger RNA (mRNA) levels correlated with changes in specific activities. Overexpression of CYP7B1 led to a marked increase in CYP7B1 mRNA levels and specific activity but no change in rates of bile acid synthesis. In conclusion, in the rat, CYP7B1 specific activity is highly regulated but does not seem to be rate limiting for bile acid synthesis. (HEPATOLOGY 2002;35:1400-1408.) onversion of cholesterol into 7␣-hydroxylated bile acids is a principal pathway of cholesterol secretion from the body. Cholesterol 7␣-hydroxylase (CYP7A1) is the initial and rate-determining enzyme in the “classic” pathway of bile acid synthesis. An
C
Abbreviations: CYP7A1, cholesterol 7␣-hydroxylase; CYP27, sterol 27-hydroxylase; CYP7B1, oxysterol 7␣-hydroxylase; cAMP, adenosine 3⬘, 5⬘-cyclic monophosphate; PMA, phorbol myristate acetate; mRNA, messenger RNA; RPA, ribonuclease protection assay; TCA, taurocholate. From the Departments of 1Medicine, 2Microbiology/Immunology, and 3Biochemistry/Molecular Biophysics, Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA. Received May 18, 2001; accepted February 27, 2002. Supported by grants from the Veterans Administration and the National Institutes of Health (P01 DK38030). †Deceased. Address reprint requests to: William M. Pandak, M.D., Veterans Affairs Medical Center, Division of Gastroenterology 111-N, 1201 Broad Rock Rd., Richmond, VA 23249. E-mail: [email protected]; fax: 804-675-5816. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3506-0014$35.00/0 doi:10.1053/jhep.2002.33200 1400
“alternative” pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (CYP27) with subsequent 7␣-hydroxylation of 27-hydroxycholesterol by oxysterol 7␣-hydroxylase (CYP7B1).1-5 CYP7A1 is highly regulated by bile acids, hormones, diurnal rhythm, cholesterol (oxysterols), cytokines, and second messengers.1,2 Like CYP7A1, CYP27 is regulated by bile acids and hormones.2 In contrast to CYP7A1, which is only found in the liver, CYP27 is also found in abundance in extrahepatic tissues, including vascular endothelium. Maybe just as importantly, initial 27-hydroxylation of cholesterol in peripheral tissues may be a mechanism for prevention of cholesterol accumulation and in the generation of a cholesterol homeostatic regulatory oxysterol in response to excess cholesterol.6-10 Oxysterols are important degradation products of cholesterol, being intermediates in the synthesis of bile acids and steroid hormones. These intermediates seem to have regulatory effects on cholesterol homeostasis, including suppression of 3-hydroxy-3-methylglutaryl– coenzyme A
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reductase and the low-density lipoprotein receptor, and transcriptional stimulation of CYP7A1 and ABC1 through ligand binding to liver X receptor.6,11-13 Recently, a complementary DNA encoding CYP7B1, capable of mediating 7␣-hydroxylation of 27-hydroxycholesterol, 25-hydroxycholesterol, dehydroepiandrosterone, and pregnenolone, was isolated and characterized.14,15 In contrast to CYP7A1, CYP7B1 is also expressed in extrahepatic tissues, including high levels in the kidney, vascular endothelium, and hippocampus.15,16 It has been suggested that 7␣ hydroxylation of 27-hydroxycholesterol may be required to metabolize oxysterols to dihydroxy metabolites with less regulatory and cytotoxic effects.17 These more water-soluble dihydroxy sterol metabolites can then be more readily excreted from peripheral cells, transported to the liver, and converted to bile acids.7 The regulation of CYP7B1, possibly a rate-determining enzyme in the alternative pathway, has not been studied in a defined system. In this study, CYP7B1 was shown to be regulated by bile acids, cholesterol, 3⬘, 5⬘-cyclic monophosphate (cAMP), and phorbol myristate acetate (PMA) in a manner similar to CYP7A1. Unlike CYP7A1, whose increased expression was associated with a dramatically increased rate of bile acid synthesis, overexpression of CYP7B1 did not significantly alter basal bile acid synthesis.
Materials and Methods Materials Thyroxine, dexamethasone, mevalonolactone, PMA, dithiothreitol, ethylene glycol-bis(-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid, phenylmethylsulfonyl fluoride, and dibutyryl cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). William’s E Medium was purchased from GIBCO-BRL (Rockville, MD). Poly Attract messenger RNA (mRNA) isolation system II was obtained from Promega (Madison, WI). Squalestatin 1 and tripotassium salt were kindly provided by Glaxo Research Group. -Cyclodextrin (Trappsol) was purchased from Cyclodextrin Technologies Development Corp. (Gainesville, FL). [4-14C]-cholesterol (59.4 mCi/mmol) and 3H-25-hydroxycholesterol were purchased from New England Nuclear (Boston, MA). 25-Hydroxycholesterol was purchased from Steraloids, Inc. (Newport, RI). Taurine conjugated bile acids were purchased from Calbiochem (La Jolla, CA). Silica gel thin-layer chromatography plates (LK6 D) were from Whatman (Clifton, NJ). Solvents were purchased from Fisher Scientific (New Lawn, NJ). All other reagents were of the highest quality commercially available. Nylon
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membranes were purchased from Micron Separation Inc. (Westborough, MA). Isolation and Culture of Primary Rat Hepatocytes Hepatocytes were isolated from male Sprague-Dawley rats (250-300 g) as previously used by us with the collagenase-perfusion technique of Bissell and Guzelian.18 Cells were routinely harvested after 72 hours of culture as previously described.19 Unless otherwise indicated, culture medium contained 0.1 mol/L dexamethasone and 1.0 mol/L L-thyroxine (T4) with culture medium changed daily. Bile acids were added 24 hours after cells were plated to achieve a 50 mol/L concentration unless indicated otherwise. Squalestatin, mevalonolactone, and cholesterol (dissolved in -cyclodextrin), when added, were added to the cells at final concentrations of 1 mol/L, 2 mmol/L, and 200 mol/L, respectively, at 48 hours of culture. Dibutyryl cAMP and PMA were added to the cells at final concentrations of 50 mol/L and 100 nmol/L, respectively, 24 hours after culturing. Actinomycin D, a transcription inhibitor, was added to cells at a final concentration of 10 g/mL 4 hours before harvesting. Toxicity was assessed as previously described.20 RNA Preparation Cells were harvested 48 hours after infection, and RNA was isolated from hepatocytes as previously described.21 RNA Quantification Northern blotting was performed as previously described21 using a mouse complementary DNA clone for mouse oxysterol 7␣-hydroxylase (a generous gift from Dr. David Russell). A ribonuclease protection assay (RPA) was used in some studies for mRNA quantitation. Briefly, an RPA probe for rat CYP7B1 mRNA was constructed. First-strand DNA synthesis for the probe was performed using the primer 5⬘-CGTGAATTCGAGCACATCATCTTGGCTTGC-3⬘ (EcoRI site in 5⬘ extension) in a reaction with reverse transcriptase and mRNA obtained from the liver of a cholestyramine-fed rat. An aliquot from the reverse-transcriptase reaction was used in a polymerase chain reaction with the first-strand primer and the primer 5⬘-CGAGATCTGGTCATTGTGTATCATTGGAGG-3⬘ (BglII site in 5⬘ extension) to produce a polymerase chain reaction product that was cloned into the EcoRI/BglII sites of pSP72 (Promega). The resulting plasmid was used for production of an [␣-32P]-uridine triphosphate–labeled RNA probe using the Maxiscript T7 in vitro transcription kit from Ambion, Inc. (Austin, TX). The RNA probe provided a 363 base protected fragment from rat CYP7B1 mRNA in the RPA assay, performed using the RPA III kit from Ambion. Rat cyclophilin com-
