Elsevier

Biochemical Pharmacology

Volume 84, Issue 12, 15 December 2012, Pages 1696-1704
Biochemical Pharmacology

Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity

https://doi.org/10.1016/j.bcp.2012.09.032Get rights and content

Abstract

20-Hydroxyvitamin D3 (20(OH)D3), the major product of CYP11A1 action on vitamin D3, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to metabolize 20(OH)D3. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)2D3 with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D3 (25(OH)D3). CYP24A1 hydroxylated 20(OH)D3 to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D3 (20,24(OH)2D3) and 20,25-dihydroxyvitamin D3 (20,25(OH)2D3). The catalytic efficiency of CYP24A1 for 20(OH)D3 metabolism was more than 10-fold lower than for either 25(OH)D3 or 1,25(OH)2D3 and no secondary metabolites were produced. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)2D3 or the parent 20(OH)D3, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D3.

Introduction

CYP24A1 catalyzes the inactivation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) with initial production of 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3), as illustrated in Fig. 1 [1], [2], [3], [4]. This product can undergo further CYP24A1-catalyzed oxidations resulting in cleavage of the side chain between C23 and C24 to produce calcitroic acid, the final product which is excreted (Fig. 1) [3], [5], [6]. Some mammalian species including the human also display a CYP24A1-catalyzed C23 oxidation pathway where initial hydroxylation is at C23 and the final product is 1,25-dihydroxyvitamin D3-26,23-lactone [6], [7], [8], [9]. The rat enzyme almost exclusively catalyzes the C24 oxidation pathway, while this pathway accounts for 79% of the metabolism of 1,25(OH)2D3 by the human enzyme [7]. CYP24A1 also catalyzes the hydroxylation of 25-hydroxyvitamin D3 (25(OH)D3) [1], [2], [9], [10] by the C24 pathway with some of the primary product, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), escaping from the enzyme and being one of the major dihydroxyvitamin D metabolites seen in human serum [11]. While this product lacks the typical activity seen for 1,25(OH)2D3, it has been reported to be active in stimulating cartilage development and bone fracture repair [12].

20S-Hydroxyvitamin D3 (20(OH)D3) is the major product of CYP11A1 action on vitamin D3 and can be further metabolized by purified CYP11A1 to a number of products with the main ones being 20,23-dihydroxyvitamin D3 (20,23(OH)2D3) and 17,20,23-trihydroxyvitamin D3, as illustrated in Fig. 1 [13], [14], [15], [16], [17]. Our most recent studies have shown that 20(OH)D3 can be produced by human placentae and rat adrenal glands fragments ex vivo through a reaction catalyzed by CYP11A1 [18], [19]. Furthermore, we have shown that this secosteroid can be produced by cultured keratinocytes both with and without the addition of vitamin D3 to the culture media, and have provided data indicating that 20(OH)D3 is present in human serum [18], [19]. Thus, there is now compelling evidence that CYP11A1 provides an alternative pathway for vitamin D3 activation via production of 20(OH)D3, separate from the classical pathway producing 1,25(OH)2D3. 20(OH)D3 shares many, but not all of the actions of 1,25(OH)2D3. It inhibits the proliferation and stimulates the differentiation of a range of normal and cancer cells including keratinocytes, melanoma and leukemia cells [20], [21], [22], [23], [24], [25]. Similar properties are seen for 20-hydroxyvitamin D2, the main product of CYP11A1-mediated metabolism of vitamin D2 [26], [27], [28]. 20(OH)D3 inhibits NF-κB activity by stimulating the expression of inhibitory IκBα protein [29], [30] and therefore serves as an excellent candidate for treatment of inflammatory diseases. Knockdown of the vitamin D receptor inhibits the effects of 20(OH)D3 indicating that like 1,25(OH)2D3, it works via the vitamin D receptor [24], [28], [30]. 20(OH)D3 also shows comparable activity to 1,25(OH)2D3 in stimulating translocation of the VDR coupled to green fluorescent protein from the cytoplasm to the nucleus [15], [28], [31]. However, unlike 1,25(OH)2D3, high concentrations of 20(OH)D3 (up to 30 μg/kg) do not raise serum calcium levels in rodents [22], [23]. Therefore, 20(OH)D3 has the potential to serve as a relatively non-toxic drug for treatment of hyperproliferative and inflammatory disorders. While 20(OH)D3 is a relatively poor substrate for 1α-hydroxylation by CYP27B1 [32], the product 1,20-dihydroxyvitamin D3, has calcemic activity although lower than that seen for 1,25(OH)2D3 [22]. Another important difference to 1,25(OH)2D3 is that 20(OH)D3 is a very poor inducer of CYP24A1 expression in a number of cell systems [15], [21], [22], [24], [28]. Therefore, if used therapeutically it may not be subject to the same rapid inactivation that occurs with 1,25(OH)2D3 and many vitamin D analogs when its levels are elevated [4].

