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J. Biol. Chem., Vol. 277, Issue 37, 34036-34041, September 13, 2002

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Human Sterol 27-Hydroxylase (CYP27) Overexpressor Transgenic Mouse Model

EVIDENCE AGAINST 27-HYDROXYCHOLESTEROL AS A CRITICAL REGULATOR OF CHOLESTEROL HOMEOSTASIS^*

Karen Meir^§, Daniel Kitsberg^§, Irit Alkalay^§, Fanny Szafer^§, Haim Rosen^¶, Shoshanna Shpitzen^§, Liat Ben Avi^§, Bart Staels, Catherine Fievet, Vardiella Meiner^**, Ingemar Björkhem, and Eran Leitersdorf^§§§¶¶

From the Departments of  Pathology, ^** Human Genetics, and ^§§ Medicine B and the ^§ Center for Research, Prevention, and Treatment of Atherosclerosis, Hadassah University Hospital, 91120 Jerusalem, Israel, the ^¶ Department of Molecular Virology, Faculty of Medicine, Hebrew University, 91120 Jerusalem, Israel, the  Department of Atherosclerosis, Institut Pasteur de Lille, 59000 Lille, France, and the  Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden

Received for publication, February 4, 2002, and in revised form, June 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sterol 27-hydroxylase (CYP27), a member of the cytochrome P450 superfamily, is primarily involved in bile acid biosynthesis (1). In the so-called classical or neutral pathway of bile acid synthesis initiated by cholesterol 7-hydroxylase (CYP7A1), CYP27 catalyzes a step in the degradation of the steroid side chain of a 7-hydroxylated intermediate. In an alternative pathway called the acidic pathway, CYP27 enzyme initiates the overall conversion of cholesterol into bile acids. The majority of the enzymes involved in bile acid biosynthesis are strictly expressed in the liver. However, the broad distribution of CYP27 in various tissues (2) is consistent with the possibility that this enzyme may have a role other than in bile acid biosynthesis. In vitro studies have shown the product of the enzyme, 27-hydroxycholesterol, to be a potent suppressor of cholesterol synthesis in cultured cells (3-6). Thus, 27-hydroxycholesterol may be an important factor in the regulation of cholesterol homeostasis. While the above in vitro results are in accordance with this hypothesis, the results of some in vivo studies have not supported it (7, 8). Since CYP27 has a broad distribution in various cells and tissues, conversion of cholesterol into 27-hydroxycholesterol and cholestenoic acid occurs not only in the liver but also in extrahepatic tissues (9). As a consequence, there is a continuous flux of 27-oxygenated cholesterol metabolites from extrahepatic organs to the liver where the metabolites are further metabolized into bile acids. Based on in vivo experiments in humans, it has been estimated that 5-10% of the total production of bile acids starts with extrahepatic 27-oxygenation of cholesterol (10, 11). Since human macrophages are able to utilize CYP27 for elimination of part of their cholesterol (12), this mechanism may have a preventive effect on the development of atherosclerosis. In accordance with this hypothesis, affected patients with the rare inherited disease cerebrotendinous xanthomatosis lack CYP27 and often develop premature atherosclerosis (13).

Previouly we developed and characterized mice lacking the cyp27 gene (14, 15). These mice were found to have a dramatic decrease in the rate of bile acid synthesis. As a consequence, there was compensatory up-regulation of CYP7A1 and of the rate-limiting enzyme in cholesterol synthesis, HMG¹-CoA reductase. Intestinal cholesterol absorption was drastically decreased (15). The mRNA for the cholesterogenic transcription factor sterol regulatory element-binding protein-2 was also increased (15). Despite these changes, the levels of cholesterol in the circulation were unaffected or only slightly affected by the lack of the cyp27 gene. Whether all of the above changes are secondary to the reduced formation of bile acids only or whether the lack of 27-hydroxycholesterol is of some importance is difficult to evaluate. In any case, the cyp27-deficient mice did not develop the symptoms found in patients with cerebrotendinous xanthomatosis, namely xanthomas, premature atherosclerosis, accumulation of cholestanol, and excretion of great amounts of bile alcohols (13). The metabolic situation in patients with cerebrotendinous xanthomatosis is complicated by the fact that the loss of CYP27 leads to one antiatherogenic effect (markedly increased degradation of cholesterol into bile alcohols) and one atherogenic effect (loss of the oxidative mechanism for removal of cholesterol from macrophages). The balance between these two effects may vary from patient to patient (9).