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plementary DNA was used as the internal loading standard for both Northern blotting and the RPA.21 Microsome Preparation Microsomes were prepared as previously described.19 Determinations of Enzyme Specific Activity CYP7B1 activity was determined according to the method of Schwarz et al.22 Briefly, 500 g of microsomal protein was incubated at 37°C for 15 minutes in a shaking water bath with 50 mmol/L Tris acetate, pH 7.4, 1 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L dithiothreitol, 0.03% Triton X-100, 1.2 mmol/L reduced nicotinamide adenine dinucleotide phosphate, and 0.06 nmol 3H-25-hydroxycholesterol in a final volume of 500 L. The reaction was stopped by addition of 6 mL methylene chloride. The organic phase was then evaporated to dryness under a nitrogen gas atmosphere, dissolved in acetone, and assayed by thin-layer chromatography in a solvent system containing toluene/ethyl acetate (2:3). The developed plate was exposed to a Tritium screen (Molecular Dynamics, San Francisco, CA), and the relative intensities of the bands were quantified and analyzed with a Phosphor Imager and the appropriate software. The validity of this assay was examined using several different approaches. These studies were undertaken in response to a study by Norlin et al. showing the ability of CYP7A1 to 7␣-hydroxylate 25-hydroxycholesterol and 27-hydroxycholesterol.23 Subsequent studies in our own laboratory confirmed these findings. In primary rat hepatocytes infected with recombinant adenovirus encoding CMV-CYP7A1, we showed that overexpression of CYP7A1 led to detection of 7␣-hydroxylated 25-hydroxycholesterol (data not shown). To ensure the changes in activity shown by the assay system used in the current study were a function of changes in CYP7B1 activity, the following control experiments were performed in rat microsomes. (1) The standard assay was performed using [14C]-cholesterol as substrate, a substrate of CYP7A1. (2) 7-Oxocholesterol, a selective inhibitor of CYP7A1, was added in optimal concentrations (10 mol/L) to the assay. (3) Assay was performed using [14C]dehydroepiandrosterone under saturating conditions (i.e., cold dehydroepiandrosterone added), a substrate of CYP7B1, but not CYP7A1. (4) CYP7B1 specific activity changes were determined in mitochondria, an organelle with no CYP7A1 activity. The results are summarized as follows. (1) Use of [14C]-cholesterol as substrate elicited no products using the CYP7B1 assay as previously described. (2) 7-Oxocholesterol (10 mol/L) did not decrease CYP7B1 activity. (3) Use of dehydroepiandrosterone under saturating conditions showed significant increases in CYP7B1
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activity after cholestyramine feeding. (4) Changes in CYP7B1 activity observed in mitochondria paralleled changes in microsomes. Of note, regulatory changes in rat mitochondrial CYP7B1 paralleled changes in microsomal CYP7B1; however, the basal CYP7B1 activity level in mitochondria was 2- to 3-fold lower than that found in microsomes. Synthesis of Bile Acids by Primary Hepatocytes The conversion of [4-14C] cholesterol into MeOH/ H2O-soluble material was determined as previously described.24 Plates were incubated with [4-14C] cholesterol (5.55 ⫻ 106 dpm/plate) 24 hours after plating (treated with virus for 2 hours before administration of [4-14C]). Media and cells were harvested 48 hours later and analyzed after Folch extraction.25 Bile acid biosynthesis rates in infected cells were expressed as percent of paired controls. Overexpression of CYP7B1 The adenovirus constructs used in this study were obtained through the Massey Cancer Center Adenovirus Shared Resource Facility of Virginia Commonwealth University. The CMV-CYP7B1 recombinant adenovirus (Ad-CMV-CYP7B1) was obtained using a pTG-CMV system as previously described.26,27 Briefly, a 1.9-kilobase murine complementary DNA clone of CYP7B114,28 was subcloned into the NotI site of pZeroTG-CMV, a plasmid containing a CMV promoter, a multiple cloning site, and a partial DNA sequence from the adenovirus construct Ad5dl324.15,28 The resulting pZeroTG-CMV/ CYP7B1 recombinant plasmid was cotransformed with ClaI-linearized pTG-CMV (containing the entire Ad5dl324 genome) into Escherichia coli. Resulting plasmids were screened for inserts before being transfected into 293 cells.29 Adenovirus DNA from resulting plaques was further screened by Southern blot for the presence of the CYP7B1 insert, confirming the Ad-CMV-CYP7B1 adenovirus. The control adenovirus contained the CMV promoter and polylinker cassette without any insert DNA. Propagation of the Ad-CMV-CYP7B1 Adenovirus. Large-scale production of recombinant virus was performed by infecting confluent monolayers of 293 cells grown in 15-cm tissue culture dishes with stock adenovirus at a multiplicity of infection of 0.1 to 1. After 2 hours of infection, the virus was removed and the dishes were fed with 15 mL Dulbecco’s modified Eagle medium and 2% fetal bovine serum. Infected monolayers were harvested by scraping when greater than 90% of the cells showed cytopathic changes and centrifuged at 2,700g for 10 minutes at 4°C. The infected cellular pellet was suspended in Dulbecco’s mod-
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ified Eagle medium/2% fetal bovine serum and subjected to 5 cycles of freeze/thaw lysis to release the virus. Cellular debris was removed by centrifugation at 7,700g for 10 minutes at 4°C. To purify, the crude viral supernatant was carefully layered over a 2-step gradient containing 3 mL CsCl (d ⫽ 1.4 g/mL) in TD buffer (0.14 mol/L NaCl, 5 mmol/L KCl, 19 mmol/L Tris, pH 7.4, and 0.7 mmol/L Na2HPO4) layered over 3 mL CsCl (d ⫽ 1.25 g/mL) in TD buffer and centrifuged at 155,000g for 1 hour at 20°C. The viral band was removed, layered over 8 mL CsCl (d ⫽ 1.33 g/mL) in TD buffer, and centrifuged at 155,000g for 18 hours at 20°C. The pure viral opalescent band was removed and dialyzed against 10 mmol/L Tris, pH 7.4, 1 mmol/L MgCl2, and 10% glycerol overnight at 4°C. The virus was aliquoted and stored at ⫺70°C. The virus titer (pfu) was determined by plaque assay, and viral particles were determined by optical density using spectrophotometry ( ⫽ 260). Infection of Cells With Replicative Defective AdCMV-CYP7B1. Primary rat hepatocytes were grown in P150-cm2 plates to confluency (⬃2.5 ⫻ 107 cells). Twenty-four hours after plating, 2.5 mL of fresh medium was added. Cells were then infected with replicative defective Ad-CMV-CYP7B1 with a multiplicity of infection of 10 (virus to cells) using unpurified virus in a volume of 14.7 L. The virus was allowed to dwell for 2 hours in minimal culture medium by shaking the plates gently every 15 minutes, after which the medium with virus was removed, 20 mL of fresh medium was added, and the plates were allowed to incubate at 37°C in 5% CO2 for an additional 48 hours. Statistics Data are reported as mean ⫾ SE. Where indicated, data were subjected to t-test analysis and determined to be significantly different if P was less than .05.
Results Effect of Dexamethasone and L-Thyroxine on CYP7B1 Activity In primary rat hepatocytes, it has been shown that dexamethasone and L-thyroxine (T4) are required for optimal expression and regulation of CYP7A1.19 Therefore, the effects of addition of dexamethasone and L-thyroxine to culture medium on CYP7B1 were explored. Both were added to culture medium (at time 0) individually and in combination. The specific activity of CYP7B1 was measured in microsomes prepared from rat hepatocyte cultures 72 hours after plating. Compared with the individual addition of optimal concentrations of dexamethasone (0.1 mol/L) or L-thyroxine (1.0 mol/L), which increased CYP7B1 activity 44% to 58% compared
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Fig. 1. Effects of squalestatin, cholesterol, and mevalonate on CYP7B1 activities in primary rat hepatocyte cultures. Culture medium contained optimal concentrations of dexamethasone (0.1 mol/L) and T4 (1.0 mol/L) and 2.5% (vol/vol) -cyclodextrin. Squalestatin (Squal; 1.0 mol/L), mevalonate (Mev, 2 mmol/L, as lactone), or cholesterol (Xol; 200 mol/L) was added to the cultures at 48 hours of incubation. Microsomes were prepared at 72 hours. CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Mean of 2 experiments for all conditions compared with paired controls.
with controls, the addition of dexamethasone (0.1 mol/L) plus L-thyroxine (1.0 mol/L) to the medium increased CYP7B1 activity 2.6-fold (n ⫽ 2; range, 123%186%) compared with controls with no hormone additions. Effect of Cholesterol on Regulation of CYP7B1 Activity The effects of cholesterol, mevalonate (cholesterol precursor), and squalestatin (inhibitor of cholesterol synthesis) on the regulation of CYP7B1 specific activity in primary rat hepatocyte cultures are shown in Fig. 1. -Cyclodextrin was used to deliver cholesterol (see Methods). The individual additions of either mevalonate (2 mmol/L) or cholesterol (200 mol/L) to culture medium resulted in a 32% (n ⫽ 2; range, 31%-38%) and 39% (n ⫽ 2; range, 30%-48%) increase, respectively, in CYP7B1 specific activity. In contrast, addition of squalestatin (1 mol/L) for 24 hours before harvest decreased CYP7B1 specific activity to 65% of paired controls (n ⫽ 2; range, 31%-38%). The addition of cholesterol to cell medium containing squalestatin not only prevented the decrease found with squalestatin alone but increased CYP7B1 activity by 37% (n ⫽ 2; range, 19%-46%) to levels to those found with cholesterol addition alone. Effect of Bile Acids on CYP7B1 Activity The ability of bile acids to down-regulate CYP7B1 was tested. Figure 2 shows the effect of the addition of increas-
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Fig. 2. Effect of TCA concentration on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated in culture for 24 hours in medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 24 hours, varying concentrations (25-200 mol/L) of TCA were added to the culture medium. Cells were harvested 48 hours after addition of TCA, microsomes were isolated, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Mean of 2 to 3 experiments for all concentrations compared with paired controls, except the 50 mol/L concentration (n ⫽ 6; 45% ⫾ 10% decrease; P ⬍ .01).
