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 3؅,5؅-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% ؎ 10% and 36% ؎ 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 “alternative” pathway of bile acid synthesis is initiated by bile acids is a principal pathway of cholesterol sterol 27-hydroxylase (CYP27) with subsequent 7␣-hy- secretion from the body. Cholesterol 7␣-hydrox- droxylation of 27-hydroxycholesterol by oxysterol 7␣-hy- C 1-5 ylase (CYP7A1) is the initial and rate-determining en- droxylase (CYP7B1). CYP7A1 is highly regulated by zyme in the “classic” pathway of bile acid synthesis. An bile acids, hormones, diurnal rhythm, cholesterol (oxysterols), cytokines, and second messengers.1,2 Like CYP7A1, CYP27 is regulated by bile acids and hor- Abbreviations: CYP7A1, cholesterol 7␣-hydroxylase; CYP27, sterol 27-hydrox- mones.2 In contrast to CYP7A1, which is only found in ylase; CYP7B1, oxysterol 7␣-hydroxylase; cAMP, adenosine 3Ј,5Ј-cyclic monophosphate; PMA, phorbol myristate acetate; mRNA, messenger RNA; RPA, ribonuclease the liver, CYP27 is also found in abundance in extrahe- protection assay; TCA, taurocholate. patic tissues, including vascular endothelium. Maybe just From the Departments of 1Medicine, 2Microbiology/Immunology, and 3Bio- as importantly, initial 27-hydroxylation of cholesterol in chemistry/Molecular Biophysics, Veterans Affairs Medical Center and Virginia peripheral tissues may be a mechanism for prevention of Commonwealth University, Richmond, VA. Received May 18, 2001; accepted February 27, 2002. cholesterol accumulation and in the generation of a cho- Supported by grants from the Veterans Administration and the National Insti- lesterol homeostatic regulatory oxysterol in response to tutes of Health (P01 DK38030). excess cholesterol.6-10 †Deceased. Address reprint requests to: William M. Pandak, M.D., Veterans Affairs Medical Oxysterols are important degradation products of cho- Center, Division of Gastroenterology 111-N, 1201 Broad Rock Rd., Richmond, VA lesterol, being intermediates in the synthesis of bile acids 23249. E-mail: [email protected]; fax: 804-675-5816. and steroid hormones. These intermediates seem to have Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3506-0014$35.00/0 regulatory effects on cholesterol homeostasis, including doi:10.1053/jhep.2002.33200 suppression of 3-hydroxy-3-methylglutaryl–coenzyme A

1400 HEPATOLOGY, Vol. 35, No. 6, 2002 PANDAK ET AL. 1401

reductase and the low-density lipoprotein receptor, and membranes were purchased from Micron Separation Inc. transcriptional stimulation of CYP7A1 and ABC1 (Westborough, MA). through ligand binding to liver X receptor.6,11-13 Recently, a complementary DNA encoding CYP7B1, Isolation and Culture of Primary Rat Hepatocytes capable of mediating 7␣-hydroxylation of 27-hydroxycho- Hepatocytes were isolated from male Sprague-Dawley lesterol, 25-hydroxycholesterol, dehydroepiandrosterone, rats (250-300 g) as previously used by us with the collag- and pregnenolone, was isolated and characterized.14,15 In enase-perfusion technique of Bissell and Guzelian.18 Cells contrast to CYP7A1, CYP7B1 is also expressed in extra- were routinely harvested after 72 hours of culture as pre- hepatic tissues, including high levels in the kidney, vascu- viously described.19 Unless otherwise indicated, culture lar endothelium, and hippocampus.15,16 It has been medium contained 0.1 ␮mol/L dexamethasone and 1.0 suggested that 7␣ hydroxylation of 27-hydroxycholes- ␮mol/L L-thyroxine (T4) with culture medium changed terol may be required to metabolize oxysterols to dihy- daily. Bile