The 25(OH)D3/1α,25(OH)2D3-24R-hydroxylase: a catabolic or biosynthetic enzyme?

doi:10.1016/S0039-128X(00)00158-6

Steroids

Volume 66, Issues 3–5, 1 March–1 May 2001, Pages 391-398

https://doi.org/10.1016/S0039-128X(00)00158-6 Get rights and content

Abstract

The kidney is the major source of the circulating dihydroxylated metabolites of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃]. The enzymes which catalyze the production of these two dihydroxylated vitamin D metabolites are the 25(OH)D₃-1α-hydroxylase (1α-hydroxylase) and –24R-hydroxylase (24R-hydroxylase), respectively. While there is no controversy regarding the fundamental importance of the 1α-hydroxylase in the production of the steroid hormone 1α,25(OH)₂D₃, the biologic significance of the 24R-hydroxylase has been the subject of ongoing discussion. Some hold that it is strictly catabolic, leading to side chain oxidation and cleavage of 25-hydroxylated vitamin D sterols, and others hold that it plays a biosynthetic role in the production of 24R,25(OH)₂D₃ which has biologic activities distinct from those of 1α,25(OH)₂D₃. The 24R-hydroxylase has properties in common with other multicatalytic steroidogenic enzymes: (1) the enzyme carries out multiple oxidative and carbon-carbon bond cleavages; (2) it utilizes two natural substrates; (3) its regulation varies depending on the cell or tissue in which it occurs. The purpose of this paper is to review the current literature relevant to the characteristics of the 24R-hydroxylase and its regulation in the context of other multicatalytic steroid hydroxylases in order to provide a perspective regarding its possible function as both a catabolic and activating enzyme in the vitamin D endocrine system.

Introduction

Vitamin D₃ is produced in the skin in response to exposure to ultraviolet light (Fig. 1). In the liver, hydroxylation of the side chain results in the circulating form of the steroid, 25-hydroxyvitamin D₃ [25(OH)D₃] which, due to the fact that it has no intrinsic biologic activity but serves as the substrate for further hydroxylation, can be considered a prohormone. The kidney is the major source of the circulating dihydroxylated metabolites of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃].

The enzymes which catalyze the production of these two dihydroxylated vitamin D metabolites are the 25(OH)D₃-1α-hydroxylase (1α-hydroxylase) and –24R-hydroxylase (24R-hydroxylase), respectively. They belong to the class of mitochondrial and microsomal cytochrome P450-dependent hydroxylases that participate in the synthetic pathways of the adrenal and gonadal steroid hormones. Both the 1α-hydroxylase and the 24R-hydroxylase are mitochondrial and share the features characteristic of mitochondrial steroid hydroxylases depicted in Fig. 2. The electrons required for the reduction of molecular oxygen are derived from NADPH and are passed by ferredoxin reductase and ferredoxin to the terminal component of the complex, cytochrome P450. It is this protein, embedded in the inner mitochondrial membrane, which confers the substrate specificity and the sterospecificity of the insertion of the hydroxyl group. As will be discussed below, several steroid hydroxylases catalyze more than one successive oxidation step on the same molecule and are therefore referred to as multicatalytic or multifunctional.

There is no controversy regarding the fundamental importance of the 1α-hydroxylase in the production of 1α,25(OH)₂D₃ by the kidney as a circulating steroid hormone and by other cells and tissues, most likely for local action. The biologic significance of the 24R-hydroxylase, however, has been the subject of continuing discussion, which has traditionally taken place in the context of beliefs regarding its major circulating product, 24R,25(OH)₂D₃. Those who believe that 24R,25(OH)₂D₃ has no intrinsic, distinctive biologic activity logically characterize the biologic function of the 24R-hydroxylase as a catalytic one initiating the side chain catabolism of both 25(OH)D₃ and more importantly, 1α,25(OH)₂D₃ in target tissues. Those who have provided experimental evidence for distinct biologic activity of 24R,25(OH)₂D₃, believe that the 24R-hydroxylase functions, in some situations, in a synthetic capacity to provide this steroid for its biologic actions.

The purpose of this paper is to review the current literature relevant to the characteristics of the 24R-hydroxylase and its regulation in the context of other multicatalytic steroid hydroxylases in order to provide a perspective regarding its possible function as both a catabolic and activating enzyme in the vitamin D endocrine system.

Section snippets

Multicatalytic steroid hydroxylases

Fig. 3 outlines the pathways of synthesis of steroid hormones as they are typified in the adrenal cortex (cortisol and aldosterone), the testis (androstenedione, testosterone) and the ovary (estradiol). The pathways are characterized steps of stereospecific hydroxylations followed by cleavage of carbon-carbon bonds. Fig. 4 presents the individual reactions of these multicatalytic cytochrome P450s in more detail.

