• Antley–Bixler syndrome;
  • bile acid homeostasis;
  • cholesterol;
  • CYP;
  • cytochrome P450;
  • knockout;
  • lipid homeostasis;
  • mouse models;
  • P450 reductase


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley–Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1−/− mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.


Antley–Bixler syndrome


Alzheimer’s disease


bile acid


cholic acid


chenodeoxycholic acid


cerebrotendinous xanthomatosis


cytochrome P450


sterol 27-hydroxylase


oxysterol 7α-hydroxylase II


cholesterol 24-hydroxylase


lanosterol 14α-demethylase


cholesterol 7α-hydroxylase


oxysterol 7α-hydroxylase


sterol 12α-hydroxylase




high-density lipoprotein


low-density lipoprotein


Online Mendelian Inheritance in Man


cytochrome P450 reductase


single nucleotide polymorphism


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

Biochemically, cholesterol plays a crucial role in eukaryotic cell growth and development. Biosynthesis of cholesterol presents the foundation for normal membrane synthesis and the regulation of membrane fluidity. Cholesterol is also an important precursor for the synthesis of biomolecules necessary for normal cell function. Furthemore, cholesterol provides building blocks for the synthesis of vitamin D, bile acids (BAs) and steroid hormones, all of which play important roles in maintaining the integrity of the organism.

Cholesterol can be obtained either from the diet or it can be synthesized de novo from acetyl coenzyme A. These two pathways are interdependent, meaning that the dietary intake and cellular requirements influence cholesterol biosynthesis in a complex feedback manner. However, daily needs can be met equally well from one or the other [1]. Cholesterol homeostasis is also maintained through its elimination pathways. The major disposal route in mammals is conversion into BAs, which are ultimately excreted from the body. As a result of their physicochemical properties, BAs stimulate biliary lipid secretion and enable the solubilization and absorption of dietary lipids and lipid soluble vitamins in the intestine [2].

In these intertwined mechanisms of cholesterol homeostasis, cytochrome P450s (CYPs) play crucial roles. Lanosterol 14α-demethylase (CYP51) is the only CYP involved in cholesterol synthesis [3]. It removes the 14α-methyl group from lanosterol and 24,25-dihydrolanosterol to produce the intermediate follicular fluid-meiosis-activating sterol [4]. Additional CYPs take part in the catabolism of cholesterol to BAs in two major pathways: the classic (‘neutral’) and the alternative (‘acidic’). The classic pathway in humans leads to two primary BAs: cholic acid (CA) and chenodeoxycholic acid (CDCA). Synthesis begins with the formation of 7α-hydroxycholesterol by cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme of the classic pathway. Several enzymatic steps follow, including sterol 12α-hydroxylation by sterol 12α-hydroxylase (CYP8B1) that directs the synthesis to CA, and sterol 27-hydroxylation by sterol 27-hydroxylase (CYP27A1). In the alternative pathway, the side-chain oxidation of cholesterol precedes the steroid ring modifications. The first step involves the oxidation of cholesterol to 27-hydroxycholesterol by CYP27A1, which is subsequently hydroxylated by oxysterol 7α-hydroxylase (CYP7B1). This pathway leads mainly to the formation of CDCA in humans [2]. Under normal conditions, the alternative pathway salvages ∼ 10% of daily BA losses [5], although it can become the major pathway when CYP7A1 activity is deficient [6,7].

The third possibility for BA synthesis is through oxidation of cholesterol to 24(S)- and 25-hydroxycholesterol derivatives. Cholesterol 24-hydroxylase (CYP46A1) is expressed mainly in the brain [8]. After 24(S)-hydroxycholesterol passes the blood–brain barrier, it is 7α-hydroxylated by oxysterol 7α-hydroxylase II (CYP39A1) in the liver. Although the contribution of this route to the overall BA synthesis is minor, 24(S)-hydroxycholesterol and other oxysterols are potent regulators of lipid homeostasis via activation of the liver X receptor [9,10].

The present review discusses CYPs that participate in cholesterol and BA synthesis. Figure 1 shows the classic and the alternative BA synthesis pathways, together with the chemical structures and names of the CYPs involved. A special focus of the present review is placed on transgenic mouse models as an invaluable in vivo system for understanding human diseases caused by deficiencies in these pathways. Table 1 shows the genotype–phenotype correlations of the cholesterol and BA synthesis CYP genes in humans and the corresponding phenotypes of the mouse knockout models.


Figure 1.  Schematic representation of cholesterol and BA biosynthesis pathways. Only steps involving CYPs are emphasized. There is a single CYP in cholesterol biosynthesis. In the classic pathway, cholic and chenodeoxycholic acid, two primary BAs in humans, are formed. In the alternative pathway, mainly chenodeoxycholic acid is formed. It usually accounts for 5–10% of daily BA losses, although it can become a major BA synthesis pathway when the classic pathway is disrupted. It also produces oxysterols, which are potent regulators of lipid homeostasis in vitro.