acids were added 24 hours after cells were droxy metabolites with less regulatory and cytotoxic plated to achieve a 50 ␮mol/L concentration unless indi- effects.17 These more water-soluble dihydroxy sterol me- cated otherwise. Squalestatin, mevalonolactone, and cho- tabolites can then be more readily excreted from periph- lesterol (dissolved in ␤-cyclodextrin), when added, were eral cells, transported to the liver, and converted to bile added to the cells at final concentrations of 1 ␮mol/L, 2 ␮ acids.7 mmol/L, and 200 mol/L, respectively, at 48 hours of The regulation of CYP7B1, possibly a rate-determin- culture. Dibutyryl cAMP and PMA were added to the ␮ ing enzyme in the alternative pathway, has not been stud- cells at final concentrations of 50 mol/L and 100 ied in a defined system. In this study, CYP7B1 was shown nmol/L, respectively, 24 hours after culturing. Actinomy- to be regulated by bile acids, cholesterol, 3Ј,5Ј-cyclic cin D, a transcription inhibitor, was added to cells at a ␮ monophosphate (cAMP), and phorbol myristate acetate final concentration of 10 g/mL 4 hours before harvest- (PMA) in a manner similar to CYP7A1. Unlike CYP7A1, ing. Toxicity was assessed as previously described.20 whose increased expression was associated with a dramatically increased rate of bile acid synthesis, overexpression RNA Preparation of CYP7B1 did not significantly alter basal bile acid syn- Cells were harvested 48 hours after infection, and RNA thesis. was isolated from hepatocytes as previously described.21

Materials and Methods RNA Quantification Northern blotting was performed as previously de- Materials scribed21 using a mouse complementary DNA clone for Thyroxine, dexamethasone, mevalonolactone, mouse oxysterol 7␣-hydroxylase (a generous gift from Dr. ␤ PMA, dithiothreitol, ethylene glycol-bis( -aminoethyl David Russell). A ribonuclease protection assay (RPA) Ј Ј ether)-N,N,N ,N -tetraacetic acid, phenylmethylsul- was used in some studies for mRNA quantitation. Briefly, fonyl fluoride, and dibutyryl cAMP were purchased an RPA probe for rat CYP7B1 mRNA was constructed. from Sigma Chemical Co. (St. Louis, MO). William’s First-strand DNA synthesis for the probe was performed E Medium was purchased from GIBCO-BRL (Rock- using the primer 5Ј-CGTGAATTCGAGCACATCAT- ville, MD). Poly Attract messenger RNA (mRNA) iso- CTTGGCTTGC-3Ј (EcoRI site in 5Ј extension) in a re- lation system II was obtained from Promega (Madison, action with reverse transcriptase and mRNA obtained WI). Squalestatin 1 and tripotassium salt were kindly from the liver of a cholestyramine-fed rat. An aliquot from provided by Glaxo Research Group. ␤-Cyclodextrin the reverse-transcriptase reaction was used in a polymer- (Trappsol) was purchased from Cyclodextrin Technol- ase chain reaction with the first-strand primer and the ogies Development Corp. (Gainesville, FL). [4-14C]-cho- primer 5Ј-CGAGATCTGGTCATTGTGTATCATT- lesterol (59.4 mCi/mmol) and 3H-25-hydroxycholesterol GGAGG-3Ј (BglII site in 5Ј extension) to produce a poly- were purchased from New England Nuclear (Boston, merase chain reaction product that was cloned into the MA). 