To further elucidate the influence of CYP27 and 27-hydroxycholesterol on the composition of bile acids, on cellular cholesterol homeostasis, and on extrahepatic cholesterol efflux as exemplified by the development of atherosclerosis, we sought to generate an animal model with marked changes in the levels of the 27-oxygenated steroids without a simultaneous marked change in bile acid biosynthesis. In the present work we describe the development and characterization of a transgenic mouse model with an overexpressed human CYP27.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Human CYP27 Overexpressor Transgenic Mice-- A 1.8-kb fragment encoding for the human CYP27 cDNA (a generous gift from Prof. D. Russell; Ref. 16) was inserted into the EcoRI restriction site in pCAGGS expression vector (kindly provided by Prof. J. Miyazaki; Ref. 17). pCAGGS, which contains the chicken -actin promoter and rabbit -globin poly(A) signal, permitted ubiquitous overexpression of the human CYP27 cDNA in all tissues. A linear purified DNA construct was microinjected into the pronuclei of fertilized mouse eggs taken from superovulated CBA females, and reimplanted into CBA × C57Bl/6J F₁ surrogate mothers. Positive transgenic mice were identified by tail DNA/PCR genotyping and by the presence of high fecal 27-hydroxycholesterol in comparison to C57Bl/6J control and cyp27 knock-out mice. Founder animals were crossed with C57Bl/6J mice (Harlan Laboratories, Jerusalem, Israel) for eight generations to create a congenic line (C57Bl/6J background >99.5%). The mice were fed a normal chow diet and housed in a sterile pathogen-free animal house under a 12-h light/dark regime. Twelve mice in the fourth backcrossed generation were fed a Western-type diet without sodium cholate (TD 88137, Harlan Teklad) for a period of 4 weeks. All experiments were performed on adult male and/or female transgenic mice obtained by backcrossing (lines 23 and 34) and on age- and sex-matched wild type C57Bl/6J animals.

Genotyping of Transgenic Mice-- Tail tip DNA was screened by PCR. Mouse tail tip was incubated overnight in 100 mM Tris, pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.2% SDS, and 100 µg of Proteinase K at 55 °C. DNA was extracted with phenol:chloroform:isoamyl alcohol (50:48:2) and precipitated with isopropanol.

PCR amplification was performed using primers common to the mouse endogenic and human transgenic CYP27 sequences. Amplification conditions were denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min repeated 35 times using 5'-CTC TAC CCT GTG GTC CCC ACA-3' as forward primer and 5'-AAC CAG GAC AAT GCG GGC CAC-3' as reverse primer. PCR products resulted in a 366-bp fragment for the human cDNA transgene sequence and a 598-bp band for the genomic mouse fragment.

Western Blot Analysis-- Total cellular extracts prepared from mouse tissues were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated for 2 h at room temperature in blocking buffer (5% bovine serum albumin in phosphate-buffered saline, 0.1% Tween) followed by incubation with anti-CYP27 antibody (a kind gift from Prof. D. Russell, University of Texas Southwestern Medical Center, Dallas, TX). Following washing with phosphate-buffered saline, 0.1% Tween, the membranes were incubated with peroxidase-conjugated protein A (Amersham Biosciences) and analyzed by the Amersham Biosciences enhanced chemiluminescence system (ECL).