Download figure to PowerPoint

Table 1.   A summary of the CYPs involved in cholesterol and BA synthesis showing their association with disease and mouse knockout models.
EnzymeGeneLocationDisease (OMIM database)PolymorphismPhenotypePhenotype of corresponding knockout mouse model
Lanosterol 14α-demethylaseCYP51A17q21.2 Exon 1 missense mutation (rs2229188)Positive correlation with HDL-cholesterol [39]Embrionally lethal (D15) with similarities to human ABS [23]
Cholesterol 7α-hydroxylaseCYP7A18q11–q12 1302–1303delTTHigh level of plasma LDL-cholesterol, increased hepatic cholesterol content and a deficient rate of BA excretion [55]Increased rate of postnatal death, fat malabsorption, wasting, skin abnormalities and vision problems [42] Hypercholesterolemia [46]
Promoter A-204C (rs3808607)Increased LDL-cholesterol level, gallstone disease, hypertriglyceridemia, hypercholesterolemia, and risk for arteriosclerosis, colorectal cancer, neuromyelitis optica [56–63]
Sterol 12α-hydroxylaseCYP8B13p22–p21.3   Lack of CA, fat malabsorption [64]
Sterol 27-hydroxylaseCYP27A12q35Cerebrotendinous xanthomatosis (213700) [77,90–93]  Reduced BA pool size [81] Hypertriglyceridemia and enlargement of the liver and adrenal glands [87] Accumulation of cholestanol in the brain and tendons [86]
Oxysterol 7α-hydroxylaseCYP7B18q21.3Congenital BA synthesis defect type 3 (613812) [99,109]  No gross phenotype, increased levels of 25- and 27-hydroxycholesterol in plasma, liver and kidneys [97]
Spastic paraplegia autosomal recessive type 5A (270800) [110,111]  
Cholesterol 24-hydroxylaseCYP46A114q32.2 Intron 2 SNP (T/C) (rs754203)Late onset sporadic AD, mild cognitive impairment [122–126]40% reduction in brain cholesterol excretion [115] Impaired learning abilities [113]
Intron 3 SNP (T/C) (rs3742376)AD [126,127]
Promoter region SNP (A/G) (rs7157609)AD [128]
Intron 3 SNP (C/T) (rs4900442)AD [128]
Oxysterol 7α-hydroxylase IICYP39A16p21.1–p11.2    

CYP51 from cholesterol synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP51 is the most evolutionarily conserved member of the CYP superfamily [11]. It has been characterized in several species, including humans and mice. Even if it is expressed ubiquitously, it appears to have tissue-specific roles [12–16]. In the liver, the gene is regulated by the sterol regulatory element-binding protein and by cyclic adenosine monophosphate signaling pathways [17–19], as well as in a circadian manner [20,21]. CYP51 is a microsomal enzyme that requires CYP reductase (POR) as an ubiquitous redox partner. The enzymatic activity (i.e. removal of the methyl group at position C14 of the sterol ring) is demethylation of lanosterol or 24,25-dihydrolanosterol by three consecutive monooxygenation reactions [4,22].

The Cyp51 knockout is embryonically lethal

We have recently developed a Cyp51 mouse knockout model that exhibited several prenatal Antley–Bixler syndrome (ABS)-like symptoms, leading to lethality at embryonic day 15 [23]. Homozygous Cyp51−/− mice do not express functional Cyp51 mRNA or CYP51 protein, leading to an accumulation of the sterol precursors lanosterol and 24,25-dihydrolanosterol. Lethality was ascribed to heart failure as a result of a variety of heart defects. As noted previously, mutations of the human CYP51A1 gene are, at present, not linked to ABS. However, the developed Cyp51 mouse knockout model encourages the reconsideration of a possible functional link between CYP51A1 defects and malformations in humans. Several developmental defects of Cyp51 knockout mice, especially skeletal and heart abnormalities, resemble the ABS-like phenotype of humans. Porter and Herman [24] reported heart ventricular defects in some ABS patients, a similar phenotype as that observed in the Cyp51 knockout mice. Syndactyly is also observed in ABS patients, whereas polydactyly resulted from the teratogenic effects of azole antimicotics. We postulate that the hypoplastic heart development in Cyp51 knockout embryos might correspond to the inhibition of CYP51 activity in a manner similar to that reported for the azoles [21,25].

The mouse knockout of Por, the obligatory redox partner of CYP51 and other microsomal CYPs

CYP reductase is a ubiquitously expressed enzyme that serves as electron donor to all endoplasmatic reticulum CYPs. Because of its general role in supporting electron transfer in microsomal monooxygenation reactions, the phenotypes of Por knockout mice were expected to be severe and pleiotropic. The two described Por knockouts [26,27] are both embrionally lethal at embryonic days 9.5–10.5. In one case [26], the deletion of one allele (Por heterozygotes) also resulted in some embryonic lethality. Cardiac development and vascularization were majorly impaired and linked to defects in retinoic acid metabolism [27]. Heterozygous mice in a study by Otto et al. [27] were apparently normal, whereas Shen et al. [26] reported some embryonic lethality. This indicates a possible influence of the mouse genetic background on the observed phenotypes. Because POR mutations in humans associate with ABS and limb and skeletal abnormalities (see below), a conditional Por knockout has been developed specifically in mouse limb bud mesenchyme [28]. Transcriptional analysis of embryonic day 12.5 mouse forelimb buds demonstrated up-regulation of the entire cholesterol biosynthesis pathway, where cholesterol deficiency may explain many aspects of the phenotype. It was thus concluded that cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development.

The importance of POR for cholesterol and BA homeostasis has been demonstrated by two independent liver conditional Por deletions [29,30]. Both models show hepatomegaly and modified cholesterol and BA metabolism. Changes in the expression of key enzymes from cholesterol and lipid homeostasis were observed, such as reduced expression of Cyp7b1, as well as elevated expression of Cyp7a1 and Cyp8b1. This provided important insights into the control of metabolic pathways by the CYP system [31].