25-Hydroxycholesterol was purchased from Ster- EcoRI/BglII sites of pSP72 (Promega). The resulting plas- aloids, Inc. (Newport, RI). Taurine conjugated bile acids mid was used for production of an [␣-32P]-uridine were purchased from Calbiochem (La Jolla, CA). Silica triphosphate–labeled RNA probe using the Maxiscript gel thin-layer chromatography plates (LK6 D) were from T7 in vitro transcription kit from Ambion, Inc. (Austin, Whatman (Clifton, NJ). Solvents were purchased from TX). The RNA probe provided a 363 base protected frag- Fisher Scientific (New Lawn, NJ). All other reagents were ment from rat CYP7B1 mRNA in the RPA assay, performed of the highest quality commercially available. Nylon using the RPA III kit from Ambion. Rat cyclophilin com- 1402 PANDAK ET AL. HEPATOLOGY, June 2002 plementary DNA was used as the internal loading stan- activity after cholestyramine feeding. (4) Changes in dard for both Northern blotting and the RPA.21 CYP7B1 activity observed in mitochondria paralleled changes in microsomes. Of note, regulatory changes in rat Microsome Preparation mitochondrial CYP7B1 paralleled changes in microsomal Microsomes were prepared as previously described.19 CYP7B1; however, the basal CYP7B1 activity level in mitochondria was 2- to 3-fold lower than that found in Determinations of Enzyme Specific Activity microsomes. CYP7B1 activity was determined according to the method of Schwarz et al.22 Briefly, 500 ␮g of microsomal Synthesis of Bile Acids by Primary Hepatocytes protein was incubated at 37°C for 15 minutes in a shaking The conversion of [4-14C] cholesterol into MeOH/ water bath with 50 mmol/L Tris acetate, pH 7.4, 1 H2O-soluble material was determined as previously de- mmol/L ethylenediaminetetraacetic acid, 2 mmol/L di- scribed.24 Plates were incubated with [4-14C] cholesterol thiothreitol, 0.03% Triton X-100, 1.2 mmol/L reduced (5.55 ϫ 106 dpm/plate) 24 hours after plating (treated nicotinamide adenine dinucleotide phosphate, and 0.06 with virus for 2 hours before administration of [4-14C]). nmol 3H-25-hydroxycholesterol in a final volume of 500 Media and cells were harvested 48 hours later and ana- ␮L. The reaction was stopped by addition of 6 mL meth- lyzed after Folch extraction.25 Bile acid biosynthesis rates ylene chloride. The organic phase was then evaporated to in infected cells were expressed as percent of paired con- dryness under a nitrogen gas atmosphere, dissolved in trols. acetone, and assayed by thin-layer chromatography in a solvent system containing toluene/ethyl acetate (2:3). Overexpression of CYP7B1 The developed plate was exposed to a Tritium screen The adenovirus constructs used in this study were ob- (Molecular Dynamics, San Francisco, CA), and the rela- tained through the Massey Cancer Center Adenovirus tive intensities of the bands were quantified and analyzed Shared Resource Facility of Virginia Commonwealth with a Phosphor Imager and the appropriate software. University. The CMV-CYP7B1 recombinant adenovirus The validity of this assay was examined using several (Ad-CMV-CYP7B1) was obtained using a pTG-CMV different approaches. These studies were undertaken in system as previously described.26,27 Briefly, a 1.9-kilobase response to a study by Norlin et al. showing the ability of murine complementary DNA clone of CYP7B114,28 was CYP7A1 to 7␣-hydroxylate 25-hydroxycholesterol and subcloned into the NotI site of pZeroTG-CMV, a plas- 27-hydroxycholesterol.23 Subsequent studies in our own mid containing a CMV promoter, a multiple cloning site, laboratory confirmed these findings. In primary rat hepa- and a partial DNA sequence from the adenovirus con- tocytes infected with recombinant adenovirus encoding struct Ad5dl324.15,28 The resulting pZeroTG-CMV/ CMV-CYP7A1, we showed that overexpression of CYP7B1 recombinant plasmid was cotransformed with CYP7A1 led to detection of 7␣-hydroxylated 25-hy- ClaI-linearized pTG-CMV (containing the entire Ad5dl324 droxycholesterol (data not shown). To ensure the changes genome) into Escherichia coli. Resulting plasmids were in activity shown by the assay system used in