Biochemical Analysis-- Animals were anesthetized with 0.5-1 ml of Avertin (tert-amyl alcohol, 2,2,2-tribromoethanol; Sigma) injected intraperitoneally. Following Avertin anesthetization, blood samples were collected from the inferior vena cava and transferred into tubes, and serum was collected and frozen in liquid nitrogen after centrifugation at 3000 × g for 5 min. Tissues were frozen in liquid nitrogen, pulverized mechanically, and extracted with chloroform:methanol (2:1, v/v). Oxysterols (7-hydroxycholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol) in serum and in various organs were analyzed by isotope dilution mass spectrometry with the use of deuterium-labeled internal standards as described previously (18). Serum levels of lathosterol, reflecting cholesterol synthesis, were also analyzed by isotope dilution mass spectrometry as described previously (19). Serum and lipoprotein lipids (cholesterol, triglycerides, and phospholipids) were determined by enzymatic assays adapted to microtiter plates using commercially available reagents (BioMerieux, Lyon, France) (cholesterol RTU, triglycerides PAP 1000, and phospholipids PAP 150). True triglycerides without glycerol interferences were measured enzymatically using a kit from Sigma Diagnostics. Fecal bile acids and neutral steroids were analyzed as described previously (14). Serum levels of apolipoprotein (apo) A-I, apoA-II, apoB, and apoC-III were measured by an immunonephelemetric assay using specific polyclonal antibodies (20).

Histology-- Organs were removed from transgenic CYP27^overexp mice and from their nontransgenic littermates (age- and gender-matched). These included the liver, kidneys, adrenals, spleen, pancreas, heart, lungs, and brain, all of which were removed and weighed. A skin and a muscle sample were also obtained from each mouse. Half of each tissue removed was fixed in 4% buffered formalin and embedded in paraffin for histological analysis. Five-micrometer-thick sections were cut and stained with hematoxylin and eosin.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of CYP27 Overexpressor Transgenic Mice-- Thirty-five pups were born from females implanted with fertilized eggs injected with the purified fragment of pCAGGS CYP27 expression construct (Fig. 1A). Five positive human CYP27 overexpressor transgenic mice were identified by tail DNA genotyping. CYP27 protein expression was examined in various tissues taken from these animals. Western blot analysis showed ubiquitous expression of human CYP27 in all organs from all five founders (not shown) as compared with control C57Bl/6J mice, which expressed the murine protein exclusively in the liver (Fig. 1B). Lines 23 and 34 were studied extensively. These animals were viable and fertile and exhibited no overt morbidity. Analysis of protein expression in the highest positive CYP27 founder transgenic mouse is shown in Fig. 1B. 27-Hydroxycholesterol content in feces of these five presumed founders was measured. In two of them, a 2-3-fold increase was observed when compared with nontransgenic control siblings (2.15-3.3 versus 0.89 µg/g of feces for the highest control value).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   A, linear pCAGGS human CYP27 expression construct. A 1.8-kb fragment encoding human CYP27 cDNA was inserted into the EcoRI restriction site in the pCAGGS plasmid. The pCAGGS vector, which contains the chicken -actin promoter and rabbit -globin poly(A) signal, permitted ubiquitous expression of human CYP27 in all tissues. B, specific overexpression of human CYP27 in various transgenic mouse organs. Total cellular extracts prepared from murine tissues were analyzed by Western blot using anti-CYP27 antibodies. C57Bl/6J, tissues from wild type (control mouse); Tg-23, tissues from the highest transgenic overexpressor founder mouse (line 23).

Line 23 (highest overexpressor) and line 34 (with lower overexpression) were crossed to C57Bl/6J mice and propagated to create congenic lines. While crossing the human CYP27 overexpressor transgenic mice, the 27-hydroxycholesterol concentration in mice serum and tissues was analyzed. Results from three to six mice, human CYP27 overexpressor and controls, from the third, fourth, and fifth backcrossed generations showed a 5-fold increase in concentration for line 34 and a 9-fold increase for line 23 (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Consistently elevated serum 27-hydroxycholesterol levels in backcrossed generations of CYP27 overexpressor mice. While crossing generations of CYP27 overexpressor transgenic mice, the 27-hydroxycholesterol concentration in serum (and tissues) was analyzed. Serum levels from three to six mice from backcross generations 3, 4, and 5 (F3, F4, and F5) showed 5-fold (line 34) and 9-fold (line 23) increases in 27-hydroxycholesterol concentrations when compared with non-overexpressor littermates. Tg, transgenic.