POR mutations in humans lead to ABS

ABS is a rare complex syndrome with pleiotropic body malformations. Although a modified sterol profile in the fluconazole-exposed patient suggested a deficiency of CYP51, mutational analysis of the CYP51A1 gene showed no obvious pathogenic mutations in exons or exon–intron boundaries [32]. Subsequently, it was revealed that the cause of the abnormal sterol profile and genital malformations lies in abnormalities of the POR gene [33]. POR is located on human chromosome 7q11.2 and has 16 exons, where exons 2–16 encode a 677 amino acid POR protein [34]. There is a single copy of the 50-kb POR gene in humans.

Another gene associated with ABS is the fibroblast growth factor receptor 2 gene, although several cases of the disease remain unexplained. The current assumption is that ABS with abnormal genitalia and/or impaired steroidogenesis is a result of POR mutations, and that the variable clinical features can be explained primarily by impaired activities of POR-dependent CYPs [35]. Inhibition of CYP51 activity as a result of mutations of POR has important implications because sterols play a regulatory role in the embryonic activation of hedgehog proteins [36]. It is thus plausible that some of the ABS patients might have functional mutations in CYP51A1. Similar to POR, the CYP51A1 gene also lies on chromosome 7 (7q21.2). It spans 22 kb, and contains ten exons and a housekeeping promoter with several transcription start sites [37]. Two polymorphisms of CYP51A1 have been described so far in unrelated studies. A C/T transition in intron 2 of CYP51A1 was discovered in a study of the association of leptin with blood pressure, although the rs6 appears to be uninformative [38]. On the other hand, a C/T change in exon 1 causes a missense mutation transforming Val to Ala [39]. Here, CYP51A1 has been placed among 14 genes where the genotypes are known to be important for the high-density lipoprotein (HDL)-cholesterol phenotype.


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP7A1 is a microsomal enzyme that catalyzes the first step in the neutral pathway of BA synthesis and is expressed only in the liver [40]. Conversion of cholesterol to 7α-hydroxycholesterol is highly regulated and a rate-limiting step in the synthesis of CA and CDCA. In general, expression of the CYP7A1 gene is increased when the BA pool size is reduced and decreased when excess BAs are present in the diet [41].

Cyp7a1 knockout mice

Disruption of the Cyp7a1 gene in mice reveals a complex phenotype, emphasizing the importance of adequate BA synthesis in early murine life [42]. Cyp7a1-null mice are subjected to an increased rate of postnatal death, fat malabsorption, wasting, skin abnormalities and vision problems. An absence of BAs in newborn animals combined with fat-soluble vitamin deficiency is considered to be the most likely explanation for this phenotype [43]. However, a small proportion of mice that do survive this early postnatal period experience a total regression of the abnormalities and are essentially indistinguishable from their wild-type littermates in later life. It was shown that, at ∼ 3 weeks of age, the alternative pathway of BA synthesis becomes active in mice and is able to compensate for the loss of Cyp7a1 [43]. Unexpectedly, serum levels of cholesterol and triglycerides are normal in Cyp7a1 knockout mice, suggesting that other regulatory mechanisms are able to effectively maintain lipid homeostasis. Further research on Cyp7a1-deficient mice [44] showed that cholesterol absorption is reduced to undetectable levels as a result of a reduced BA pool size; however, the source of sterols is replaced by an increased de novo synthesis in the liver and intestine. Interestingly, alternative pathways of BA synthesis are not up-regulated in Cyp7a1-null mice, and they remain unresponsive to marked increases in the enterohepatic flux of cholesterol or BAs [45].

By contrast to the studies described above, another study on Cyp7a1-deficient mice showed a milder phenotype with a much lower mortality rate but with elevated levels of serum cholesterol or hypercholesterolemia [46]. The observed increase in the expression of Cyp27a1 is suggestive of a secondary induction of the acidic pathway of BA synthesis. There is also no increase in hepatic de novo cholesterol synthesis in these mice.

The discrepancies in the studies noted above were ultimately ascribed to differences in the genetic background or the environment of the mice [46]. Of note, the latter Cyp7a1 knockout mouse model is more representative of the human CYP7A1 deficiency, as will be described subsequently.

Overexpression of CYP7A1 in mouse models

To further elucidate the role of Cyp7a1 in vivo, mice that overexpress human CYP7A1 were generated. These mouse models provide valuable information on cholesterol and BA homeostasis, as well as an informative approach for understanding human diseases.

One of the first studies investigating the overexpression of CYP7A1 was performed in LDLr-deficient mice [47], which suffer from overtly elevated serum low-density lipoprotein (LDL) concentrations [48]. Interventions that accelerate the conversion of cholesterol to BAs have been widely used to reduce plasma LDL concentrations mainly through the mechanism of increased expression of the LDL receptor pathway [49]. Interestingly, even in the absence of LDL receptors, expression of CYP7A1 in mice results in a lowering of plasma LDL concentrations [47], an effect that has been ascribed to a decrease in the rate of LDL entry into the plasma space. Use of high-fat cholesterol-enriched diet in this type of mice showed a marked resistance to diet-induced hypercholesterolemia [50].

In studies on normal mice with a stable expression of CYP7A1 [51], it was shown that the production of apolipoprotein B-containing lipoproteins is increased. However, plasma levels of triglycerides and cholesterol remain unchanged as a result of the counter-effect of an increased expression of LDL receptors. This type of transgenic mice is also protected from atherosclerosis and gallstone formation when fed an atherogenic diet rich in BAs because there is no possibility of dietary suppression of CYP7A1. In this case, cholesterol homeostasis is effectively maintained, which makes CYP7A1-overexpressing mice resistant to diet-induced dyslipidemia [52]. Moreover, in a recent study, the overexpression of CYP7A1 in mice prevented high-fat diet-induced obesity, fatty liver and insulin resistance [53]. Most likely, enhanced enzyme activity and the activation of cellular BA signaling as a result of an expanded BA pool size both contribute to this phenomenon [54].