the current screened for inserts before being transfected into 293 study were a function of changes in CYP7B1 activity, the cells.29 Adenovirus DNA from resulting plaques was fur- following control experiments were performed in rat mi- ther screened by Southern blot for the presence of the crosomes. (1) The standard assay was performed using CYP7B1 insert, confirming the Ad-CMV-CYP7B1 ade- [14C]-cholesterol as substrate, a substrate of CYP7A1. (2) novirus. The control adenovirus contained the CMV pro- 7-Oxocholesterol, a selective inhibitor of CYP7A1, was moter and polylinker cassette without any insert DNA. added in optimal concentrations (10 ␮mol/L) to the as- Propagation of the Ad-CMV-CYP7B1 Adenovirus. say. (3) Assay was performed using [14C]dehydroepi- Large-scale production of recombinant virus was per- androsterone under saturating conditions (i.e., cold formed by infecting confluent monolayers of 293 cells dehydroepiandrosterone added), a substrate of CYP7B1, grown in 15-cm tissue culture dishes with stock adenovi- but not CYP7A1. (4) CYP7B1 specific activity changes rus at a multiplicity of infection of 0.1 to 1. After 2 hours were determined in mitochondria, an organelle with no of infection, the virus was removed and the dishes were CYP7A1 activity. The results are summarized as follows. fed with 15 mL Dulbecco’s modified Eagle medium and (1)Useof[14C]-cholesterol as substrate elicited no prod- 2% fetal bovine serum. ucts using the CYP7B1 assay as previously described. (2) Infected monolayers were harvested by scraping when 7-Oxocholesterol (10 ␮mol/L) did not decrease CYP7B1 greater than 90% of the cells showed cytopathic changes activity. (3) Use of dehydroepiandrosterone under satu- and centrifuged at 2,700g for 10 minutes at 4°C. The rating conditions showed significant increases in CYP7B1 infected cellular pellet was suspended in Dulbecco’s mod- HEPATOLOGY, Vol. 35, No. 6, 2002 PANDAK ET AL. 1403 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 ϭ 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 ϭ 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 ϭ 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 Ϫ70°C. The Fig. 1. Effects of squalestatin, cholesterol, and mevalonate on virus titer (pfu) was determined by plaque assay, and viral CYP7B1 activities in primary rat hepatocyte cultures. Culture medium contained optimal concentrations of dexamethasone (0.1 ␮mol/L) and particles were determined by optical density using spec- T4 (1.0 ␮mol/L) and 2.5% (vol/vol) ␤-cyclodextrin. Squalestatin (Squal; trophotometry (␭ ϭ 260). 1.0 ␮mol/L), mevalonate (Mev, 2 mmol/L, as lactone), or cholesterol Infection of Cells With Replicative Defective Ad- (Xol; 200 ␮mol/L) was added to the cultures at 48 hours of incubation. CMV-CYP7B1. Primary rat hepatocytes were grown Microsomes were prepared at 72 hours. CYP7B1 activities were determined using thin-layer chromatography as described in Materials and 2 ϳ ϫ 7 in P150-cm plates to confluency ( 2.5 10 cells). Methods. Mean of 2 experiments for all conditions compared with paired Twenty-four hours after plating, 2.5 mL of fresh medium controls. was added. Cells were then infected with replicative defective Ad-CMV-CYP7B1 with a multiplicity of infec- with controls, the addition of dexamethasone (0.1 tion of 10 (virus to cells) using unpurified virus in a ␮ ␮ ␮ mol/L) plus L-thyroxine (1.0 mol/L) to the medium volume of 14.7 L. The virus was allowed to dwell for 2 increased CYP7B1 activity 2.6-fold (n ϭ 2; range, 123%- hours in minimal culture medium by shaking the plates 186%) compared with controls with no hormone