Multicatalytic nature

As demonstrated by Akiyoshi-Shibata et al. [11] and confirmed and extended by Beckman et al. [12], CYP24 not only catalyzes the 24R-hydroxylation of 25(OH)D₃ and 1α,25(OH)₂D₃, but also the subsequent oxidation of this hydroxyl to a ketone group, hydroxylation at the C-23 position and cleavage of the C23-C24 bond. These steps, depicted in Fig. 5 , were shown to be catalyzed by rat and human 24R-hydroxylases expressed in E. coli and Sf21 insect cells, respectively. Thus the 24R-hydroxylase is

Summary

A consideration of the characteristics and regulation of the 25(OH)D₃/1α,25(OH)₂D₃ - 24R-hydroxylase in the context of other multicatalytic hormones emphasizes that it shares the following features with that group of enzymes: (1) the enzyme carries out multiple oxidative and carbon-carbon bond cleavages; (2) it utilizes two natural substrates; (3) its regulation varies depending on the cell or tissue in which it occurs. The interpretation which embraces the multiplicity of observations made on

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Therefore, the biologically-active levels of 24,25D3 or 1,24,25D3 fully depend on the velocity of the subsequent steps in the degradation pathway.[240] It is of no surprise the biological significance of 24-hydroxylase has been continuously discussed because of its dual role first as a catalytic enzyme initiating the side chain catabolism of both 25(OH)D3 and more importantly 1α,25(OH)2D3 in target tissues and second as an enzyme with a synthetic capacity since, in some situations, it is activated to produce 24,25(OH)2D3.[241] Chronic kidney disease (CKD) is characterized by a state of active vitamin D deficiency.
Vitamin D, an important hormone with a central role in calcium and phosphate homeostasis, is required for bone and muscle development as well as preservation of musculoskeletal function. The most abundant vitamin D metabolite is 25-hydroxyvitamin D [25(OH)D], which is currently considered the best marker to evaluate overall vitamin D status. 25(OH)D is therefore the most commonly measured metabolite in clinical practice. However, several other metabolites, although not broadly measured, are useful in certain clinical situations. Vitamin D and all its metabolites are circulating in blood bound to vitamin D binding protein, (VDBP). This highly polymorphic protein is not only the major transport protein which, along with albumin, binds over 99% of the circulating vitamin D metabolites, but also participates in the transport of the 25(OH)D into the cell via a megalin/cubilin complex.
The accurate measurement of 25(OH)D has proved a difficult task. Although a reference method and standardization program are available for 25(OH)D, the other vitamin D metabolites still lack this. Interpretation of results, creation of clinical supplementation, and generation of therapeutic guidelines require not only accurate measurements of vitamin D metabolites, but also the accurate measurements of several other “molecules” related with bone metabolism.
IFCC understood this priority and a committee has been established with the task to support and continue the standardization processes of vitamin D metabolites along with other bone-related biomarkers.
In this review, we present the position of this IFCC Committee on Bone Metabolism on the latest developments concerning the measurement and standardization of vitamin D metabolites and its binding protein, as well as clinical indications for their measurement and interpretation of the results.
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Boron is increasingly added to food supplements due to multiple effects that have been reported in mammals after boric acid administration. Among these effects are inflammatory process control, bone and muscle strength enhancement, protein expression regulation, and a decreased risk of developing some pathologies in which these processes are key, such as osteoporosis, dermatological inflammatory non-infectious maladies and diseases affecting the central nervous system. Experimental data have suggested that steroid hormone levels in plasma change after boric acid administration, but a clear mechanism behind these variations has not been established. We analyzed possibilities for these changes and hypothesized that boric acid disrupts the interactions between steroid hormones and several carriers in plasma. In particular, we proposed that there is an uncoupling of the interactions between sex hormone binding globulin (SHBG) and estrogens and testosterone and that there are alterations in the binding of hydrophobic ligands by other carrier proteins in plasma. Further experimental and computational studies are required to support the hypothesis that boric acid and probably other boron-containing compounds can displace steroid hormones from their plasma carriers. If such phenomena are confirmed, boron administration with a clear mechanism could be employed as a therapeutic agent in several diseases or physiological events that require modulation of steroid hormone levels in plasma.
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The production of the hormone 1α,25(OH)₂ vitamin D₃ (1,25D₃) begins in the body by the processing of 7-dehydrocholesterol to vitamin D₃ (VD3), where the B-ring of the molecule opens. This increases the molecular space the D3 A-ring can sample. The addition of the 25-OH and 1-OH groups, D3, adds H-bonding character to the molecule. It is these features of the 1,25D₃ molecule, in addition to the innate flexible of the side-chain atoms, which determine what proteins 1,25D₃ will bind to and perhaps more importantly the strength and dynamics of the interaction(s). The nuclear vitamin D receptor (VDR) is an intracellular protein that binds with very strong affinity to 1,25D₃. In binding to the VDR, 1,25D₃ can directly regulate gene transcription and lipid signaling and the activity and/or localization of enzymes and ion channels. This chapter reviews how the dynamic interactions between 1,25D₃ and the VDR can regulate the pluripotent cellular functions that have been associated with the two molecules.