CYP7A1 defects in humans

In humans, CYP7A1 lies on chromosome 8q11–q12 and spans almost 10 kb, containing five introns and six exons, which encode a 504 amino acid protein. Despite the importance of CYP7A1, its absence is not lethal in humans; however, deficiencies are linked to a hyper-cholesterolemic phenotype.

A 2-bp deletion in exon 6 (1302–1303delTT; numbering of nucleotides in the cDNA from the transcription start site) in the CYP7A1 gene results in a frameshift: L413fsX414. This mutation causes a Leu[RIGHTWARDS ARROW]Arg substitution at codon 413 followed immediately by a premature stop codon, resulting in a truncated protein lacking the C-terminal 91 residues with a loss of the heme-binding domain, which is essential for activity. In this case, the loss of activity resulted in high level of plasma LDL-cholesterol, doubled hepatic cholesterol content and a markedly deficient rate of BA excretion. In addition, the alternative BA pathway was up-regulated in this patient [55].

Genetic variations in the CYP7A1 gene associated with disorders of cholesterol and BA metabolism have been studied extensively in different laboratories. Most studies have focused on a single nucleotide polymorphism (SNP) in the promoter region of the CYP7A1 gene (rs3808607). The association of an A/C transversion polymorphism (−278 from the translation initiation or −204 from the transcriptional start site) with plasma lipid levels, hypertriglyceridemia, hypercholesterolemia, the risk of arteriosclerosis, gallstone disease and colorectal cancer has been studied in adults and children in Caucasian and Asian populations, with conflicting results being reported [56]. This polymorphism was initially associated with LDL-cholesterol levels; namely, plasma LDL-cholesterol concentrations were significantly higher in –278C homozygotes than in –278A homozygotes [57].

Further studies confirmed that plasma total cholesterol and LDL-cholesterol were lower in patients with an A containing-allele (AA homozygote or AC heterozygote) than in CC homozygotes, although the A allele might be considered as a risk factor for gallstone disease [58]. A allele carriers have shown a better response to atorvastatin treatment in terms of total cholesterol and a LDL-cholesterol-lowering effect [59]. Studies investigating the response to plant sterols in humans suggest that the promoter A-204C variant is associated with enhanced CYP7A1 enzyme activity. Increased intestinal BA levels, which lead to more efficient cholesterol absorption, may explain why C allele carriers (AC heterozygotes and CC homozygotes) show enhanced cholesterol-lowering and increased feedback cholesterol synthesis in response to an intervention by plant sterols [60].

The CC variant of the CYP7A1 promoter polymorphism increases the progression of atherosclerosis and possibly the risk of a new clinical event [61]. On the other hand, a colon cancer study reported a lower risk for the CYP7A1 CC genotype with respect to proximal colon cancer, although not for distal colon or rectal cancer [62]. The CC genotype of this common CYP7A1 promoter polymorphism was also associated with a higher protective effect regarding the risk of neuromyelitis optica, which is a severe idiopathic inflammatory disease of the central nervous system primarily affecting the optic nerves and spinal cord [63].


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP8B1 is a microsomal CYP that catalyzes the addition of hydroxyl group at position 12 of various sterol intermediates destined to become CA in the BA synthesis pathways [64]. Activity of CYP8B1 thus effectively determines the ratio of CDCA to CA formed in the neutral pathway [2].

Cyp8b1 knockout mice

As expected, the disruption of the Cyp8b1 gene in mice results in a complete lack of CA, which is replaced mostly by muricholates (muricholates are considered to be metabolites of CDCA) [65] and CDCA [64]. In mice, β-muricholic acid and CA are the dominating BAs as opposed to CDCA and CA in humans. Cyp8b1-null mice also have an enlarged BA pool size as a consequence of the up-regulation of Cyp7a1. However, the absorption of dietary cholesterol and lipids is reduced (steatorrhea), suggesting that the composition of BA pool is more important than its size. Loss in cholesterol absorption is compensated with increased de novo synthesis by the liver, resulting in unchanged serum levels of cholesterol and triglycerides. There is also no change in expression of Cyp7b1, Cyp27a1 and Cyp39a1. The addition of CA to the diet suppresses Cyp7a1 and several cholesterogenic genes in Cyp8b1-null mice. At the same time, the intestinal cholesterol absorption is normalized, suggesting an important regulative function of this BA in lipid homeostasis [66,67].

The absence of CA synthesis was also studied in apolipoprotein E knockout mice, a common animal model for atherosclerosis [68]. Given the fact that CA is involved in multiple sites of cholesterol homeostasis, such as in promoting cholesterol absorption and inhibiting BA synthesis, the lack of CA might prove beneficial against cholesterol-driven diseases. In general, the phenotype of Cyp8b1 gene ablation is preserved in ApoE and Cyp8b1 double knockout mice, resulting in a 50% reduction of atherosclerotic lesions compared to ApoE knockout mice [69].

Cholic acid as a key molecule for effective cholesterol absorption

The aforementioned studies established that CA is a key factor in the formation of micelles (together with phospholipids) as a means to achieve an efficient absorption of cholesterol. In mice, CA also functions as a signaling molecule that down-regulates the expression of Cyp7a1, as well as Cyp8b1 [66]. The combined effects of CA have a great impact on maintaining optimal cholesterol balance, which is probably one of the reasons why the expression of Cyp8b1 is so highly regulated [70,71]. The therapeutic approach of decreasing CA synthesis appears to represent a promising strategy for atherosclerosis susceptible patients. Unfortunately, it is difficult to anticipate all the effects of such therapeutic intervention because, in humans, CDCA instead of CA appears to be the primary ligand for farnesoid X receptor, through which BAs act as signaling molecules [72]. Nevertheless, the inhibition of Cyp8b1 might also be considered for treating gallstone disease because gallstone formation can be averted with depletion of CA [67,69].