addi- gently every 15 minutes, after which the medium with tions. virus was removed, 20 mL of fresh medium was added, and the plates were allowed to incubate at 37°Cin5% Effect of Cholesterol on Regulation of CYP7B1 CO2 for an additional 48 hours. Activity The effects of cholesterol, mevalonate (cholesterol pre- Statistics cursor), and squalestatin (inhibitor of cholesterol synthe- Ϯ Data are reported as mean SE. Where indicated, sis) on the regulation of CYP7B1 specific activity in data were subjected to t-test analysis and determined to be primary rat hepatocyte cultures are shown in Fig. 1. ␤-Cy- significantly different if P was less than .05. clodextrin was used to deliver cholesterol (see Methods). The individual additions of either mevalonate (2 Results mmol/L) or cholesterol (200 ␮mol/L) to culture medium Effect of Dexamethasone and L-Thyroxine on resulted in a 32% (n ϭ 2; range, 31%-38%) and 39% CYP7B1 Activity (n ϭ 2; range, 30%-48%) increase, respectively, in In primary rat hepatocytes, it has been shown that CYP7B1 specific activity. In contrast, addition of squal- dexamethasone and L-thyroxine (T4) are required for op- estatin (1 ␮mol/L) for 24 hours before harvest decreased timal expression and regulation of CYP7A1.19 Therefore, CYP7B1 specific activity to 65% of paired controls (n ϭ the effects of addition of dexamethasone and L-thyroxine 2; range, 31%-38%). The addition of cholesterol to cell to culture medium on CYP7B1 were explored. Both were medium containing squalestatin not only prevented the added to culture medium (at time 0) individually and in decrease found with squalestatin alone but increased combination. The specific activity of CYP7B1 was mea- CYP7B1 activity by 37% (n ϭ 2; range, 19%-46%) to sured in microsomes prepared from rat hepatocyte cul- levels to those found with cholesterol addition alone. tures 72 hours after plating. Compared with the individual addition of optimal concentrations of dexa- Effect of Bile Acids on CYP7B1 Activity methasone (0.1 ␮mol/L) or L-thyroxine (1.0 ␮mol/L), The ability of bile acids to down-regulate CYP7B1 was which increased CYP7B1 activity 44% to 58% compared tested. Figure 2 shows the effect of the addition of increas- 1404 PANDAK ET AL. HEPATOLOGY, June 2002

Fig. 2. Effect of TCA concentration on CYP7B1 activity in primary cultures of rat hepatocytes. Hepatocytes were incubated in culture for 24 Fig. 3. Effect of bile acids on CYP7B1 activity. Hepatocytes were incu- hours in medium containing optimal concentrations of T4 (1.0 ␮mol/L) bated in culture medium containing optimal concentrations of T4 (1.0 and dexamethasone (0.1 ␮mol/L). At 24 hours, varying concentrations ␮mol/L) and dexamethasone (0.1 ␮mol/L). Bile acids (50 ␮mol/L) were (25-200 ␮mol/L) of TCA were added to the culture medium. Cells were added to culture medium 24 hours after plating, and microsomes were harvested 48 hours after addition of TCA, microsomes were isolated, and harvested at 72 hours. CYP7B1 activities were determined using thin-layer CYP7B1 activities were determined using thin-layer chromatography as chromatography as described in Materials and Methods. Changes in CYP7B1 described in Materials and Methods. Mean of 2 to 3 experiments for all activity are expressed as percent of control cultures without bile acid concentrations compared with paired controls, except the 50 ␮mol/L additions. Mean of 4 (taurodeoxycholate [TDCA], taurochenodeoxycholate concentration (n ϭ 6; 45% Ϯ 10% decrease; P Ͻ .01). [TCDCA], and tauroursodeoxycholate [TUDCA]) or more (taurocholate [TCA]) experiments Ϯ SE. P Ͻ .05 compared with paired controls.