ing concentrations of taurocholate (TCA) on the specific activity of microsomal CYP7B1 in hepatocyte cultures containing optimal concentrations of L-thyroxine and dexamethasone. Increasing concentrations of TCA led to increased suppression of CYP7B1 activity. In separate studies, actinomycin D (transcriptional inhibitor) was added to the culture medium alone or in combination with TCA. The half-life of CYP7B1 was similar in both experiments, suggesting that bile acid repression of CYP7B1 is mediated at the transcriptional level (data not shown). The effect of other bile acids on CYP7B1 activity was also examined (Fig. 3). Taurodeoxycholic acid led to repression (36% ⫾ 7% decrease; P ⬍ .05) of CYP7B1 activity to levels similar to that induced by TCA (45% ⫾ 10% decrease; P ⬍ .01). Neither taurochenodeoxycholic acid nor tauroursodeoxycholic acid led to any significant suppression of CYP7B1 activity. Previously, we have shown that taurochenodeoxycholic acid in culture is rapidly metabolized to muricholic acid, a relatively hydrophilic bile acid.20 Therefore, as previously shown with CYP7A1 in primary rat hepatocyte cultures, only relatively hydrophobic bile acids seem to act as repressors. Effect of cAMP and PMA on CYP7B1 Once culture conditions had been established that could maintain CYP7B1 activity at nearly in vivo levels, the effect of activators of cell signaling pathways on the
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Fig. 3. Effect of bile acids on CYP7B1 activity. Hepatocytes were incubated in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). Bile acids (50 mol/L) were added to culture medium 24 hours after plating, and microsomes were harvested at 72 hours. CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Changes in CYP7B1 activity are expressed as percent of control cultures without bile acid additions. Mean of 4 (taurodeoxycholate [TDCA], taurochenodeoxycholate [TCDCA], and tauroursodeoxycholate [TUDCA]) or more (taurocholate [TCA]) experiments ⫾ SE. P ⬍ .05 compared with paired controls.
regulation of CYP7B1 activity could be examined. The addition of dibutyryl cAMP (50 mol/L) to primary rat hepatocytes rapidly decreased cholesterol CYP7B1 activity (Fig. 4). Although the degree of suppression of CYP7B1 by
Fig. 4. Time course of the effects of cAMP on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated for 48 hours in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 48 hours, dibutyryl cAMP (50 mol/L) was added to the culture medium. Cells were harvested at various time points after the addition of dibutyryl cAMP, microsomes were isolated, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Repression of CYP7B1 activity is expressed as percent of control cultures without addition of cAMP (n ⫽ 1 for each time point).
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Table 1. Effect of TCA, cAMP, and Squalestatin on CYP7B1 mRNA Levels Condition
TCA (50 mol/L) cAMP (50 mol/L) Squalestatin (1 mol/L)
n
mRNA Levels (% control)
3 3 3
73 ⫾ 3 (227) 53 ⫾ 7 (247) 67 ⫾ 11 (233)
NOTE. Data expressed as mean ⫾ SE. cAMP levels determined 3 hours after addition. P⬍ .05 for all conditions.
Fig. 5. Time course of the effects of PMA on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). PMA (100 nmol/L) was added to hepatocyte culture medium at the indicated times before harvest, beginning 4 hours after plating. All cultures were harvested for microsomes 28 hours after plating, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Repression of CYP7B1 activity is expressed as percent of control cultures without addition of PMA (n ⫽ 1 for each time point).
cAMP was not as great as that of CYP7A1, the time over which the effect was observed was similar.2,30 PMA (100 nmol/L) was added to hepatocyte culture medium at the indicated times (1.5, 3, 6, and 24 hours) before harvest, beginning 4 hours after plating. Microsomes were harvested from all cells 28 hours after plating. The effects of PMA were biphasic (Fig. 5). Initially, PMA caused a rapid decline in CYP7B1 activity to 40% of control cultures 3 hours after addition. This decline was followed by a recovery to control levels approximately 20 hours after addition of PMA, with an overshoot to 127% of control at 24 hours. Regulation of CYP7B1 mRNA Levels CYP7B1 mRNA levels after the addition of TCA, cAMP, and squalestatin to the culture medium were all decreased. TCA (50 mol/L) led to a 27% ⫾ 3% (P ⬍ .01) decrease in CYP7B1 mRNA levels. Addition of cAMP (50 mol/L) and squalestatin (1 mol/L) decreased CYP7B1 mRNA levels 47% ⫾ 7% (P ⬍ .01) and 33% ⫾ 11% (P ⬍ .05), respectively (Table 1). Changes in CYP7B1 mRNA levels after the addition of PMA followed a course similar to that of CYP7B1 activity levels (data not shown). Overexpression of CYP7B1 The rate of bile acid synthesis through the alternative (i.e., “acidic”) pathway is believed to be controlled by
CYP27, the initial step of the pathway. Recent evidence from our laboratory has shown in HepG2 cells and in primary human hepatocytes that overexpression of CYP27 can increase bile acid synthesis by ⬃60%.31 Overexpression of CYP27 in primary rat hepatocytes led to a similar increase (43% ⫾ 17%; P ⬍ .05) in bile acid synthesis compared with paired controls. These findings suggest that, under normal physiologic conditions, 27hydroxylation of cholesterol may be the rate-determining step in this pathway. In an attempt to further define the role of CYP7B1 in the rate of bile acid synthesis, CYP7B1 was overexpressed using a recombinant adenovirus encoding the gene for CYP7B1. Northern blot analysis (Fig. 6) shows the dramatic increase in CYP7B1 mRNA levels after infection of hepatocytes with recombinant adenovirus encoding CMV-CYP7B1. In contrast to earlier cultures, primary rat hepatocytes were initially maintained in the absence of L-thyroxine. Under these culture conditions, we have previously shown that levels of CYP7A1 are
Fig. 6. Representative Northern blot of CYP7B1 mRNA levels in primary rat hepatocytes before and after infection with recombinant adenovirus encoding CMV-CYP7B1. Hepatocytes were incubated for 24 hours in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 24 hours, cells were infected with control virus or recombinant adenovirus encoding CMVCYP7B1 as described in Materials and Methods. Cells were harvested 48 hours after infection, and mRNA levels for CYP7B1 and cyclophilin (loading standard) were determined.
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Fig. 7. Effect of overexpression of CYP7B1 and CYP7A1 with recombinant adenoviruses encoding CMV-CYP7B1 and CMV-CYP7A1 on bile acid synthesis in primary rat hepatocyte cultures. Hepatocytes were incubated for 24 hours in culture medium containing 0.1 mol/L dexamethasone. At 24 hours, cells were infected with control (null) virus or recombinant adenovirus with the multiplicity of infection as described in Materials and Methods. Bile acid synthesis was measured as conversion of 14C-labeled cholesterol into water-soluble products (14C-labeled methanol/water-extractable counts). Data are expressed as a percentage of controls (mean ⫾ SE).
undetectable and that only the acidic pathway of bile acid synthesis is functional.19,32 A greater than 3-fold increase (221% ⫾ 64%; P ⬍ .02) in CYP7B1 specific activity was observed compared with paired controls (cells infected with control virus). Despite this dramatic increase in CYP7B1 activity, bile acid synthesis rates were not significantly changed (86% ⫾ 15%; P ⫽ NS) (Fig. 7). This is in contrast to CYP7A1 overexpression in primary rat hepatocytes, in which overexpression of CYP7A1 led to a greater than 10-fold increase (P ⬍ .001) in the rate of bile acid synthesis (Fig. 7). Overexpression of CYP7B1 in the presence of L-thyroxine with dexamethasone (i.e., functional CYP7A1 and neutral bile acid synthesis pathway) also led to no increase in bile acid synthesis (116% ⫾ 29%; P ⫽ NS), whereas CYP7A1 overexpression led to a greater than 4-fold (n ⫽ 3; P ⬍ .001) increase in bile acid synthesis.