Defects of the CYP8B1 gene in humans

The CYP8B1 gene was mapped to chromosome 3p22–p21.3; it spans 3949 bp encoding 504 amino acids protein and lacks introns. The human CYP8B1 has a broad specificity towards steroids; however, its main activity is the conversion of 7α-hydroxy-4-cholesten-3-one into 7α,12α-dihydroxy-4-cholesten-3-one. Thus, CYP8B1 activity determines the primary BA composition [73]. In human bile, CA and CDCA, representing two major primary BAs, occur in a molar ratio of ∼ 2 : 1 [74].

The loss of activity of CYP8B1 leads to the production of CDCA over CA, which results in the modification of BA biosynthesis and changes in the excretion of cholesterol [75]. Polymorphism in the coding part of the CYP8B1 gene does not explain the marked differences in the ratio of CA and CDCA in human bile [76]. To the best of our knowledge, polymorphisms in CYP8B1 have not yet been associated with any disease.


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP27A1 is located on the inner membranes of the mitochondria and is expressed in almost all cells of the body [77]. It catalyzes the first step in the alternative BA synthesis pathway, and also plays a significant role in the classic synthesis pathway, where it catalyzes the 27-hydroxylation of BA intermediates. Approximately 25–30% of all synthesized BAs come from the alternative pathway in mice and rats; however, in humans, it accounts for only 5–10% of the daily losses [40]. Because of the universal expression of CYP27A1 and the general presence of cholesterol throughout the body, minor amounts of 27-hydroxycholesterol and cholestenoic acid are formed in the extrahepatic tissues, and their flux to the liver could be regarded as an alternative mechanism to the common HDL-dependent reversed cholesterol transport [78]. 27-Hydroxycholesterol was also found to be a strong inhibitor of cholesterol synthesis in vitro [79]. A deficiency of CYP27A1 in humans is associated with a disease called cerebrotendinous xanthomatosis (CTX), which is characterized by dementia, ataxia, cataracts and xanthomas of the tendons and the nervous system [80].

Cyp27a1 knockout in mice

Mice with a disrupted Cyp27a1 gene are normal in appearance with no gross histological abnormalities. As expected, the total amount of BAs is severely reduced, with CA predominating in the remaining BA pool size [81]. Interestingly, BA synthesis is not completely disrupted, as might be expected, because Cyp27a1 is involved in both the neutral and acidic pathways of the BA synthesis. Low levels of BA synthesis are maintained through the induction of microsomal 25-hydroxylase pathway [82]; however, compensatory synthesis via bile alcohols as intermediates is unable to fully compensate for the absence of Cyp27a1. Because of the decreased BA pool size, there is a compensatory up-regulation of Cyp7a1 as a result of a lack of negative feedback suppression. Low levels of BAs also result in a decreased cholesterol absorption, which leads to a secondary increase of de novo synthesis to maintain normal serum cholesterol levels. As opposed to humans, in Cyp27-null mice, there is no hepatic or bloodstream accumulation of cholestanol, a substrate formed mainly from the neutral pathway intermediate 7α-hydroxy-4-cholesten-3-one [83]. Although the intermediates in the early steps of the classic BA synthesis and 25-hydroxylated bile alcohols are increased in Cyp27 knockout mice compared to wild-type mice, the levels are much lower than in CTX patients [83]. The explanation of these differences is probably a result of the Cyp7a1 pathway being much less up-regulated in conjunction with the marked increase of CYP3A11 enzyme in mice [83,84]. CYP3A was shown to be capable of carrying out 25- and other side chain hydroxylations on various BA intermediates in mice [83], as well as in humans [85]. In CTX patients, however, CYP3A4 is not induced. A more recent study identified a marked accumulation of cholestanol in the brain and tendons of Cyp27a1 knockout mice [86]. Despite the fact that the contents of cholestanol can reach as much as 10% of the sterol fraction in the brain, no xanthomas (i.e. the most specific feature of CTX) are formed as a consequence.

Further investigation of CYP27A1 deficiency in mice identified hypertriglyceridemia and a significant enlargement of the liver and adrenal glands [87]. Hepatic fatty acid synthesis was also found to be increased, indicating a more global role of Cyp27a1 in the whole body lipid metabolism than previously assumed. Because of a decrease in cholesterol absorption, a compensatory up-regulation of cholesterol synthesis can be seen in various organs, especially the liver and adrenal glands. Histological observations indicated a cortical cell hypertrophy and, presumably, the increase in adrenal cholesterol synthesis and content is necessary to maintain normal levels of corticosterone [87].

Several differences can also be observed between homozygous and heterozygous Cyp27a1 knockout mice. The phenotype of the heterozygotes is generally milder compared to the homozygotes, and there are two distinct features between them. First, heterozygous mice have increased plasma cholesterol compared to homozygotes, indicating that a partial deficiency of Cyp27a1 may be a greater risk factor for atherosclerosis than its complete absence. Second, heterozygotes show no increase in Cyp7a1 expression and could thus represent a model of Cyp27a1 deficiency that is uncomplicated by the effects of a secondary up-regulation of the CYP7A1 pathway [88].