ing concentrations of taurocholate (TCA) on the speciﬁc activity of microsomal CYP7B1 in hepatocyte cultures regulation of CYP7B1 activity could be examined. The ␮ containing optimal concentrations of L-thyroxine and addition of dibutyryl cAMP (50 mol/L) to primary rat dexamethasone. Increasing concentrations of TCA led to hepatocytes rapidly decreased cholesterol CYP7B1 activity increased suppression of CYP7B1 activity. In separate (Fig. 4). Although the degree of suppression of CYP7B1 by 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% Ϯ 7% decrease; P Ͻ .05) of CYP7B1 activity to levels similar to that induced by TCA (45% Ϯ 10% decrease; P Ͻ .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 hydro- philic bile acid.20 Therefore, as previously shown with Fig. 4. Time course of the effects of cAMP on CYP7B1 activity in CYP7A1 in primary rat hepatocyte cultures, only rela- primary cultures of rat hepatocytes. Hepatocytes were incubated for 48 tively hydrophobic bile acids seem to act as repressors. 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 Effect of cAMP and PMA on CYP7B1 various time points after the addition of dibutyryl cAMP, microsomes were Once culture conditions had been established that isolated, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Repression of could maintain CYP7B1 activity at nearly in vivo levels, CYP7B1 activity is expressed as percent of control cultures without the effect of activators of cell signaling pathways on the addition of cAMP (n ϭ 1 for each time point). HEPATOLOGY, Vol. 35, No. 6, 2002 PANDAK ET AL. 1405

Table 1. Effect of TCA, cAMP, and Squalestatin on CYP7B1 mRNA Levels

mRNA Levels Condition n (% control)

TCA (50 ␮mol/L) 3 73 Ϯ 3(227) cAMP (50 ␮mol/L) 3 53 Ϯ 7(247) Squalestatin (1 ␮mol/L) 3 67 Ϯ 11 (233)

NOTE. Data expressed as mean Ϯ SE. cAMP levels determined 3 hours after addition. PϽ .05 for all conditions.

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 ϳ60%.31 Over- Fig. 5. Time course of the effects of PMA on CYP7B1 activity in expression of CYP27 in primary rat hepatocytes led to a primary cultures of rat hepatocytes. Hepatocytes were incubated in similar increase (43% Ϯ 17%; P Ͻ .05) in bile acid syn- culture medium containing optimal concentrations of T4 (1.0 ␮mol/L) and dexamethasone (0.1 ␮mol/L). PMA (100 nmol/L) was added to thesis compared with paired controls. These ﬁndings sug- hepatocyte culture medium at the indicated times before harvest, be- gest that, under normal physiologic conditions, 27- ginning 4 hours after plating. All cultures were harvested for microsomes hydroxylation of cholesterol may be the rate-determining 28 hours after plating, and CYP7B1 activities were determined using thin-layer chromatography as described in Materials and Methods. Re- step in this pathway. In an attempt to further deﬁne the pression of CYP7B1 activity is expressed as percent of control cultures role of CYP7B1 in the rate of bile acid synthesis, CYP7B1 without addition of PMA (n ϭ 1 for each time point). was overexpressed using a recombinant adenovirus encoding the gene for CYP7B1. Northern blot analysis (Fig. 6) shows the dramatic increase in CYP7B1 mRNA levels cAMP was not as great as that of CYP7A1, the time over after infection of hepatocytes with recombinant adenovi- which the effect was observed was similar.2,30 rus encoding CMV-CYP7B1. In contrast to earlier cul- PMA (100 nmol/L) was added to hepatocyte culture tures, primary rat hepatocytes were initially maintained in medium at the indicated times (1.5, 3, 6, and 24 hours) the absence of L-thyroxine. Under these culture condi- before harvest, beginning 4 hours after plating. Micro- tions, we have previously shown that levels of CYP7A1 are somes 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% Ϯ 3% (P Ͻ .01) decrease in CYP7B1 mRNA levels. Addition of cAMP (50 ␮mol/L) and squalestatin (1 ␮mol/L) decreased CYP7B1 mRNA levels 47% Ϯ 7% (P Ͻ .01) and 33% Ϯ 11% (P Ͻ .05), respectively (Table 1). Changes in CYP7B1 mRNA levels after the addition of PMA fol- Fig. 6. Representative Northern blot of CYP7B1 mRNA levels