Discussion The results of this study performed in primary rat hepatocytes describe the effects of bile acids, cholesterol, hormones, cAMP, and PMA on the regulation of CYP7B1 mRNA and activity. The addition of L-thyroxine plus dexamethasone increased CYP7B1 activity levels, with optimal concentrations of 1.0 mol/L and 0.1 mol/L, respectively. These concentrations were similar to those found to optimize primary rat hepatocyte culture conditions for CYP7A1 expression.19 Furthermore, the
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addition of bile acids, cholesterol, cAMP, or PMA all led to changes in CYP7B1 activity in a fashion previously observed for CYP7A1 but to a lesser degree.20 Addition of TCA, taurodeoxycholic acid, cAMP, and PMA to culture medium suppressed CYP7B1 activity, whereas the addition of cholesterol had a stimulatory effect. The changes observed in CYP7B1 specific activities were paralleled by changes in mRNA levels. The importance of tightly regulating CYP7B1 is not clearly defined but could be crucial for regulating cellular oxysterol levels. Oxysterols have been presented as probable regulators of cholesterol homeostasis, with in vitro effects such as repression of 3-hydroxy-3-methylglutaryl– coenzyme A reductase.13 The 7␣-hydroxylation of 27hydroxycholesterol mediated through overexpression of CYP7B1 activity in CHO cells has been shown to be capable of preventing the repressive effects of this oxysterol on 3-hydroxy-3-methylglutaryl– coenzyme A reductase.10 However, in Cyp7b1⫺/⫺ mice, in which plasma levels of 25-hydroxycholesterol and 27-hydroxycholesterol are increased, no major differences in cholesterol homeostasis have been observed.33 Based on these findings, it was hypothesized that commensurate pathways of bile acid synthesis may be able to be stimulated in the mouse. However, the absence of generalized suppression of sterol synthesis in the presence of the increased oxysterols, 25-hydroxycholesterol and 27-hydroxycholesterol, is unclear. In a rare, naturally occurring mutation of CYP7B1 in a human neonate with cholestasis, no 7␣hydroxylated bile acids were formed.17 Acidic intermediates in the bile acid biosynthetic pathway accumulated despite the CYP7A1 gene being normal. These findings suggest, at least in humans early in life, that CPY7B1 is critical for formation of 7␣-hydroxylated bile acids. The fact that both dexamethasone and L-thyroxine were found to be optimal culture conditions for CYP7B1 in a manner similar to CYP7A1 is interesting and implies similarities in the hormonal effects on CYP7B1 and CYP7A1. L-thyroxine elicits little effect in primary rat hepatocytes on CYP27, the initial step in the alternative pathway of bile acid synthesis, with dexamethasone required for its optimal expression.4 In contrast, sterol 12␣-hydroxylase (CYP8B1) expression is dramatically suppressed by L-thyroxine, demonstrating a hormonal role in the regulation of the ratio of cholic to chenodeoxycholic acid.32 The ability of cholesterol to up-regulate CYP7B1 is also similar to CYP7A1. In support of this type of regulation is the identification of an SRE-like site in the human CYP7B1 promoter.1,34 Whether this represents a true sterol regulatory site mediated through previously defined pathways is uncertain.
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The original concept that feedback repression of bile acid biosynthesis was mediated solely through the regulation of CYP7A1 is being modified. It has been shown in Cyp7a1⫺/⫺ mice that bile acid synthesis continues to occur, demonstrating the ability of at least one alternative pathway to 7␣-hydroxylate cholesterol and generate bile acids.22,35 Furthermore, CYP7B1 enzyme activity, protein, and mRNA levels in Cyp7a1⫺/⫺ mice were induced. Although the relative importance of these alternative pathways may be species and/or disease state dependent, the question of their ability to be regulated, and by what, has begun to be explored.2,36-38 CYP27, the initial step in the alternative (i.e., “acidic”) pathway, has now been shown to be regulated.2 However, the regulation of the subsequent enzymatic step in the pathway, the 7␣-hydroxylation of the oxysterol, has been given little attention. In rat liver, Toll et al. failed to see any stimulation of 7␣-hydroxylation of oxysterol activity by feeding cholestyramine.39 In the mouse, Schwarz et al. also showed no effects of cholestyramine feeding on CYP7B1 mRNA expression but found an approximately 50% decrease in CYP7B1 mRNA levels with cholate feeding.15,16 The results of this study in primary rat hepatocytes not only show a suppression of CYP7B1 mRNA levels with bile acids but suppression of CYP7B1 activity as well. These findings have been corroborated in whole animals in which bile acid feeding repressed CYP7B1 mRNA and specific activity levels; conversely, CYP7B1 mRNA levels and specific activity levels were increased with chronic biliary diversion and cholestyramine feeding (under review). cAMP was able to repress CYP7B1, like CYP7A1 but to a lesser degree. The mechanism by which cAMP regulates CYP7A1 has not been elucidated but represents a plausible explanation for why bile acid synthesis remains low in fasting despite low circulating levels of bile acids as it is during fasting that glucagon levels increase, stimulating an increase in cAMP levels.2 The mechanism, however, is different from that of PMA, which activates different cell signaling cascades resulting in repression of CYP7A1.30,40 An understanding of this mechanism of regulation of CYP7A1 has laid the groundwork for the more recent observation by Gupta et al. that bile acids can repress CYP7A1 via the JNK/c-Jun pathway, which in turn appears able to repress CYP7A1 transcription via SHP induction.40 The relevance of these signaling pathways to the regulation to CYP7B1 has not yet been explored. Overexpression of CYP7B1 using a recombinant adenovirus containing CMV-CYP7B1 led to a dramatic increase in CYP7B1 activity (data not shown) but no increase in bile acid synthesis rates. This is in contrast to
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findings following CYP7A1 overexpression, where a dramatic increase in bile acid synthesis was observed. Increased CYP27 expression in HepG2 cells also resulted in a significant increase in bile acid synthesis (⬃50%).31 Overexpression of CYP7B1 in combination with CYP27 also did not increase bile acid synthesis any further than overexpression of CYP27 alone.31 These findings suggest that CYP7B1, under normal physiologic conditions, is not the rate-determining step in the “acidic” pathway of bile acid biosynthesis. In summary, in the rat, CYP7B1, like CYP7A1, is a highly regulated enzyme. As with CYP7A1, these regulatory effects seem to down-regulate CYP7B1 at the level of gene transcription. Interestingly, overexpression of CYP7B1 did not increase bile acid synthesis as has been previously observed with CYP7A1, suggesting that the 7␣-hydroxylation of oxysterols is not a rate-limiting step in the alternative pathway of bile acid synthesis under physiologic conditions. Acknowledgment: The authors thank Li Jun Zhao and Pat Bohdan for technical assistance.
References 1. Chiang JYL. Regulation of bile acid synthesis. Front Biosci 1998; 3:D176-D193. 2. Vlahcevic ZR, Pandak WM, Stravitz RT. Regulation of bile acid biosynthesis. Gastroenterol Clin North Am 1999;28:1-25, v. 3. Dahlback-Sjoberg H, Bjorkhem I, Princen HM. Selective inhibition of mitochondrial 27-hydroxylation of bile acid intermediates and 25-hydroxylation of vitamin D3 by cyclosporin A. Biochem J 1993;293:203-206. 4. Stravitz RT, Vlahcevic ZR, Russell TL, Heizer ML, Avadhani NG, Hylemon PB. Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes. J Steroid Biochem Mol Biol 1996;57:337-347. 5. Vlahcevic ZR, Stravitz RT, Heuman DM, Hylemon PB, Pandak WM. Quantitative estimations of the contribution of different bile acid pathways to total bile acid synthesis in the rat. Gastroenterology 1997;113:1949-1957. 6. Axelson M, Larsson O, Zhang J, Shoda J, Sjovall J. Structural specificity in the suppression of HMG-CoA reductase in human fibroblasts by intermediates in bile acid biosynthesis. J Lipid Res 1995;36:290-298. 7. Lund E, Andersson O, Zhang J, Babiker A, Ahlborg G, Diczfalusy U, Einarsson K, et al. Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans. Arterioscler Thromb Vasc Biol 1996;16:208-212. 8. Babiker A, Andersson O, Lund E, Xiu RJ, Deeb S, Reshef A, Leitersdorf E, et al. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport. J Biol Chem 1997;272:26253-26261. 9. Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan C, Lund E. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc Natl Acad Sci U S A 1994;91:85928596.
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10. Martin KO, Reiss AB, Lathe R, Javitt NB. 7 alpha-hydroxylation of 27-hydroxycholesterol: biologic role in the regulation of cholesterol synthesis. J Lipid Res 1997;38:1053-1058. 11. Repa JJ, Mangelsdorf DJ. The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu Rev Cell Dev Biol 2000;16:459-481. 12. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B, et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 2000;289:1524-1529. 13. Brown AJ, Jessup W. Oxysterols and atherosclerosis. Atherosclerosis 1999;142:1-28. 14. Rose KA, Stapleton G, Dott K, Kieny MP, Best R, Schwarz M, Russell DW, et al. Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone. Proc Natl Acad Sci U S A 1997;94:4925-4930. 15. Schwarz M, Lund EG, Lathe R, Bjorkhem I, Russell DW. Identification and characterization of a mouse oxysterol 7alpha-hydroxylase cDNA. J Biol Chem 1997;272:23995-24001. 16. Schwarz M, Lund EG, Russell DW. Two 7 alpha-hydroxylase enzymes in bile acid biosynthesis. Curr Opin Lipidol 1998;9:113118. 17. Setchell KD, Schwarz M, O’Connell NC, Lund EG, Davis DL, Lathe R, Thompson HR, et al. Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alphahydroxylase gene causes severe neonatal liver disease. J Clin Invest 1998;102:1690-1703. 18. Bissell DM, Guzelian PS. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci 1980; 349:85-98. 19. Hylemon PB, Gurley EC, Stravitz RT, Litz JS, Pandak WM, Chiang JY, Vlahcevic ZR, et al. Hormonal regulation of cholesterol 7 alpha-hydroxylase mRNA levels and transcriptional activity in primary rat hepatocyte cultures. J Biol Chem 1992;267: 16866-16871. 20. 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. 21. 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. 22. Schwarz M, Lund EG, Setchell KD, Kayden HJ, Zerwekh JE, Bjorkhem I, Herz J, et al. Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase. J Biol Chem 1996; 271:18024-18031. 23. Norlin M, Andersson U, Bjorkhem I, Wikvall K. Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A). J Biol Chem 2000;275:34046-34053. 24. Hylemon PB, Studer EJ, Pandak WM, Heuman DM, Vlahcevic ZR, Chiang JY. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate. Anal Biochem 1989;182: 212-216.