Overexpression of CYP27A1 in mice

To gain further insights into the physiological role of CYP27A1 in regulating cholesterol metabolism, transgenic mice overexpressing the human CYP27A1 gene were generated [89]. The expected increased output from the acidic pathway of BA synthesis does not manifest in these mice. Also, there are no changes in cholesterol metabolism; an observation that does not favor the hypothesis of 27-hydroxycholesterol being a potent signaling molecule in vivo.

Defects of CYP27A1 in humans

In humans, CYP27A1 was mapped to the long arm of chromosome locus 2q35 and spans almost 33.5 kb, containing nine exons and eight introns that encode for 531 amino acids of the protein, which consists of a 498 amino acid mature enzyme and a 33 amino acid mitochondrial signal sequence. Subsequent to 1991, when CYP27A1 was reported to be associated with CTX [Online Mendelian Inheritance in Man (OMIM) database: 606530] [90], the gene was (and still is) under investigation. To date, over 50 mutations have been reported to be associated with CTX, which is a rare autosomal recessive disease characterized by an accumulation of cholesterol and cholestanol in the brain and tendons. Although the disorder is rare, its incidence is substantially greater than previously recognized. A greater awareness of CTX is important because specific treatments are available.

The first patient with CTX was reported in 1937 [91]. The patient suffered from dementia, ataxia, cataracts and xanthomas of the tendons and the brain. Subsequently, several hundred patients have been diagnosed, with clusters reported from Japan, Israel and the Netherlands [92]. In general, CTX patients have normal levels of cholesterol in the blood but increased levels in tissues. By contrast to the xanthomas in patients with familiar hypercholesterolemia, xanthomas from CTX patients contain up to 30% of cholestanol, the 5α-saturated analogue of cholesterol. The typical onset of the disease consists of early bilateral cataracts and diarrhea, followed by cerebellar and pyramidal signs, mental retardation and xanthomas. The most serious consequence of the disease is the development of xanthomas in the brain and consequent neurological symptoms. The preferential site of brain xanthomas is the white matter of the cerebellum [77].

Various mutations in all nine exons and introns 2, 4, 6 and 7 have been associated with CTX. Approximately 50% of mutations in the CYP27A1 gene were found in the region of exons 6–8 and 16% and 14% in regions of exons 2 and 4, respectively. Deletion, insertion and nonsense mutations cause a truncation of CYP27A1 mRNA, resulting in the lack of heme-binding and adrenodoxin-binding sites. Splice site mutations lead to aberrant splicing and exon skipping. Missense mutations could influence the stability and catalytic activity of the enzyme. These mutations are inferred to be pathogenic when they disrupt heme-binding and adrenodoxin-binding domains, or when they identify a potential substrate binding or other protein contact site. Mutation analysis showed that 45% of variations are missense mutations; 20% are reported as nonsense mutations; and splice site mutations, deletions and insertions constitute 18%, 14% and 2% of reported mutations, respectively [93].


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP7B1 is presumably located in the endoplasmic reticulum and is present in many organs, with the highest expression in the brain and kidneys. In mice, the expression of Cyp7b1 is restricted mainly to the liver and lungs, although it can also be found in the kidneys, brain and reproductive tract [94]. It acts as an inactivating enzyme for 25- and 27-hydroxycholesterol, producing 7α-hydroxylated oxysterols, which are substrates for BA synthesis. CYP7B1 is also active towards steroid hormones and two studies using knockout mouse models have been reported [95,96].

Cyp7b1 knockout mice

Interestingly, knocking out Cyp7b1 in mice does not have an effect on the levels of plasma cholesterol and triglycerides, tissue cholesterol, composition and BA pool size, and intestinal cholesterol absorption [97]. The loss of the alternative BA synthetic pathway is presumably fully compensated by the CYP7A1 pathway, as suggested by the elevated CYP7A1 protein levels in knockout mice. On the other hand, levels of 25- and 27-hydroxycholesterol in the plasma, liver and kidneys are markedly increased. Unexpectedly, the accumulation of these oxysterols does not affect cholesterol synthesis in contrast to their described regulatory effects on cholesterol homeostasis in vitro [9,10]. Apparently, the major physiological role of CYP7B1 is to inactivate oxysterols as a means to produce intermediates in the BA synthesis pathway [94]. It is likely to have an important role in steroid metabolism and the regulation of lipid metabolism [98].

Physiological functions of CYP7B1 in humans

The human CYP7B1 gene spans ∼ 220 kb on chromosome 8q21.3 and encompasses six exons separated by five introns, which encode a 506 amino acid protein. CYP7B1 is involved not only in an alternative BA synthesis pathway, but also in steroid hormone metabolism, metabolism of estrogen receptor ligands and immunoglobulin production in humans. As a result of expression in different tissues (liver, brain and reproductive tract) and a wide spectrum of substrates, CYP7B1 performs different physiological functions in each tissue [94].

In the liver, CYP7B1 catalyzes the 7α-hydroxylation of two major oxysterols; 25-hydroxycholesterol and 27-hydroxycholesterol [45,99,100].

In the brain, CYP7B1 is involved in steroid hormone metabolism, with activity toward two neurosteroids: pregnenolone and dehydroepiandrosterone (DHEA) [95,101].

The 7α-hydroxydehydroepiandrosterone, a product of DHEA hydroxylation by CYP7B1, was proposed to be an activator of estrogen β subtype in human prostatic cells, although conflicting results have been reported [102,103]. The other CYP7B1 substrate, 27-hydroxycholesterol, is known to be a selective estrogen receptor modulator, antagonizing estrogen-mediated activation in the vascular wall [104,105] and activating the receptor in breast cancer and other cell lines [106]. Another steroid substrate of CYP7B1 is 5α-androstane-3β,17β-diol, an agonist for the estrogen receptor, which, unlike the others, is inactivated by 6α-hydroxylation catalyzed by CYP7B1 [94].

