The Journal of Steroid Biochemistry and Molecular Biology
Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1
Highlights
► CYP27A1 metabolizes cholesterol and vitamin D3 in phospholipid vesicles. ► Comparable Km values observed for the metabolism of vitamin D3 and cholesterol. ► CYP27A1 metabolizes CYP11A1-derived 20-hydroxyvitamin D3 to two major products. ► Products identified as 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3. ► 20-Hydroxyvitamin D3 is metabolized more efficiently than vitamin D3.
Introduction
CYP27A1 is a multifunctional enzyme involved in the initial activation of vitamin D3, producing 25-hydroxyvitamin D3 (25(OH)D3), as well as in the biosynthesis of acidic and neutral bile acids. In the acidic bile acid pathway, CYP27A1 is responsible for the rate limiting step of 26-hydroxylation of cholesterol forming 26-hydroxycholesterol. Furthermore it has the ability to subsequently hydroxylate carbon 26 several times to yield 3β-hydroxy-5-cholestenoic acid [1], [2], [3]. In the neutral bile acid pathway, CYP27A1 serves to hydroxylate bile acid intermediates, 5β-cholestane-3α,7α-diol and 5β-cholestane-3α,7α,12α-triol, to initiate side chain cleavage, forming cholic acid and chenodeoxycholic acid, respectively [4]. Although primarily expressed in the liver, CYP27A1 has also been detected in keratinocytes, dermal fibroblasts, osteoblasts, arterial endothelium, parathyroid gland, ovaries and duodenum, where it could play a role in the local synthesis of 25-hydroxyvitamin D3 [5], [6], [7], [8], [9], [10].
Once formed, 25(OH)D3 is further activated by the mitochondrial 1α-hydroxylase (CYP27B1) to produce 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3. 1,25(OH)2D3 is essential for calcium and phosphorous homeostasis and thus skeletal integrity [11], [12]. In addition, 1,25(OH)2D3 has tumorostatic and anti-carcinogenic properties, where it promotes differentiation in normal and transformed cells including melanoma, leukemia, prostate, breast, keratinocytes and hematopoietic cells [13], [14]. As a result 1,25(OH)2D3 has the potential to treat hyperproliferative diseases such as psoriasis and cancer [14]. However, supraphysiological doses of 1,25(OH)2D3 are needed and this has limited its therapeutic use due to the resulting calcemic effect. As a result there is considerable interest in finding vitamin D analogs which retain the anti-proliferative property but are non-calcemic. One source of vitamin D analogs with these properties is from the metabolism of vitamin D by CYP11A1, with the major metabolite being 20-hydroxyvitamin D3 (20(OH)D3) [15], [16], [17]. This product as well as its sequential metabolites are biologically active exhibiting anti-proliferative and pro-differentiation effects on a range of cell lines including keratinocytes, leukemic and myeloid cells [18], [19], [20]. It also inhibits NF-κB activity [21] but shows no calcemic activity in rats at doses as high as 4 μg/kg [18]. Structurally similar 20(OH)D2 shows similar properties [22]. Thus, 20(OH)D3 has the potential to be used as a therapeutic drug for the treatment of hyperproliferative and inflammatory disorders. The addition of a 1α-hydroxyl group to 20(OH)D3 by CYP27B1, produces 1,20-dihydroxyvitamin D3, which exhibits moderate calcemic activity when administered at comparable doses to 20(OH)D3 [18]. However, it remains to be determined if 20(OH)D3 can undergo 25-hydroxylation by CYP27A1 or other P450s, and whether these novel products have an altered biological activity.
CYP27A1 belongs to the mitochondrial type I cytochrome P450 family, which receives its electrons from NADPH via adrenodoxin reductase and its redox partner adrenodoxin [23], [24]. CYP27A1 interacts with the matrix side of the inner mitochondrial membrane [25]. The F-G loop and the N-terminal part of the G helix have been identified as the sites of membrane attachment, similar to what has been reported for CYP24 and CYP11A1 [26], [27], [28]. As membrane bound P450s acquire their hydrophobic substrates such as vitamin D3 from the membrane phase of the phospholipid bilayer, it is important to characterize P450 activity in a membrane environment. Murtazina et al. [29] found that the activity of CYP27A1 was altered according to the presence of different phospholipid species, such as phosphatidylglycerol and phosphatidylethanolamine. However, these phospholipids are found predominantly in bacterial membranes and while they can influence the properties of the purified expressed enzyme, they are not representative of phospholipids of the inner mitochondrial membrane. Recently, unilamellar phospholipid vesicles have been used to characterize the kinetics of vitamin D metabolism by CYP11A1 and CYP27B1 [30], [31], [32]. This membrane system uses dioleoyl phosphatidylcholine and cardiolipin to closely mimic the composition of the inner mitochondrial membrane [33].
While CYP27A1 can metabolize a range of substrates including cholesterol, oxysterols and vitamin D, kinetic comparisons of the ability of CYP27A1 to metabolize different substrates are lacking. Even though one study did show that the activity of CYP27A1 towards cholesterol was about 4-fold higher than that for vitamin D3, the incubation conditions were not identical for both substrates and were not under initial rate conditions [34]. In the current study we address this deficiency by comparing the kinetic parameters for vitamin D3 and cholesterol metabolism in the phospholipid vesicle system. In addition, we describe the ability of CYP27A1 to hydroxylate the novel non-calcemic vitamin D3 analog, 20(OH)D3.
Section snippets
Materials
20(OH)D3 was enzymatically synthesized by the action of CYP11A1 on vitamin D3 and purified as described before [15]. Vitamin D3, 2-hydroxypropyl-β-cyclodextrin (cyclodextrin), NADPH, dioleoyl phosphatidylcholine, bovine heart cardiolipin and cholesterol were from Sigma-Aldrich Pty. Ltd. (Sydney, Australia). The pGro7 plasmid was from Takara Bio Inc. (Shiga, Japan). The silica gel plates were from Alugram Sil G, Macherey-Nagel, Inc. (Easton, PA). The [4-14C]cholesterol and emulsifier safe
Metabolism of cholesterol and vitamin D3 incorporated in phospholipid vesicles
Phospholipid vesicles provide a means of mimicking the inner mitochondrial membrane environment of mitochondrial P450s. Both cholesterol and vitamin D3 partition exclusively into the bilayer of phospholipid vesicles prepared in aqueous buffer [31], [42]. 25(OH)D3 has also been shown to partition greater than 97% into phospholipid vesicles [30]. As expected, the major product of vitamin D3 metabolism was identified as 25(OH)D3 based upon its identical HPLC retention time to authentic 25(OH)D3,
Discussion
In this study we have shown that purified human CYP27A1 is catalytically active towards substrates that have been incorporated into phospholipid membranes. Kinetic analysis shows that vitamin D3 metabolism by CYP27A1 has a kcat of 2.09 min−1, which is 10-fold higher than what Sawada et al. [43] reported using bacterial membranes. Our study reports the highest kcat for the 25-hydroxylation of vitamin D3 by any human cytochrome P450. Kinetic assays using membrane fractions containing CYP2R1
Acknowledgments
This work was supported by NIH [Grant R01AR052190] to AS, by the University of Western Australia and by the College of Pharmacy at the University of Tennessee Health Science Center. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
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