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25. Folch J. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497-509. 26. Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A, Mehtali M. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 1996;70:4805-4810. 27. Valerie K, Brust D, Farnsworth J, Amir C, Taher MM, Hershey C, Feden J. Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir. Cancer Gene Ther 2000;7: 879-884. 28. Stapleton G, Steel M, Richardson M, Mason JO, Rose KA, Morris RG, Lathe R. A novel cytochrome P450 expressed primarily in brain. J Biol Chem 1995;270:29739-29745. 29. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977;36:59-74. 30. 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:G293-G303. 31. Hall E, Hylemon P, Vlahcevic Z, Mallonee D, Valerie K, Avadhani N, Pandak W. Overexpression of CYP27 in hepatic and extrahepatic cells: role in the regulation of cholesterol homeostasis. Am J Physiol 2001;281:G293-G301. 32. Li-Hawkins J, Lund EG, Turley SD, Russell DW. Disruption of the oxysterol 7alpha-hydroxylase gene in mice. J Biol Chem 2000; 275:16536-16542. 33. Pandak WM, Bohdan P, Franklund C, Mallonee DH, Eggertsen G, Bjorkhem I, Gil G, et al. Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo. Gastroenterology 2001;120:1801-1809. 34. Wu Z, Martin KO, Javitt NB, Chiang JY. Structure and functions of human oxysterol 7alpha-hydroxylase cDNAs and gene CYP7B1. J Lipid Res 1999;40:2195-2203. 35. Ishibashi S, Schwarz M, Frykman PK, Herz J, Russell DW. Disruption of cholesterol 7alpha-hydroxylase gene in mice. I. Postnatal lethality reversed by bile acid and vitamin supplementation. J Biol Chem 1996;271:18017-18023. 36. Axelson M, Mork B, Aly A, Wisen O, Sjovall J. Concentrations of cholestenoic acids in plasma from patients with liver disease. J Lipid Res 1989;30:1877-1882. 37. Axelson M, Sjovall J. Potential bile acid precursors in plasma— possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man. J Steroid Biochem 1990;36:631640. 38. Nguyen LB, Xu G, Shefer S, Tint GS, Batta A, Salen G. Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7alpha-hydroxylase activities in the rat, guinea pig, and rabbit: effects of cholesterol and bile acids. Metabolism 1999;48: 1542-1548. 39. Toll A, Wikvall K, Sudjana-Sugiaman E, Kondo KH, Bjorkhem I. 7 Alpha hydroxylation of 25-hydroxycholesterol in liver microsomes. Evidence that the enzyme involved is different from cholesterol 7 alpha-hydroxylase. Eur J Biochem 1994;224:309-316. 40. Gupta S, Stravitz RT, Dent P, Hylemon PB. Down-regulation of cholesterol 7alpha -hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun Nterminal kinase pathway. J Biol Chem 2001;276:15816-15822.
C
Abbreviations: CYP7A1, cholesterol 7␣-hydroxylase; CYP27, sterol 27-hydroxylase; CYP7B1, oxysterol 7␣-hydroxylase; cAMP, adenosine 3⬘, 5⬘-cyclic monophosphate; PMA, phorbol myristate acetate; mRNA, messenger RNA; RPA, ribonuclease protection assay; TCA, taurocholate. From the Departments of 1Medicine, 2Microbiology/Immunology, and 3Biochemistry/Molecular Biophysics, Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA. Received May 18, 2001; accepted February 27, 2002. Supported by grants from the Veterans Administration and the National Institutes of Health (P01 DK38030). †Deceased. Address reprint requests to: William M. Pandak, M.D., Veterans Affairs Medical Center, Division of Gastroenterology 111-N, 1201 Broad Rock Rd., Richmond, VA 23249. E-mail: [email protected]; fax: 804-675-5816. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3506-0014$35.00/0 doi:10.1053/jhep.2002.33200 1400
“alternative” pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (CYP27) with subsequent 7␣-hydroxylation of 27-hydroxycholesterol by oxysterol 7␣-hydroxylase (CYP7B1).1-5 CYP7A1 is highly regulated by bile acids, hormones, diurnal rhythm, cholesterol (oxysterols), cytokines, and second messengers.1,2 Like CYP7A1, CYP27 is regulated by bile acids and hormones.2 In contrast to CYP7A1, which is only found in the liver, CYP27 is also found in abundance in extrahepatic tissues, including vascular endothelium. Maybe just as importantly, initial 27-hydroxylation of cholesterol in peripheral tissues may be a mechanism for prevention of cholesterol accumulation and in the generation of a cholesterol homeostatic regulatory oxysterol in response to excess cholesterol.6-10 Oxysterols are important degradation products of cholesterol, being intermediates in the synthesis of bile acids and steroid hormones. These intermediates seem to have regulatory effects on cholesterol homeostasis, including suppression of 3-hydroxy-3-methylglutaryl– coenzyme A
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reductase and the low-density lipoprotein receptor, and transcriptional stimulation of CYP7A1 and ABC1 through ligand binding to liver X receptor.6,11-13 Recently, a complementary DNA encoding CYP7B1, capable of mediating 7␣-hydroxylation of 27-hydroxycholesterol, 25-hydroxycholesterol, dehydroepiandrosterone, and pregnenolone, was isolated and characterized.14,15 In contrast to CYP7A1, CYP7B1 is also expressed in extrahepatic tissues, including high levels in the kidney, vascular endothelium, and hippocampus.15,16 It has been suggested that 7␣ hydroxylation of 27-hydroxycholesterol may be required to metabolize oxysterols to dihydroxy metabolites with less regulatory and cytotoxic effects.17 These more water-soluble dihydroxy sterol metabolites can then be more readily excreted from peripheral cells, transported to the liver, and converted to bile acids.7 The regulation of CYP7B1, possibly a rate-determining enzyme in the alternative pathway, has not been studied in a defined system. In this study, CYP7B1 was shown to be regulated by bile acids, cholesterol, 3⬘, 5⬘-cyclic monophosphate (cAMP), and phorbol myristate acetate (PMA) in a manner similar to CYP7A1. Unlike CYP7A1, whose increased expression was associated with a dramatically increased rate of bile acid synthesis, overexpression of CYP7B1 did not significantly alter basal bile acid synthesis.