Activation of macrophages via Toll-like receptors induces the production of 25-hydroxycholesterol, comprising an oxysterol that suppresses rearrangement of the Ig heavy chain locus through class switch recombination in naive B cells, which consequently suppresses the production of immunoglobulin A. The 25-hydroxycholesterol is one of the CYP7B1 substrates and a loss of function of CYP7B1 leads to the accumulation of 25-hydroxycholesterol and significantly reduced IgA levels in the serum [107].

CYP7B1 expression in the heart was also reported; however, its main function remains unclear [108].

CYP7B1 defects in humans

Because of the wide spectrum of substrates and multiple physiological functions of CYP7B1 in humans, defects in the CYP7B1 gene give rise to two different diseases: liver failure in children as a result of congenital BA synthesis defect type 3 (OMIM database: 613812) and neuropathy in adults of a spastic paraplegia autosomal recessive type 5A (OMIM database: 270800) [94].

Both in human and mouse livers, CYP7B1 catalyzes hydroxylation of two substrates: 25-hydroxycholesterol and 27-hydroxycholesterol. Loss of function of CYP7B1 leads to the accumulation of total oxysterols both in the serum and urine, including 24-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol and poly-hydroxy cholesterols. Liver damage in children was caused by a high concentration of abnormal BA 3β-hydroxy-5-cholen-24-oic acid detected both in serum and urine. In these patients, normal BAs were detected in small amounts or were not detected at all. Such a clinical presentation can result from two different mutations in the CYP7B1 gene: a homozygous nonsense mutation at codon 388 of exon 5 [99] or a homozygous nonsense mutation at codon 112 in exon 3 [109].

The spastic paraplegias represent a clinically heterogeneous group of disorders characterized by lower limb spasticity and weakness that is associated with the degradation of motor neuron axons in the spinal cord [110], and sometimes with ataxia, mental retardation and other neurological symptoms. To date, mutations in more than 17 different genes have been associated with spastic paraplegia, including the CYP7B1 gene. Eighteen different mutations in the six exons of CYP7B1, including missense, nonsense and frame-shift mutations, have been linked to spastic paraplegia [111].


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

The major organ of cholesterol metabolism is the liver. Because all cells depend on adequate levels of cholesterol to function normally, an efficient transport system has evolved by which peripheral tissues turn over cholesterol. The exchange of cholesterol-rich and cholesterol-poor lipoprotein particles takes place on the surface of the cells. An exception to this rule is the brain. The protective blood–brain barrier prevents the transfer of cholesterol in and out of brain cells. To overcome this problem, cholesterol in the brain is utilized via an alternative pathway involving 24-hydroxylation of cholesterol by CYP46A1. 24(S)-hydroxycholesterol is then able to pass the blood–brain barrier and is cleared by the liver via the BA synthesis pathway [112]. In addition to the brain, CYP46A1 is also expressed in the testes and liver, although at much lower levels [8].

Cyp46a1 knockout and CYP46A1 knock-in mouse models

An important role of CYP46A1 in the brain cholesterol turnover was established by a study of Cyp46a1 knockout mice [112]. The disruption of the gene causes a 40% reduction in brain cholesterol excretion in mice; however, this decrease is compensated for by a reduction of de novo synthesis, such that cholesterol turnover in the brain is effectively maintained. Unexpectedly, cholesterol synthesis is not completely abolished, indicating the existence of other routes of cholesterol removal from the brain. Behavioral studies on mice lacking Cyp46a1 showed that their learning abilities are impaired [113]. The cholesterol synthesis pathway provides the cells not only with its final product cholesterol, but also with metabolites that lead to the synthesis of other important molecules, such as dolichol, ubiquinine or heme A [114]. One important aspect involves the supply of different isoprenoids. An observed reduction in the brain de novo cholesterol synthesis was shown to limit the supply of the polyisoprenoid geranylgeraniol, which is apparently required for normal learning processes [113].

The effect of the reduced cholesterol synthesis in the brain was also investigated in transgenic mice that develop AD-like pathology. The 24-hydroxylase was found to be active in the hippocampal and cortical neurons that are affected by AD [115,116]. Disruption of the Cyp46a1 gene in these mice does not affect the rate or the extent of amyloid plaque formation, which is the most prominent feature of the disease. Interestingly, as a result of the ablation of the 24-hydroxylase gene, longevity is prolonged in this strain of mice [117].

24(S)-hydroxycholesterol was established as the most potent ligand for liver X receptor among the oxysterols in vitro [118]. To investigate whether it has the same effect on lipid homeostasis in vivo, a mouse model with a stable expression of human CYP46A1 was engineered. The expression of CYP46A1 mRNA in several organs and systemic levels of 24(S)-hydroxycholesterol were found to be significantly elevated in these mice [119]. Similar to the case of overexpression of CYP27A1 [89], there is no significant impact on cholesterol metabolism in the mouse liver, again discarding the notion of oxysterols being potent regulators of cholesterol homeostasis in vivo.

Defects in CYP46A1 in humans

CYP46A1 is located on chromosome 14q32.2 and spans over 42.8 kbp, containing 15 exons and 14 introns that encode for a 500 amino acid protein.

Some studies have provided evidence showing that cholesterol metabolism has an important role in the pathogenesis of AD. A high serum cholesterol concentration increases the risk of AD [120,121]. CYP46 is a key protein in brain cholesterol metabolism and might represent a genetic risk factor of AD.