in primary rat hepatocytes before and after infection with recombinant ade- lowed a course similar to that of CYP7B1 activity levels novirus encoding CMV-CYP7B1. Hepatocytes were incubated for 24 (data not shown). hours in culture medium containing optimal concentrations of T4 (1.0 ␮mol/L) and dexamethasone (0.1 ␮mol/L). At 24 hours, cells were Overexpression of CYP7B1 infected with control virus or recombinant adenovirus encoding CMV- CYP7B1 as described in Materials and Methods. Cells were harvested 48 The rate of bile acid synthesis through the alternative hours after infection, and mRNA levels for CYP7B1 and cyclophilin (i.e., “acidic”) pathway is believed to be controlled by (loading standard) were determined. 1406 PANDAK ET AL. HEPATOLOGY, June 2002

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 prob- able regulators of cholesterol homeostasis, with in vitro effects such as repression of 3-hydroxy-3-methylglutaryl– coenzyme A reductase.13 The 7␣-hydroxylation of 27- Fig. 7. Effect of overexpression of CYP7B1 and CYP7A1 with recom- hydroxycholesterol mediated through overexpression of binant adenoviruses encoding CMV-CYP7B1 and CMV-CYP7A1 on bile CYP7B1 activity in CHO cells has been shown to be acid synthesis in primary rat hepatocyte cultures. Hepatocytes were capable of preventing the repressive effects of this oxy- incubated for 24 hours in culture medium containing 0.1 ␮mol/L dexamethasone. At 24 hours, cells were infected with control (null) virus sterol on 3-hydroxy-3-methylglutaryl–coenzyme A re- Ϫ Ϫ or recombinant adenovirus with the multiplicity of infection as described ductase.10 However, in Cyp7b1 / mice, in which plasma in Materials and Methods. Bile acid synthesis was measured as conver- levels of 25-hydroxycholesterol and 27-hydroxycholes- sion of 14C-labeled cholesterol into water-soluble products (14C-labeled methanol/water-extractable counts). Data are expressed as a percentage terol are increased, no major differences in cholesterol of controls (mean Ϯ SE). 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 undetectable and that only the acidic pathway of bile acid mouse. However, the absence of generalized suppression 19,32 synthesis is functional. A greater than 3-fold increase of sterol synthesis in the presence of the increased oxy- Ϯ Ͻ (221% 64%; P .02) in CYP7B1 specific activity was sterols, 25-hydroxycholesterol and 27-hydroxycholes- observed compared with paired controls (cells infected terol, is unclear. In a rare, naturally occurring mutation of with control virus). Despite this dramatic increase in CYP7B1 in a human neonate with cholestasis, no 7␣- CYP7B1 activity, bile acid synthesis rates were not signif- hydroxylated bile acids were formed.17 Acidic intermedi- Ϯ ϭ icantly changed (86% 15%; P NS) (Fig. 7). This is in ates in the bile acid biosynthetic pathway accumulated contrast to CYP7A1 overexpression in primary rat hepa- despite the CYP7A1 gene being normal. These findings tocytes, in which overexpression of CYP7A1 led to a suggest, at least in humans early in life, that CPY7B1 is Ͻ greater than 10-fold increase (P .001) in the rate of bile critical for formation of 7␣-hydroxylated bile acids. acid synthesis (Fig. 7). Overexpression of CYP7B1 in the The fact that both dexamethasone and L-thyroxine presence of L-thyroxine with dexamethasone (i.e., func- were found to be optimal culture conditions for CYP7B1 tional CYP7A1 and neutral bile acid synthesis pathway) in a manner similar to CYP7A1 is interesting and implies Ϯ also led to no increase in bile acid synthesis (116% similarities in the hormonal effects on CYP7B1 and ϭ 29%; P NS), whereas CYP7A1 overexpression led to a CYP7A1. L-thyroxine elicits little effect in primary rat ϭ Ͻ greater than 4-fold (n 3; P .001) increase in bile acid hepatocytes on CYP27, the initial step in the alternative synthesis. pathway of bile acid synthesis, with dexamethasone re- 4 Discussion quired for its optimal expression. In contrast, sterol 12␣-hydroxylase (CYP8B1) expression is dramatically The results of this study performed in primary rat suppressed by