Materials and Methods Materials Thyroxine, dexamethasone, mevalonolactone, PMA, dithiothreitol, ethylene glycol-bis(-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid, phenylmethylsulfonyl fluoride, and dibutyryl cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). William’s E Medium was purchased from GIBCO-BRL (Rockville, MD). Poly Attract messenger RNA (mRNA) isolation system II was obtained from Promega (Madison, WI). Squalestatin 1 and tripotassium salt were kindly provided by Glaxo Research Group. -Cyclodextrin (Trappsol) was purchased from Cyclodextrin Technologies Development Corp. (Gainesville, FL). [4-14C]-cholesterol (59.4 mCi/mmol) and 3H-25-hydroxycholesterol were purchased from New England Nuclear (Boston, MA). 25-Hydroxycholesterol was purchased from Steraloids, Inc. (Newport, RI). Taurine conjugated bile acids were purchased from Calbiochem (La Jolla, CA). Silica gel thin-layer chromatography plates (LK6 D) were from Whatman (Clifton, NJ). Solvents were purchased from Fisher Scientific (New Lawn, NJ). All other reagents were of the highest quality commercially available. Nylon
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membranes were purchased from Micron Separation Inc. (Westborough, MA). Isolation and Culture of Primary Rat Hepatocytes Hepatocytes were isolated from male Sprague-Dawley rats (250-300 g) as previously used by us with the collagenase-perfusion technique of Bissell and Guzelian.18 Cells were routinely harvested after 72 hours of culture as previously described.19 Unless otherwise indicated, culture medium contained 0.1 mol/L dexamethasone and 1.0 mol/L L-thyroxine (T4) with culture medium changed daily. Bile acids were added 24 hours after cells were plated to achieve a 50 mol/L concentration unless indicated otherwise. Squalestatin, mevalonolactone, and cholesterol (dissolved in -cyclodextrin), when added, were added to the cells at final concentrations of 1 mol/L, 2 mmol/L, and 200 mol/L, respectively, at 48 hours of culture. Dibutyryl cAMP and PMA were added to the cells at final concentrations of 50 mol/L and 100 nmol/L, respectively, 24 hours after culturing. Actinomycin D, a transcription inhibitor, was added to cells at a final concentration of 10 g/mL 4 hours before harvesting. Toxicity was assessed as previously described.20 RNA Preparation Cells were harvested 48 hours after infection, and RNA was isolated from hepatocytes as previously described.21 RNA Quantification Northern blotting was performed as previously described21 using a mouse complementary DNA clone for mouse oxysterol 7␣-hydroxylase (a generous gift from Dr. David Russell). A ribonuclease protection assay (RPA) was used in some studies for mRNA quantitation. Briefly, an RPA probe for rat CYP7B1 mRNA was constructed. First-strand DNA synthesis for the probe was performed using the primer 5⬘-CGTGAATTCGAGCACATCATCTTGGCTTGC-3⬘ (EcoRI site in 5⬘ extension) in a reaction with reverse transcriptase and mRNA obtained from the liver of a cholestyramine-fed rat. An aliquot from the reverse-transcriptase reaction was used in a polymerase chain reaction with the first-strand primer and the primer 5⬘-CGAGATCTGGTCATTGTGTATCATTGGAGG-3⬘ (BglII site in 5⬘ extension) to produce a polymerase chain reaction product that was cloned into the EcoRI/BglII sites of pSP72 (Promega). The resulting plasmid was used for production of an [␣-32P]-uridine triphosphate–labeled RNA probe using the Maxiscript T7 in vitro transcription kit from Ambion, Inc. (Austin, TX). The RNA probe provided a 363 base protected fragment from rat CYP7B1 mRNA in the RPA assay, performed using the RPA III kit from Ambion. Rat cyclophilin com-
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plementary DNA was used as the internal loading standard for both Northern blotting and the RPA.21 Microsome Preparation Microsomes were prepared as previously described.19 Determinations of Enzyme Specific Activity CYP7B1 activity was determined according to the method of Schwarz et al.22 Briefly, 500 g of microsomal protein was incubated at 37°C for 15 minutes in a shaking water bath with 50 mmol/L Tris acetate, pH 7.4, 1 mmol/L ethylenediaminetetraacetic acid, 2 mmol/L dithiothreitol, 0.03% Triton X-100, 1.2 mmol/L reduced nicotinamide adenine dinucleotide phosphate, and 0.06 nmol 3H-25-hydroxycholesterol in a final volume of 500 L. The reaction was stopped by addition of 6 mL methylene chloride. The organic phase was then evaporated to dryness under a nitrogen gas atmosphere, dissolved in acetone, and assayed by thin-layer chromatography in a solvent system containing toluene/ethyl acetate (2:3). The developed plate was exposed to a Tritium screen (Molecular Dynamics, San Francisco, CA), and the relative intensities of the bands were quantified and analyzed with a Phosphor Imager and the appropriate software. The validity of this assay was examined using several different approaches. These studies were undertaken in response to a study by Norlin et al. showing the ability of CYP7A1 to 7␣-hydroxylate 25-hydroxycholesterol and 27-hydroxycholesterol.23 Subsequent studies in our own laboratory confirmed these findings. In primary rat hepatocytes infected with recombinant adenovirus encoding CMV-CYP7A1, we showed that overexpression of CYP7A1 led to detection of 7␣-hydroxylated 25-hydroxycholesterol (data not shown). To ensure the changes in activity shown by the assay system used in the current study were a function of changes in CYP7B1 activity, the following control experiments were performed in rat microsomes. (1) The standard assay was performed using [14C]-cholesterol as substrate, a substrate of CYP7A1. (2) 7-Oxocholesterol, a selective inhibitor of CYP7A1, was added in optimal concentrations (10 mol/L) to the assay. (3) Assay was performed using [14C]dehydroepiandrosterone under saturating conditions (i.e., cold dehydroepiandrosterone added), a substrate of CYP7B1, but not CYP7A1. (4) CYP7B1 specific activity changes were determined in mitochondria, an organelle with no CYP7A1 activity. The results are summarized as follows. (1) Use of [14C]-cholesterol as substrate elicited no products using the CYP7B1 assay as previously described. (2) 7-Oxocholesterol (10 mol/L) did not decrease CYP7B1 activity. (3) Use of dehydroepiandrosterone under saturating conditions showed significant increases in CYP7B1
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activity after cholestyramine feeding. (4) Changes in CYP7B1 activity observed in mitochondria paralleled changes in microsomes. Of note, regulatory changes in rat mitochondrial CYP7B1 paralleled changes in microsomal CYP7B1; however, the basal CYP7B1 activity level in mitochondria was 2- to 3-fold lower than that found in microsomes. Synthesis of Bile Acids by Primary Hepatocytes The conversion of [4-14C] cholesterol into MeOH/ H2O-soluble material was determined as previously described.24 Plates were incubated with [4-14C] cholesterol (5.55 ⫻ 106 dpm/plate) 24 hours after plating (treated with virus for 2 hours before administration of [4-14C]). Media and cells were harvested 48 hours later and analyzed after Folch extraction.25 Bile acid biosynthesis rates in infected cells were expressed as percent of paired controls. Overexpression of CYP7B1 The adenovirus constructs used in this study were obtained through the Massey Cancer Center Adenovirus Shared Resource Facility of Virginia Commonwealth University. The CMV-CYP7B1 recombinant adenovirus (Ad-CMV-CYP7B1) was obtained using a pTG-CMV system as previously described.26,27 Briefly, a 1.9-kilobase murine complementary DNA clone of CYP7B114,28 was subcloned into the NotI site of pZeroTG-CMV, a plasmid containing a CMV promoter, a multiple cloning site, and a partial DNA sequence from the adenovirus construct Ad5dl324.15,28 The resulting pZeroTG-CMV/ CYP7B1 recombinant plasmid was cotransformed with ClaI-linearized pTG-CMV (containing the entire Ad5dl324 genome) into Escherichia coli. Resulting plasmids were screened for inserts before being transfected into 293 cells.29 Adenovirus DNA from resulting plaques was further screened by Southern blot for the presence of the CYP7B1 insert, confirming the Ad-CMV-CYP7B1 adenovirus. The control adenovirus contained the CMV promoter and polylinker cassette without any insert DNA. Propagation of the Ad-CMV-CYP7B1 Adenovirus. Large-scale production of recombinant virus was performed by infecting confluent monolayers of 293 cells grown in 15-cm tissue culture dishes with stock adenovirus at a multiplicity of infection of 0.1 to 1. After 2 hours of infection, the virus was removed and the dishes were fed with 15 mL Dulbecco’s modified Eagle medium and 2% fetal bovine serum. Infected monolayers were harvested by scraping when greater than 90% of the cells showed cytopathic changes and centrifuged at 2,700g for 10 minutes at 4°C. The infected cellular pellet was suspended in Dulbecco’s mod-
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ified Eagle medium/2% fetal bovine serum and subjected to 5 cycles of freeze/thaw lysis to release the virus. Cellular debris was removed by centrifugation at 7,700g for 10 minutes at 4°C. To purify, the crude viral supernatant was carefully layered over a 2-step gradient containing 3 mL CsCl (d ⫽ 1.4 g/mL) in TD buffer (0.14 mol/L NaCl, 5 mmol/L KCl, 19 mmol/L Tris, pH 7.4, and 0.7 mmol/L Na2HPO4) layered over 3 mL CsCl (d ⫽ 1.25 g/mL) in TD buffer and centrifuged at 155,000g for 1 hour at 20°C. The viral band was removed, layered over 8 mL CsCl (d ⫽ 1.33 g/mL) in TD buffer, and centrifuged at 155,000g for 18 hours at 20°C. The pure viral opalescent band was removed and dialyzed against 10 mmol/L Tris, pH 7.4, 1 mmol/L MgCl2, and 10% glycerol overnight at 4°C. The virus was aliquoted and stored at ⫺70°C. The virus titer (pfu) was determined by plaque assay, and viral particles were determined by optical density using spectrophotometry ( ⫽ 260). Infection of Cells With Replicative Defective AdCMV-CYP7B1. Primary rat hepatocytes were grown in P150-cm2 plates to confluency (⬃2.5 ⫻ 107 cells). Twenty-four hours after plating, 2.5 mL of fresh medium was added. Cells were then infected with replicative defective Ad-CMV-CYP7B1 with a multiplicity of infection of 10 (virus to cells) using unpurified virus in a volume of 14.7 L. The virus was allowed to dwell for 2 hours in minimal culture medium by shaking the plates gently every 15 minutes, after which the medium with virus was removed, 20 mL of fresh medium was added, and the plates were allowed to incubate at 37°C in 5% CO2 for an additional 48 hours. Statistics Data are reported as mean ⫾ SE. Where indicated, data were subjected to t-test analysis and determined to be significantly different if P was less than .05.