Recently, all reported polymorphisms in CYP46A1 were summarized [122]. A single nucleotide polymorphism (rs754203) in intron 2 of the CYP46A1 gene has been studied, with very conflicting results. Different reports associate both the T and C variant with an increased risk for late onset sporadic AD. The frequency of the CYP46A1 T allele and TT genotype was significantly higher in AD patients from Switzerland, Greece and Italy compared to controls [123]. The same association was observed in patients from Spain [124], although it was absent in Swedish and Scottish patients [125]. Studies in Chinese patients have shown no association with AD [126], although the T allele was associated with mild cognitive impairment. On the other hand, seven independent studies of SNP rs754203 report an association of the C allele and CC genotype with AD in patients from Italy, Spain, Finland, China and Poland and, in two studies, no significant association with AD was found in German and French patients carrying the C allele [122]. A single nucleotide polymorphism (T/C) (rs3742376) in intron 3 of CYP46A1 was also reported, and the C allele variant has been associated with AD in patients from China [126,127].

A recent study in a German population investigated 16 SNPs in CYP46A1 and found two of them to be associated with AD risk. Both the G allele of rs7157609 SNP in the promoter region (and the C allele of rs4900442 SNP in intron 3) of the CYP46A1 gene are associated with an increased risk of AD and decreased cerebrospinal fluid levels of cholesterol and 24S-hydroxycholesterol [128].


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

CYP39A1 exists in humans and mice and is specific for 24-hydroxycholesterol. It is a poorly characterized enzyme, expressed only in the endoplasmic reticulum in the liver. Its physiological role is considered to be restricted to BA synthesis. Similar to the case of Cyp7b1, the expression of Cyp39a1 is sexually dimorphic, with higher expression in females [100]. To the best of our knowledge, no results of mouse models of either disrupting or overexpressing the Cyp39a1 gene have been published to date. In a study on Cyp7b1 knockout mice [97], the expression of Cyp39a1 was found to be essentially unchanged, indicating that this pathway of BA synthesis is constitutively active, although probably of minor importance.

Li-Hawkins et al. [100] showed that the CYP39A1 gene contains 12 exons and spans ∼ 150 kb of genomic DNA. They mapped the gene to chromosome 6p21.1–p11.2, whereas the mouse Cyp39a1 gene maps to a region of syntenic homology on chromosome 17.

CYP39 is poorly characterized also in humans. The enzyme was identified in the liver and was initially assumed to be expressed exclusively in this organ [100]. However, subsequent reports reported expression in the prostate and heart [108]. To the best of our knowledge, CYP39 polymorphisms have yet to be be associated with diseases or other defects in humans.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

Despite the fact that the best model for humans is a human, our understanding of complex human diseases (including those associated with CYP genes of cholesterol and BA synthesis) would be much slower and less complete without animal models. This is especially true in cases where specific human mutations are created in mice, providing an informative approach for understanding human diseases. In the personalized medicine era, we are aware of important subtle differences in individual human genomes, as well as how these differences potentially affect disease progression and phenotypes. Here, mouse knockout models can be very helpful for understanding the different pathologies associated with a disease. The examples included in the present review comprise Cyp7a1 knockout mice, where the milder phenotype is similar to the observed phenotypes in humans. However, different genetic backgrounds resulted in Cyp7a1 knockout mice with a much more severe phenotype and an increased rate of postnatal death. Accordingly, it might be reasonable to screen for the Cyp7a1 genotype in a more general manner to avoid some of the unexplained infant deaths. Another example of the importance of animal models is the recent Cyp51 mouse knockout, which led us to revisit the possibility that human CYP51A1 mutations are linked to ABS. It is important to note that, so far, mutations in only two CYPs described in the present review (CYP7B1 and CYP27A1) are linked to human disorders listed in the OMIM database. One possible explanation lies in the difference between cholesterol and BA homeostasis in humans and rodents. Consequently, the disease phenotypes of transgenic mice might differ from diseases in humans. For example, cholic and chenodeoxycholic acid are the primary human BAs, whereas, in rodents, alternative hydroxylations lead to muricholic acids [129]. This chemical diversity is further increased by the action of human and rodent intestinal bacteria (which differ between the species), leading to a different spectrum of secondary BAs. BAs represent important natural ligands of nuclear farnesoid X receptor [129] and are sensed by pregnane X receptor and constitutive androstane receptor [130,131], which regulate BA homeostasis. The different spectrum of BAs in humans and rodents, combined with the different specificities of human versus rodent nuclear receptors, might result in the observed phenotype variations. Interestingly, only mice can compensate for secondary BA metabolism, showing less severe phenotypes in Cyp27a1 and Cyp7b1 knockouts compared to mutations of homologous genes in humans. Nevertheless, it is expected that further progress with respect to providing and characterizing mouse models, as well as high-throughput sequencing of individual genes/genomes, will soon reveal additional links between mutations of CYP genes in cholesterol and BA synthesis and disease phenotypes. From the 57 genes for human CYP, most are reasonably well characterized. CYP39 appears to remain an ‘orphan’ CYP where the availability of a mouse model could play a crucial role in revealing its physiological roles in vivo.


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References

This work was supported by the Slovenian Research Agency program grants P1-0104 and J7-4053. G. Lorbek is funded as a young researcher by the Slovenian Research Agency. M. Lewinska is a FP7 ITN Marie Curie student paid by the ‘FightingDrugFailure’ grant #238132.


  1. Top of page
  2. Abstract
  3. Introduction
  4. CYP51 from cholesterol synthesis
  5. CYP7A1
  6. CYP8B1
  7. CYP27A1
  8. CYP7B1
  9. CYP46A1
  10. CYP39A1
  11. Concluding remarks
  12. Acknowledgements
  13. References
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