L-thyroxine, demonstrating a hormonal hepatocytes describe the effects of bile acids, cholesterol, role in the regulation of the ratio of cholic to chenodeoxy- hormones, cAMP, and PMA on the regulation of cholic acid.32 CYP7B1 mRNA and activity. The addition of L-thyrox- The ability of cholesterol to up-regulate CYP7B1 is ine plus dexamethasone increased CYP7B1 activity levels, also similar to CYP7A1. In support of this type of regu- with optimal concentrations of 1.0 ␮mol/L and 0.1 lation is the identification of an SRE-like site in the hu- ␮mol/L, respectively. These concentrations were similar man CYP7B1 promoter.1,34 Whether this represents a to those found to optimize primary rat hepatocyte culture true sterol regulatory site mediated through previously conditions for CYP7A1 expression.19 Furthermore, the defined pathways is uncertain. HEPATOLOGY, Vol. 35, No. 6, 2002 PANDAK ET AL. 1407

The original concept that feedback repression of bile findings following CYP7A1 overexpression, where a dra- acid biosynthesis was mediated solely through the regula- matic increase in bile acid synthesis was observed. In- tion of CYP7A1 is being modified. It has been shown in creased CYP27 expression in HepG2 cells also resulted in Cyp7a1Ϫ/Ϫ mice that bile acid synthesis continues to oc- a significant increase in bile acid synthesis (ϳ50%).31 cur, demonstrating the ability of at least one alternative Overexpression of CYP7B1 in combination with CYP27 pathway to 7␣-hydroxylate cholesterol and generate bile also did not increase bile acid synthesis any further than acids.22,35 Furthermore, CYP7B1 enzyme activity, pro- overexpression of CYP27 alone.31 These findings suggest tein, and mRNA levels in Cyp7a1Ϫ/Ϫ mice were induced. that CYP7B1, under normal physiologic conditions, is Although the relative importance of these alternative not the rate-determining step in the “acidic” pathway of pathways may be species and/or disease state dependent, bile acid biosynthesis. the question of their ability to be regulated, and by what, In summary, in the rat, CYP7B1, like CYP7A1, is a has begun to be explored.2,36-38 CYP27, the initial step in highly regulated enzyme. As with CYP7A1, these regula- the alternative (i.e., “acidic”) pathway, has now been tory effects seem to down-regulate CYP7B1 at the level of shown to be regulated.2 However, the regulation of the gene transcription. Interestingly, overexpression of subsequent enzymatic step in the pathway, the 7␣-hy- CYP7B1 did not increase bile acid synthesis as has been droxylation of the oxysterol, has been given little atten- previously observed with CYP7A1, suggesting that the tion. In rat liver, Toll et al. failed to see any stimulation of 7␣-hydroxylation of oxysterols is not a rate-limiting step 7␣-hydroxylation of oxysterol activity by feeding cho- in the alternative pathway of bile acid synthesis under lestyramine.39 In the mouse, Schwarz et al. also showed no physiologic conditions. effects of cholestyramine feeding on CYP7B1 mRNA ex- Acknowledgment: The authors thank Li Jun Zhao pression but found an approximately 50% decrease in and Pat Bohdan for technical assistance. CYP7B1 mRNA levels with cholate feeding.15,16 The results of this study in primary rat hepatocytes not only References show a suppression of CYP7B1 mRNA levels with bile 1. Chiang JYL. Regulation of bile acid synthesis. Front Biosci 1998; acids but suppression of CYP7B1 activity as well. These 3:D176-D193. findings have been corroborated in whole animals in 2. Vlahcevic ZR, Pandak WM, Stravitz RT. Regulation of bile acid which bile acid feeding repressed CYP7B1 mRNA and biosynthesis. Gastroenterol Clin North Am 1999;28:1-25, v. specific activity levels; conversely, CYP7B1 mRNA levels 3. Dahlback-Sjoberg H, Bjorkhem I, Princen HM. Selective inhibition of mitochondrial 27-hydroxylation of bile acid intermediates and specific activity levels were increased with chronic and 25-hydroxylation of vitamin D3 by cyclosporin A. Biochem J biliary diversion and cholestyramine feeding (under re- 1993;293:203-206. view). 4. 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