Genetically homogeneous mice (>99.5%) on a C57Bl/6J background were physically and biochemically characterized. Twelve-week-old (females and males) CYP27^overexp mice and control siblings fed a normal chow diet were sacrificed.

Physical Characterization-- No macroscopic differences in external or internal general appearance were observed between CYP27^overexp transgenic and control siblings. Total body weight and most internal organ weights were similar in transgenic CYP27^overexp and control mice. The adrenal glands of the overexpressor males were significantly larger than those of control males (0.2 ± 0.01 versus 0.1 ± 0.03 mg/g of total body weight, respectively; p = 0.001; see Table I).

Histological Characterization-- None of the sampled organs showed any gross morphological changes. No obvious histological differences were observed between the groups. Hematoxylin- and eosin-stained preparations did not reveal an excess of macrophages in the overexpressor mice lungs that might explain the high amounts of 27-hydroxycholesterol in that tissue.

Plasma Lipids and Lipoproteins-- Mice serum lipid content was analyzed in 10 transgenic CYP27^overexp mice and nine control littermates. Total cholesterol in serum was similar in both groups: 0.78 ± 0.09 (S.D.) mg/ml in transgenic mice and 0.78 ± 0.12 mg/ml in the control group. Similar values for total triglycerides, true triglycerides, and phospholipid concentration were found in both groups. Values of 1.06 ± 0.31 mg/ml for total triglycerides, 0.24 ± 0.14 mg/ml for true triglycerides, and 1.49 ± 0.18 mg/ml for phospholipid were measured in the transgenic group compared with 0.83 ± 0.17, 0.15 ± 0.05, and 1.52 ± 0.22 mg/ml, respectively, in controls (Fig. 3). Similarly, when males and females were compared separately, there were no significant differences between the values for any of the above parameters.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Serum lipid concentrations in CYP27 overexpressor transgenic mice. Total cholesterol, triglycerides, true triglycerides, and phospholipids were measured in the sera of 10 overexpressor mice and nine control littermates. Total cholesterol, 0.78 ± 0.03 mg/ml (overexpressor) versus 0.81 + 0.05 mg/ml (control), nonsignificant; total triglycerides; 1.06 ± 0.1 mg/ml (overexpressor) versus 0.83 ± 0.26 mg/ml (control), nonsignificant; true triglycerides, 0.24 ± 0.14 mg/ml (overexpressor) versus 0.15 ± 0.06 mg/ml (control), nonsignificant; phospholipids, 1.49 ± 0.18 mg/ml (overexpressor) versus 1.52 ± 0.22 mg/ml (control), nonsignificant. Values are expressed as mean ± S.D. (Student's t test). Tg, transgenic.

Measurement of apolipoprotein concentrations in serum from CYP27^overexp and control groups did not show any significant differences between groups. ApoA-I (CYP27^overexp, 0.38 ± 0.03 mg/ml; control, 0.37 ± 0.02 mg/ml), apoA-II (CYP27^overexp, 0.32 ± 0.02 mg/ml; control, 0.32 ± 0.02 mg/ml), apoB (CYP27^overexp, 0.13 ± 0.01 mg/ml; control, 0.12 ± 0.01 mg/ml), and apoC-III (CYP27^overexp, 0.32 ± 0.02 mg/ml; control, 0.30 ± 0.02 mg/ml) concentrations were similar when groups were composed of males and females combined. As males outnumbered females in both control and transgenic groups, comparisons of apolipoprotein concentrations between male groups only showed no significant differences (data not shown).