Results Effect of Dexamethasone and L-Thyroxine on CYP7B1 Activity In primary rat hepatocytes, it has been shown that dexamethasone and L-thyroxine (T4) are required for optimal expression and regulation of CYP7A1.19 Therefore, the effects of addition of dexamethasone and L-thyroxine to culture medium on CYP7B1 were explored. Both were added to culture medium (at time 0) individually and in combination. The specific activity of CYP7B1 was measured in microsomes prepared from rat hepatocyte cultures 72 hours after plating. Compared with the individual addition of optimal concentrations of dexamethasone (0.1 mol/L) or L-thyroxine (1.0 mol/L), which increased CYP7B1 activity 44% to 58% compared
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Fig. 1. Effects of squalestatin, cholesterol, and mevalonate on CYP7B1 activities in primary rat hepatocyte cultures. Culture medium contained optimal concentrations of dexamethasone (0.1 mol/L) and T4 (1.0 mol/L) and 2.5% (vol/vol) -cyclodextrin. Squalestatin (Squal; 1.0 mol/L), mevalonate (Mev, 2 mmol/L, as lactone), or cholesterol (Xol; 200 mol/L) was added to the cultures at 48 hours of incubation. Microsomes were prepared at 72 hours. CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Mean of 2 experiments for all conditions compared with paired controls.
with controls, the addition of dexamethasone (0.1 mol/L) plus L-thyroxine (1.0 mol/L) to the medium increased CYP7B1 activity 2.6-fold (n ⫽ 2; range, 123%186%) compared with controls with no hormone additions. Effect of Cholesterol on Regulation of CYP7B1 Activity The effects of cholesterol, mevalonate (cholesterol precursor), and squalestatin (inhibitor of cholesterol synthesis) on the regulation of CYP7B1 specific activity in primary rat hepatocyte cultures are shown in Fig. 1. -Cyclodextrin was used to deliver cholesterol (see Methods). The individual additions of either mevalonate (2 mmol/L) or cholesterol (200 mol/L) to culture medium resulted in a 32% (n ⫽ 2; range, 31%-38%) and 39% (n ⫽ 2; range, 30%-48%) increase, respectively, in CYP7B1 specific activity. In contrast, addition of squalestatin (1 mol/L) for 24 hours before harvest decreased CYP7B1 specific activity to 65% of paired controls (n ⫽ 2; range, 31%-38%). The addition of cholesterol to cell medium containing squalestatin not only prevented the decrease found with squalestatin alone but increased CYP7B1 activity by 37% (n ⫽ 2; range, 19%-46%) to levels to those found with cholesterol addition alone. Effect of Bile Acids on CYP7B1 Activity The ability of bile acids to down-regulate CYP7B1 was tested. Figure 2 shows the effect of the addition of increas-
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Fig. 2. Effect of TCA concentration on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated in culture for 24 hours in medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 24 hours, varying concentrations (25-200 mol/L) of TCA were added to the culture medium. Cells were harvested 48 hours after addition of TCA, microsomes were isolated, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Mean of 2 to 3 experiments for all concentrations compared with paired controls, except the 50 mol/L concentration (n ⫽ 6; 45% ⫾ 10% decrease; P ⬍ .01).
ing concentrations of taurocholate (TCA) on the specific activity of microsomal CYP7B1 in hepatocyte cultures containing optimal concentrations of L-thyroxine and dexamethasone. Increasing concentrations of TCA led to increased suppression of CYP7B1 activity. In separate studies, actinomycin D (transcriptional inhibitor) was added to the culture medium alone or in combination with TCA. The half-life of CYP7B1 was similar in both experiments, suggesting that bile acid repression of CYP7B1 is mediated at the transcriptional level (data not shown). The effect of other bile acids on CYP7B1 activity was also examined (Fig. 3). Taurodeoxycholic acid led to repression (36% ⫾ 7% decrease; P ⬍ .05) of CYP7B1 activity to levels similar to that induced by TCA (45% ⫾ 10% decrease; P ⬍ .01). Neither taurochenodeoxycholic acid nor tauroursodeoxycholic acid led to any significant suppression of CYP7B1 activity. Previously, we have shown that taurochenodeoxycholic acid in culture is rapidly metabolized to muricholic acid, a relatively hydrophilic bile acid.20 Therefore, as previously shown with CYP7A1 in primary rat hepatocyte cultures, only relatively hydrophobic bile acids seem to act as repressors. Effect of cAMP and PMA on CYP7B1 Once culture conditions had been established that could maintain CYP7B1 activity at nearly in vivo levels, the effect of activators of cell signaling pathways on the
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Fig. 3. Effect of bile acids on CYP7B1 activity. Hepatocytes were incubated in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). Bile acids (50 mol/L) were added to culture medium 24 hours after plating, and microsomes were harvested at 72 hours. CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Changes in CYP7B1 activity are expressed as percent of control cultures without bile acid additions. Mean of 4 (taurodeoxycholate [TDCA], taurochenodeoxycholate [TCDCA], and tauroursodeoxycholate [TUDCA]) or more (taurocholate [TCA]) experiments ⫾ SE. P ⬍ .05 compared with paired controls.
regulation of CYP7B1 activity could be examined. The addition of dibutyryl cAMP (50 mol/L) to primary rat hepatocytes rapidly decreased cholesterol CYP7B1 activity (Fig. 4). Although the degree of suppression of CYP7B1 by
Fig. 4. Time course of the effects of cAMP on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated for 48 hours in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 48 hours, dibutyryl cAMP (50 mol/L) was added to the culture medium. Cells were harvested at various time points after the addition of dibutyryl cAMP, microsomes were isolated, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Repression of CYP7B1 activity is expressed as percent of control cultures without addition of cAMP (n ⫽ 1 for each time point).
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Table 1. Effect of TCA, cAMP, and Squalestatin on CYP7B1 mRNA Levels Condition
TCA (50 mol/L) cAMP (50 mol/L) Squalestatin (1 mol/L)
n
mRNA Levels (% control)
3 3 3
73 ⫾ 3 (227) 53 ⫾ 7 (247) 67 ⫾ 11 (233)
NOTE. Data expressed as mean ⫾ SE. cAMP levels determined 3 hours after addition. P⬍ .05 for all conditions.
Fig. 5. Time course of the effects of PMA on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). PMA (100 nmol/L) was added to hepatocyte culture medium at the indicated times before harvest, beginning 4 hours after plating. All cultures were harvested for microsomes 28 hours after plating, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Repression of CYP7B1 activity is expressed as percent of control cultures without addition of PMA (n ⫽ 1 for each time point).
cAMP was not as great as that of CYP7A1, the time over which the effect was observed was similar.2,30 PMA (100 nmol/L) was added to hepatocyte culture medium at the indicated times (1.5, 3, 6, and 24 hours) before harvest, beginning 4 hours after plating. Microsomes were harvested from all cells 28 hours after plating. The effects of PMA were biphasic (Fig. 5). Initially, PMA caused a rapid decline in CYP7B1 activity to 40% of control cultures 3 hours after addition. This decline was followed by a recovery to control levels approximately 20 hours after addition of PMA, with an overshoot to 127% of control at 24 hours. Regulation of CYP7B1 mRNA Levels CYP7B1 mRNA levels after the addition of TCA, cAMP, and squalestatin to the culture medium were all decreased. TCA (50 mol/L) led to a 27% ⫾ 3% (P ⬍ .01) decrease in CYP7B1 mRNA levels. Addition of cAMP (50 mol/L) and squalestatin (1 mol/L) decreased CYP7B1 mRNA levels 47% ⫾ 7% (P ⬍ .01) and 33% ⫾ 11% (P ⬍ .05), respectively (Table 1). Changes in CYP7B1 mRNA levels after the addition of PMA followed a course similar to that of CYP7B1 activity levels (data not shown). Overexpression of CYP7B1 The rate of bile acid synthesis through the alternative (i.e., “acidic”) pathway is believed to be controlled by
CYP27, the initial step of the pathway. Recent evidence from our laboratory has shown in HepG2 cells and in primary human hepatocytes that overexpression of CYP27 can increase bile acid synthesis by ⬃60%.31 Overexpression of CYP27 in primary rat hepatocytes led to a similar increase (43% ⫾ 17%; P ⬍ .05) in bile acid synthesis compared with paired controls. These findings suggest that, under normal physiologic conditions, 27hydroxylation of cholesterol may be the rate-determining step in this pathway. In an attempt to further define the role of CYP7B1 in the rate of bile acid synthesis, CYP7B1 was overexpressed using a recombinant adenovirus encoding the gene for CYP7B1. Northern blot analysis (Fig. 6) shows the dramatic increase in CYP7B1 mRNA levels after infection of hepatocytes with recombinant adenovirus encoding CMV-CYP7B1. In contrast to earlier cultures, primary rat hepatocytes were initially maintained in the absence of L-thyroxine. Under these culture conditions, we have previously shown that levels of CYP7A1 are
Fig. 6. Representative Northern blot of CYP7B1 mRNA levels in primary rat hepatocytes before and after infection with recombinant adenovirus encoding CMV-CYP7B1. Hepatocytes were incubated for 24 hours in culture medium containing optimal concentrations of T4 (1.0 mol/L) and dexamethasone (0.1 mol/L). At 24 hours, cells were infected with control virus or recombinant adenovirus encoding CMVCYP7B1 as described in Materials and Methods. Cells were harvested 48 hours after infection, and mRNA levels for CYP7B1 and cyclophilin (loading standard) were determined.