Metabolism of 20(OH)D3 to 20,23(OH)2D3 and other products by CYP11A1 occurs with low catalytic efficiency and the metabolites retain strong biological activity [15], [22], [25], [29], [31]. Since CYP24A1 is the enzyme that inactivates 1,25(OH)2D3, it is important to know whether CYP24A1 can metabolize 20(OH)D3 and whether the resulting products are also biologically inactive. We have addressed these questions in the current study using purified rat CYP24A1 expressed in Escherichia coli. The rat enzyme was chosen for these studies because it is easier to express and purify than the less stable human enzyme [33], plus we have used rodents to test the toxicity and other effects of 20(OH)D3 [22], [23] where potential metabolism by CYP24A1 is of importance. The rat and human enzymes share 90% amino acid sequence identity [34] and both metabolize 25(OH)D3 and 1,25(OH)2D3 predominantly by the C24 oxidation pathway, therefore data for 20(OH)D3 metabolism for the rat enzyme should provide a useful model for 20(OH)D3 metabolism by human CYP24A1.

The anti-melanoma activity of 1,25(OH)2D3 is well established (reviewed in [35], [36], [37]), but its clinical use is limited by its calcemic activity. Since 20(OH)D3 is non-calcemic and non-toxic at high concentrations [22], [23], it is a good candidate for treatment of melanoma with comparable anti-melanoma activity to 1,25(OH)2D3, mediated via binding to the vitamin D receptor [20], [25], [28]. These effects of 20(OH)D3 include differential inhibition of melanoma growth compared to melanocytes [25]. Since inhibition of melanoma colony formation in soft agar by 20(OH)D3 is well characterized and represents a good in vitro measure of anti-tumorigenic activity, we have chosen this system for the preliminary biological testing of the products of CYP24A1 action on 20(OH)D3 and have shown that both of the major products, 20,24(OH)2D3 and 20,25(OH)2D3, display enhanced, rather than reduced, anti-melanoma activity.

Section snippets

Materials

20(OH)D3 was synthesized enzymatically from the action of CYP11A1 on vitamin D3 and was purified by TLC and reverse phase HPLC as before [15], [16]. 20,26-Dihydroxyvitamin D3 (20,26(OH)2D3) was produced by the action of human CYP27A1 on 20(OH)D3 [38]. 25(OH)D3 and NADPH were purchased from Merck (Darmstadt, Germany). Vitamin D3, dioleoyl phosphatidylcholine, bovine heart cardiolipin, cyclodextrin, and glucose-6-phosphate were purchased from Sigma (St. Louis, MO, USA). Glucose-6-phosphate

Metabolism of 20(OH)D3 in phospholipid vesicles by rat CYP24A1

CYP24A1 activity was measured with 20(OH)D3 incorporated into phospholipid vesicles as this system mimics the native environment of the cytochrome in the inner mitochondrial membrane [1], [46] and has been observed to give high catalytic activity for other mitochondrial cytochromes P450 [17], [32], [38], [42], [43], [47], [48]. Incubation of 20(OH)D3 in phospholipid vesicles, with rat CYP24A1, resulted in two major products and several minor ones (Fig. 2). The same products were seen with

Discussion

Analysis of the crystal structure of CYP24A1 [46] indicates that substrate enters the active site of CYP24A1 from an access channel open to the hydrophobic domain of the membrane bilayer. Entry of substrate into the active site from the membrane phase is also supported from studies on other mitochondrial P450s [17], [38], [42], [43], [47], [50]. Strong partitioning of the substrates for CYP24A1 such as 25(OH)D3 into the phospholipid bilayer has also been demonstrated [42]. To replicate this in

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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