Levels of Lathosterol-- Serum levels of lathosterol did not differ between the two groups. Lathosterol levels were found to be 24 ± 6 ng/ml in the controls (n = 4) and 24 ± 5 ng/ml in CYP27^overexp mice (n = 3).

Pattern of Bile Acids in Bile-- Table II summarizes the results of the measurements of bile acids in bile of the CYP27^overexp animals and their controls. The relative concentration of cholic acid was slightly lower in bile of female CYP27^overexp than in that of their controls (p < 0.05), but this was not the case in the male overexpressors. The relative concentration of -muricholic acid in bile of female overexpressors was higher than in that of their controls (p < 0.05), but again this was not true of bile of males. The ratio between 12-hydroxylated bile acids and non-12-hydroxylated bile acids in bile was slightly lower in female CYP27 overexpressors than in controls, but this difference was not statistically significant (p > 0.05). In male mice the above ratio was almost identical in the two groups of animals (Table II).

Excretion of Neutral Steroids in Feces-- Neutral steroids were quantitated in pools of feces collected from four CYP27^overexp female mice during 3 days and from six female control mice during the same time period. A similar collection was performed for three male control mice and three male overexpressor mice. The amount of feces produced per day and animal was not different in the two cases. The amounts of cholesterol, coprostanol, and cholestanol excreted by the control female mice were slightly but significantly lower than in CYP27^overexp mice (0.93 ± 0.04 and 1.10 ± 0.01 mg/g of feces, respectively (p < 0.001)). In male mice, the difference between the amounts of the above neutral steroids excreted by overexpressors versus controls was not significant (1.1 ± 0.0 and 1.2 ± 0.1 mg/g of feces, respectively).

Levels of Oxysterols-- CYP27 ubiquitous overexpression was corroborated by the presence of high amounts of 27-hydroxycholesterol in serum, liver, and lungs of the CYP27^overexp group. 27-Hydroxycholesterol levels in serum were 2.7 times higher in CYP27^overexp mice than in their normal littermates (113 and 41 ng/ml, respectively). In the liver (122 versus 29 ng/100 mg of tissue) and lungs (125 versus 25.2 ng/100 mg of tissue) these levels were markedly increased (4.2 and 5 times higher, respectively). In general, the levels were higher in the female groups despite the fact that they had lower body weight than the males (see Table III for transgene status gender-specific results). Analysis of 24-hydroxycholesterol in the liver and lungs revealed no significant differences between overexpressors and controls. The serum levels of 24-hydroxycholesterol were, however, significantly lower in the transgenic animals. This effect was most accentuated in the female group. The selective increase of 27-hydroxycholesterol in combination with the normal levels of the other oxysterols confirm the specific overexpression of CYP27 by the ubiquitous promoter.

Effect of a High Fat Diet-- Seven CYP27^overexp mice and five control littermates (females and males from the fourth backcrossed generation) were challenged with a high fat diet for a period of 3 weeks. Overall average weight gain was similar in the two groups: 7.8 ± 0.79 g in CYP27^overexp and 8.8 ± 1.39 g in controls, although female transgenic mice demonstrated somewhat less weight gain compared with controls (6.2 versus 8.5 g, respectively). Serum analysis of the fourth backcrossed generation (CYP27^overexp × C57Bl/6J) showed constant 7-hydroxycholesterol concentration in both groups (Table IV). Histologically the high fat diet caused striking centrilobular microvesicular steatosis and mild periportal macrovesicular steatosis in the livers of all males regardless of CYP27 expression level. In female mice, periportal macrovesicular steatosis appeared more prominent than in males, but this too appeared unrelated to the transgene. The histological appearance of the lungs was normal and unaffected by high fat diet challenge.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Overexpression of CYP27 is likely to cause effects in the liver different from those in peripheral cells. In the liver, the overexpression is expected to lead to increased utilization of the acid pathway of bile acid biosynthesis both as a consequence of the increased amount of CYP27 in the liver and as a consequence of the greater flux of 27-oxygenated metabolites to the liver. It was recently reported by Hall et al. (21) that infection of HepG2 cells with adenovirus encoding CYP27 is associated with increased bile acid synthesis and increased levels of HMG-CoA reductase and low density lipoprotein receptor activity. The latter two effects may have been secondary to cholesterol depletion in hepatocytes as a consequence of rapid bile acid synthesis. If increased bile acid synthesis in the liver of the present mice with overexpressed CYP27 occurs, this increase does not reduce the levels of cholesterol in the liver or the rate of synthesis of cholesterol. A change in the rate of bile acid synthesis mediated by cholesterol 7-hydroxylase would be expected to lead to changed levels of 7-hydroxycholesterol in the circulation. No such changes were seen, however. If there had been a markedly increased utilization of the acid pathway with an overall increase in the total production of bile acids, the negative feedback mechanism for regulation of CYP7A1 would have been expected to lead to reduced activity of this enzyme and reduced levels of 7-hydroxycholesterol in the circulation.