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Fig. 7. Effect of overexpression of CYP7B1 and CYP7A1 with recombinant adenoviruses encoding CMV-CYP7B1 and CMV-CYP7A1 on bile acid synthesis in primary rat hepatocyte cultures. Hepatocytes were incubated for 24 hours in culture medium containing 0.1 mol/L dexamethasone. At 24 hours, cells were infected with control (null) virus or recombinant adenovirus with the multiplicity of infection as described in Materials and Methods. Bile acid synthesis was measured as conversion of 14C-labeled cholesterol into water-soluble products (14C-labeled methanol/water-extractable counts). Data are expressed as a percentage of controls (mean ⫾ SE).
undetectable and that only the acidic pathway of bile acid synthesis is functional.19,32 A greater than 3-fold increase (221% ⫾ 64%; P ⬍ .02) in CYP7B1 specific activity was observed compared with paired controls (cells infected with control virus). Despite this dramatic increase in CYP7B1 activity, bile acid synthesis rates were not significantly changed (86% ⫾ 15%; P ⫽ NS) (Fig. 7). This is in contrast to CYP7A1 overexpression in primary rat hepatocytes, in which overexpression of CYP7A1 led to a greater than 10-fold increase (P ⬍ .001) in the rate of bile acid synthesis (Fig. 7). Overexpression of CYP7B1 in the presence of L-thyroxine with dexamethasone (i.e., functional CYP7A1 and neutral bile acid synthesis pathway) also led to no increase in bile acid synthesis (116% ⫾ 29%; P ⫽ NS), whereas CYP7A1 overexpression led to a greater than 4-fold (n ⫽ 3; P ⬍ .001) increase in bile acid synthesis.
Discussion The results of this study performed in primary rat hepatocytes describe the effects of bile acids, cholesterol, hormones, cAMP, and PMA on the regulation of CYP7B1 mRNA and activity. The addition of L-thyroxine plus dexamethasone increased CYP7B1 activity levels, with optimal concentrations of 1.0 mol/L and 0.1 mol/L, respectively. These concentrations were similar to those found to optimize primary rat hepatocyte culture conditions for CYP7A1 expression.19 Furthermore, the
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addition of bile acids, cholesterol, cAMP, or PMA all led to changes in CYP7B1 activity in a fashion previously observed for CYP7A1 but to a lesser degree.20 Addition of TCA, taurodeoxycholic acid, cAMP, and PMA to culture medium suppressed CYP7B1 activity, whereas the addition of cholesterol had a stimulatory effect. The changes observed in CYP7B1 specific activities were paralleled by changes in mRNA levels. The importance of tightly regulating CYP7B1 is not clearly defined but could be crucial for regulating cellular oxysterol levels. Oxysterols have been presented as probable regulators of cholesterol homeostasis, with in vitro effects such as repression of 3-hydroxy-3-methylglutaryl– coenzyme A reductase.13 The 7␣-hydroxylation of 27hydroxycholesterol mediated through overexpression of CYP7B1 activity in CHO cells has been shown to be capable of preventing the repressive effects of this oxysterol on 3-hydroxy-3-methylglutaryl– coenzyme A reductase.10 However, in Cyp7b1⫺/⫺ mice, in which plasma levels of 25-hydroxycholesterol and 27-hydroxycholesterol are increased, no major differences in cholesterol homeostasis have been observed.33 Based on these findings, it was hypothesized that commensurate pathways of bile acid synthesis may be able to be stimulated in the mouse. However, the absence of generalized suppression of sterol synthesis in the presence of the increased oxysterols, 25-hydroxycholesterol and 27-hydroxycholesterol, is unclear. In a rare, naturally occurring mutation of CYP7B1 in a human neonate with cholestasis, no 7␣hydroxylated bile acids were formed.17 Acidic intermediates in the bile acid biosynthetic pathway accumulated despite the CYP7A1 gene being normal. These findings suggest, at least in humans early in life, that CPY7B1 is critical for formation of 7␣-hydroxylated bile acids. The fact that both dexamethasone and L-thyroxine were found to be optimal culture conditions for CYP7B1 in a manner similar to CYP7A1 is interesting and implies similarities in the hormonal effects on CYP7B1 and CYP7A1. L-thyroxine elicits little effect in primary rat hepatocytes on CYP27, the initial step in the alternative pathway of bile acid synthesis, with dexamethasone required for its optimal expression.4 In contrast, sterol 12␣-hydroxylase (CYP8B1) expression is dramatically suppressed by L-thyroxine, demonstrating a hormonal role in the regulation of the ratio of cholic to chenodeoxycholic acid.32 The ability of cholesterol to up-regulate CYP7B1 is also similar to CYP7A1. In support of this type of regulation is the identification of an SRE-like site in the human CYP7B1 promoter.1,34 Whether this represents a true sterol regulatory site mediated through previously defined pathways is uncertain.
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The original concept that feedback repression of bile acid biosynthesis was mediated solely through the regulation of CYP7A1 is being modified. It has been shown in Cyp7a1⫺/⫺ mice that bile acid synthesis continues to occur, demonstrating the ability of at least one alternative pathway to 7␣-hydroxylate cholesterol and generate bile acids.22,35 Furthermore, CYP7B1 enzyme activity, protein, and mRNA levels in Cyp7a1⫺/⫺ mice were induced. Although the relative importance of these alternative pathways may be species and/or disease state dependent, the question of their ability to be regulated, and by what, has begun to be explored.2,36-38 CYP27, the initial step in the alternative (i.e., “acidic”) pathway, has now been shown to be regulated.2 However, the regulation of the subsequent enzymatic step in the pathway, the 7␣-hydroxylation of the oxysterol, has been given little attention. In rat liver, Toll et al. failed to see any stimulation of 7␣-hydroxylation of oxysterol activity by feeding cholestyramine.39 In the mouse, Schwarz et al. also showed no effects of cholestyramine feeding on CYP7B1 mRNA expression but found an approximately 50% decrease in CYP7B1 mRNA levels with cholate feeding.15,16 The results of this study in primary rat hepatocytes not only show a suppression of CYP7B1 mRNA levels with bile acids but suppression of CYP7B1 activity as well. These findings have been corroborated in whole animals in which bile acid feeding repressed CYP7B1 mRNA and specific activity levels; conversely, CYP7B1 mRNA levels and specific activity levels were increased with chronic biliary diversion and cholestyramine feeding (under review). cAMP was able to repress CYP7B1, like CYP7A1 but to a lesser degree. The mechanism by which cAMP regulates CYP7A1 has not been elucidated but represents a plausible explanation for why bile acid synthesis remains low in fasting despite low circulating levels of bile acids as it is during fasting that glucagon levels increase, stimulating an increase in cAMP levels.2 The mechanism, however, is different from that of PMA, which activates different cell signaling cascades resulting in repression of CYP7A1.30,40 An understanding of this mechanism of regulation of CYP7A1 has laid the groundwork for the more recent observation by Gupta et al. that bile acids can repress CYP7A1 via the JNK/c-Jun pathway, which in turn appears able to repress CYP7A1 transcription via SHP induction.40 The relevance of these signaling pathways to the regulation to CYP7B1 has not yet been explored. Overexpression of CYP7B1 using a recombinant adenovirus containing CMV-CYP7B1 led to a dramatic increase in CYP7B1 activity (data not shown) but no increase in bile acid synthesis rates. This is in contrast to
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findings following CYP7A1 overexpression, where a dramatic increase in bile acid synthesis was observed. Increased CYP27 expression in HepG2 cells also resulted in a significant increase in bile acid synthesis (⬃50%).31 Overexpression of CYP7B1 in combination with CYP27 also did not increase bile acid synthesis any further than overexpression of CYP27 alone.31 These findings suggest that CYP7B1, under normal physiologic conditions, is not the rate-determining step in the “acidic” pathway of bile acid biosynthesis. In summary, in the rat, CYP7B1, like CYP7A1, is a highly regulated enzyme. As with CYP7A1, these regulatory effects seem to down-regulate CYP7B1 at the level of gene transcription. Interestingly, overexpression of CYP7B1 did not increase bile acid synthesis as has been previously observed with CYP7A1, suggesting that the 7␣-hydroxylation of oxysterols is not a rate-limiting step in the alternative pathway of bile acid synthesis under physiologic conditions. Acknowledgment: The authors thank Li Jun Zhao and Pat Bohdan for technical assistance.
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