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At the same time, FGF-23 and 1α,25-(OH)2D synergize also in increasing the activity of the renal 24R-hydroxylase [28], which converts 25-(OH) D and 1α,25-(OH)2D into the 24-hydroxylated inactive metabolites (Fig. 2). The 24R-hydroxylase (CYP24A1 or CYP24), also named 1,25-hydroxyvitamin D3 24-hydroxylase, is a cytochrome P450-dependent is a cytochrome P450-dependent multicatalytic enzyme, using either two substrates, namely 25-(OH)D3 or 1α,25-(OH)D3 [15,29]. The 24R-hydroxylase activity is inversely related to 1α-hydroxylase activity, mainly because of the opposing effects of 1α,25-(OH)D on the expression of the two enzymes (induction of 24R-hydroxylase and inhibition of 1α-hydroxylase), but it also involves suppressive effects of PTH on the transcription of the CYP24 gene in the kidney [29].
Despite a wide number of studies performed on the general population, little is known about the Vitamin D status of athletes. A particular influence of many factors, including skin pigmentation, early- or late-day training, indoor training, geographic location and extensive sunscreen use, has been observed in this specific population. The need of supplementation with Vitamin D in athletes is not defined or, when supplementation is needed, even the optimal amount of Vitamin D to be used is not specified. The periodic measurement of Vitamin D is the only procedure capable to define athletes' status. Although various methods for the measurement of Vitamin D are routinely used, they often give discordant and poorly reproducible results; thus, it is necessary to standardize the various methods, in order to have comparable results.
In conclusion, current available data indicate both that little is known about the Vitamin D status of athletes and that is still unclear if supplementation could be desirable. Finally, it must be pointed out that all the papers about Vitamin D status should indicate in detail the method used for really allowing a correct interpretation of data.
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The impact of tumor CYP24 expression on the local elimination of 1,25(OH)2D3 in vivo has not yet been determined. Typically, CYP24 transcription is increased by 1,25(OH)2D3 in target tissues including the kidney, colon, intestine, and bone [11–13]. Physiologically, CYP24 may be induced by 1,25(OH)2D3 to attenuate its action and prevent accumulation of toxic concentrations which would result in hypercalcemia.
The vitamin D₃ catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)₂D₃ in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC–MS/MS assay that permits absolute quantitation of 1,25(OH)₂D₃ in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)₂D₃ by a CYP24-dependent process in vitro, and 1,25(OH)₂D₃ rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)₂D₃ reached a maximum of 21.6 and 1.70 ng/mL, respectively, following intraperitoneal dosing (20 μg/kg 1,25(OH)₂D₃). When co-administered with the CYP24 selective inhibitor CTA091 (250 μg/kg), 1,25(OH)₂D₃ plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)₂D₃ AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)₂D₃ independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)₂D₃ concentration versus time data confirmed that: 1,25(OH)₂D₃ was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)₂D₃ by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)₂D₃.
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Further studies will undoubtedly lead to an elucidation of the details of these autocrine/paracrine interactions between the peripheral synthesis and the local actions of 1,25D3. The 24R-hydroxylase, a cytochrome P450-dependent enzyme also known as CYP24A1 or CYP24, is, as are several other hydroxylases involved with the synthesis of steroid hormones, a multicatalytic enzyme.55 That is, it catalyzes more than one successive step at the same active site of the enzyme.
Fundamental to understanding the way in which perturbations in the vitamin D endocrine system can affect human health is an appreciation of the steps involved in the production of the well-recognized active hormonal form, 1,25-dihydroxyvitamin D₃. Thus this paper focuses first on the nature and regulation of the two enzymes responsible for the production of 1,25-dihydroxyvitamin D₃, the 25-hydroxylase in the liver and the 1α-hydroxylase in the kidney. The most important regulators of the 1α-hydroxylase in the kidney are 1,25-dihydroxyvitamin D₃ itself, parathyroid hormone and FGF23. The extent and importance of extra-renal, 1,25-dihydroxyvitamin D₃ synthesis is then considered. Finally the features of the 24R-hydroxylase, which produces 24R,25-dihydroxyvitamin D₃inthe kidney and is induced by and inactivated, 1,25-dihydroxyvitamin D₃in target cells are described.

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The 25(OH)D3/1α,25(OH)2D3-24R-hydroxylase: a catabolic or biosynthetic enzyme?

Abstract

Introduction

Section snippets

Multicatalytic steroid hydroxylases

Multicatalytic nature

Summary

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