Since 27-hydroxylated intermediates in bile acid biosynthesis seem to be less efficient substrates for the sterol 12-hydroxylase at least in rats (22), increased utilization of the acid pathway would be expected to lead to increased synthesis of chenodeoxycholic acid and decreased synthesis of cholic acid. A tendency to such an effect was observed in female overexpressed mice, whereas the overexpression had no effect at all on the composition of bile acids in bile of male mice. Whether the lack of effect of CYP27 overexpression on bile acid composition is due to a broad substrate specificity of sterol 12-hydroxylase or due to some other factor cannot be evaluated at the present state of knowledge.

With respect to the increased flux of 27-oxygenated cholesterol metabolites to the liver, a recent study by Souidi et al. (23) is of interest. When feeding high amounts of 27-hydroxycholesterol to hamsters, there was a marked parallel depression of CYP7A1 and HMG-CoA reductase without any effect on the cholesterol levels in the circulation. An effect on CYP7A1 in our animals would have resulted in a change in the levels of 7-hydroxycholesterol in the circulation. No such effect was seen, however.

In peripheral tissues the overexpression of CYP27 is expected to result in increased efflux of 27-oxygenated cholesterol metabolites from cells. In itself, such a flux is likely to cause a compensatory increase in cholesterol synthesis and an increased expression of the low density lipoprotein receptor. Since 27-hydroxycholesterol is an inhibitor of HMG-CoA reductase (3-6), the markedly increased levels of 27-hydroxycholesterol in the tissues may counteract the above stimulatory effect on cholesterol synthesis. Due to less extensive metabolism, the levels of 27-hydroxycholesterol in the peripheral cells would be expected to be higher than those in the liver. In accordance with this, overexpression of CYP27 led to a relatively higher increase in the levels of 27-hydroxycholesterol in the lung than in the liver of our mice. In this regard, an experiment by Hall et al. (21) is of interest. Overexpression of CYP27 in Chinese hamster ovary cells by use of the adenovirus vector approach resulted in decreased HMG-CoA reductase activity and increased expression of low density lipoprotein receptor activity.

Overexpression of a specific gene in a cell culture is more likely to cause significant effects than is general overexpression of the same gene in an intact animal. The latter situation is more likely to reflect the physiological importance of the gene product. On the other hand it is evident that there may be a number of mechanisms available in the intact animals that may balance or counteract any specific biochemical change.

An intriguing feature in male CYP27^overexp mice was an increased adrenal size as compared with control littermates. It remains to be shown whether this reflects a nonspecific finding with no physiological consequences or whether corticosterone content and adrenal sterol composition were also affected.

The results obtained with the present animal model are of particular interest in relation to the hypothesis that 27-hydroxycholesterol is an important regulator of cholesterol homeostasis (3-6). The normal levels of cholesterol and lathosterol despite the markedly increased levels of 27-hydroxycholesterol do not favor this hypothesis. Similar to the present animal model, mice with a deletion of the gene coding for cyp7b (oxysterol 7-hydroxylase) have been reported to have markedly increased levels of 27-hydroxycholesterol (24). On a normal diet, such mice have normal levels of cholesterol in the circulation, and with the exception of the male kidneys, the in vivo sterol biosynthetic rates are unaffected by the deletion. The results of the present investigation together with the results of the previous investigations with CYP7b- and CYP27-deficient mice show that 27-hydroxycholesterol is not likely to be of major importance in the regulation of cholesterol homeostasis. Based on a carefully performed in vitro study of levels of various oxysterols in subcellular fractions of livers of mice exposed to dietary cholesterol, Zhang et al. (25) also recently concluded that 27-hydroxycholesterol is unlikely to be an important regulator of cholesterol homeostasis at least not at a transcriptional level. In contrast to another oxysterol, 24,25-epoxycholesterol, there was no nuclear accumulation of 27-hydroxycholesterol after administration of labeled cholesterol. This finding does not support the contention that 27-hydroxycholesterol binds to a nuclear receptor. The possibility that 27-hydroxycholesterol may be a ligand for the nuclear receptors liver X receptor  and liver X receptor  has been discussed, and very recently experimental evidence that production of 27-hydroxycholesterol in cholesterol-loaded human cells may be a driving force in liver X receptor-mediated stimulation of ATP-binding cassette transporters has been presented (26). According to two other studies, however, 27-hydroxycholesterol is not an effective liver X receptor ligand (27, 28). Thus, three of the above four studies are consistent with our conclusion that CYP27 and 27-hydroxycholesterol are less important as general regulators of cholesterol homeostasis.

Regardless of whether formation and levels of 27-hydroxycholesterol appear to be of lesser importance in overall cholesterol homeostasis, CYP27 may be of utmost importance for cholesterol turnover in some specific cells or tissues. The best illustration of this is that patients with cerebrotendinous xanthomatosis lacking CYP27 may develop premature atherosclerosis despite normal levels of cholesterol in the circulation (13). Whether or not overexpression of CYP27 has a protective effect on development of atherosclerosis in our mouse model is now under investigation.

One unexpected effect of the overexpression of CYP27 in the mice was a marked reduction of the levels of the oxysterol (24S)-hydroxycholesterol. In humans most of this oxysterol originates in the brain (29, 30), whereas in mice a considerable fraction originates in extracerebral sources (31). Very recently evidence was presented that a glucuronidated and sulfated form of 5-cholestene-3,24(S)-diol is a major metabolite of (24S)-hydroxycholesterol in humans (32). If the situation is similar in mice, overexpression of CYP27 would be expected to lead to increased metabolism of (24S)-hydroxycholesterol with reduced levels of it in the circulation.

	ACKNOWLEDGEMENTS

pCAGGS expression vector was kindly provided by Dr. J. Miyazaki from the Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka, Japan. Human CYP27 cDNA, pCMV2-H26, and antibodies toward human CYP27 were kindly provided by Dr. D. W. Russell from the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX. The skillful technical assistance of Manfred Held and Anita Lövgren-Sandblom is gratefully acknowledged.

	FOOTNOTES

^* This research was supported by grants from the Israel Science Foundation (Grant 510/98-1 to E. L.), the Hurvitz Foundation, and the Swedish Medical Research Council and by the Swedish Heart and Lung Foundation (to I. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶¶ To whom correspondence should be addressed: Dept. of Medicine B, Center for Research, Prevention, and Treatment of Atherosclerosis, Hadassah University Hospital, 91120 Jerusalem, Israel. Tel.: 972-2-677-8029; Fax: 972-2-641-1136; E-mail: eranl@hadassah.org.il.

Published, JBC Papers in Press, July 15, 2002, DOI 10.1074/jbc.M201122200

	ABBREVIATIONS

The abbreviations used are: HMG, 3-hydroxy-3-methylglutaryl; apo, apolipoprotein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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