Monocyte-derived cells express CYP27A1 and convert vitamin D3 into its active metabolite

CYP27A1 catalyses hydroxylations in the biosynthesis of bile acids and the bioactivation of vitamin D3. We investigated the expression of CYP27A1 in human monocytes, monocyte-derived macrophages, and dendritic cells on mRNA and protein levels as well as its enzymatic activity in comparison with the expression of CYP27B1 and CYP24A1. Macrophages showed a strong expression of CYP27A1, whereas monocytes and dendritic cells expressed low levels of CYP27A1 mRNA. Immunohistochemistry revealed CYP27A1 and CYP27B1 protein expression in macrophages. Accordingly, macrophages converted vitamin D3 into the active metabolite 1,25(OH)2D3. Dendritic cells also metabolized vitamin D3 although to a lesser extent. This could be due to the high expression of CYP24A1, the enzyme that degrades 25(OH)D3 and 1,25(OH)2D3. Our results show that macrophages and dendritic cells are capable to perform both hydroxylation steps of the vitamin D3 metabolism suggesting a possible role of local 1,25(OH)2D3 synthesis by myeloid cells in the skin and gut.     

Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES

The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Expression,structure-function,and molecular modeling of vitamin D P450s

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.     

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25‐hydroxyvitamin D₃ (25D) to hormonal 1,25‐dihydroxyvitamin D₃ (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish cyp27b1 cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25‐fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.     

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Altered expression of key players in vitamin D metabolism and signaling in malignant and benign thyroid tumors

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, mediates antitumor effects in various cancers. The expression of key players in vitamin D signaling in thyroid tumors was investigated. Vitamin D receptor (VDR) and CYP27B1 and CYP24A1 (respectively activating and catabolizing vitamin D) expression was studied (RT-PCR, immunohistochemistry) in normal thyroid, follicular adenoma (FA), differentiated thyroid cancer (DTC) consisting of the papillary (PTC) and follicular (FTC) subtype, and anaplastic thyroid cancer (ATC). VDR, CYP27B1, and CYP24A1 expression was increased in FA and DTC compared with normal thyroid. However, in PTC with lymph node metastasis, VDR and CYP24A1 were decreased compared with non-metastasized PTC. In ATC, VDR expression was often lost, whereas CYP27B1/CYP24A1 expression was comparable to DTC. Moreover, ATC with high Ki67 expression (>30%) or distant metastases at diagnosis was characterized by more negative VDR/CYP24A1/CYP27B1 staining. In conclusion, increased expression of key players involved in local 1,25(OH)(2)D(3) signaling was demonstrated in benign and differentiated malignant thyroid tumors, but a decrease was observed for local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)(2)D(3) in early cancer stages. These findings advocate further studies with 1,25(OH)(2)D(3) and analogs in persistent and recurrent iodine-refractory DTC.     

Regulation of extrarenal synthesis of 1,25-dihydroxyvitamin D3--relevance for colonic cancer prevention and therapy

Epidemiological studies have demonstrated a high incidence of colonic tumors in populations living in areas of low sunlight exposure. This suggests 1,25-dihydroxyvitamin D3, an antimitotic prodifferentiating steroid hormone, as a potentially preventive factor since levels of the precursor 25-hydroxyvitamin D3 in serum are, to a major part, dependent upon sun exposure. Conversion into the active metabolite from the precursor is effected by CYP27B1, and degradation by CYP24. Both p450 hydroxylases are known to be located in the kidney. However, we were able to demonstrate presence, and activity of both enzymes also in the colon. We have shown also that during early tumor progression expression of CYP27B1 and of the vitamin D receptor is upregulated. Therefore the vitamin D system may function as a potent physiological defense against further tumor progression in cancer patients. We suggest that estrogenic substances, and also phytoestrogens present in soy food could, by increasing tumor tissue-located CYP27B1 activity and decreasing degradative CYP24 activity, augment tumor-localized 1,25-dihydroxyvitamin D3 levels and activity.     

Selective inhibition of vitamin D hydroxylases in human keratinocytes

Human keratinocytes convert 25(OH)D(3) to hormonally active 1alpha,25(OH)(2)D(3) and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1alpha,25(OH)(2)D(3) are short-lived. 1alpha,25(OH)(2)D(3) induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1alpha,25(OH)(2)D(3) synthesis, catalyzed by 1alpha-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1alpha-hydroxylase, (3)H-25(OH)D(3) (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89-443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1alpha-hydroxylated products generated from (3)H-25(OH)D(3) and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1alpha,25(OH)(2)D(3) to 1alpha,25(OH)(2)-3epi-D(3). As spin-off from our program, selective inhibitors of 1alpha-hydroxylase were also found (e.g. SDZ 88-357). Using (3)H-25(OH)D(3) as substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1alpha-hydroxylated metabolites over abundant 25(OH)D(3). In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxylation of (3)H-25(OH)D(3). Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.     

Metabolism of vitamin D by human microsomal CYP2R1

The activation of vitamin D requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation. Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase. Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D(3) 25-hydroxylase. A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D(2). CYP2R1 hydroxylated vitamin D(2) at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27. The K(m) and k(cat) values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D(3) were 0.45microM and 0.97min(-1), respectively. The k(cat)/K(m) value of CYP2R1 was 26-fold higher than that of CYP27A1. These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans.     

UVB-induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo-2 cells and in THP-1 macrophages pretreated with a sterol Delta7-reductase inhibitor

Epidermal keratinocytes are able to produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and induce vitamin D activity upon UVB irradiation. To find out whether this property is keratinocyte specific, we investigated this characteristic in two other cell types, namely intestinal CaCo-2 cells and the macrophage-like differentiated THP-1 cells. THP-1 macrophages and preconfluent CaCo-2 cells contain the vitamin D receptor (VDR), possess 25-hydroxylase (CYP2R1 and CYP27A1) and 1alpha-hydroxylase (CYP27B1) activity, and survive the low UVB doses essential for vitamin D3 photoproduction. Upon irradiation, 24-hydroxylase (CYP24) mRNA is induced in both cell types pretreated with the sterol Delta7-reductase inhibitor BM15766 whereby the 7-dehydrocholesterol (7-DHC) content was increased. Transfection studies in CaCo-2 cells with a vitamin D response element-containing construct revealed the involvement of the VDR in this UVB-dependent CYP24 induction. The CYP24 inducing activity in BM15766-pretreated UVB-irradiated CaCo-2 cells and THP-1 macrophages was identified as 1,25(OH)2D3 by combined high-performance liquid chromatography radioimmunoassay. Addition of vitamin D binding protein to the CaCo-2 cells attenuated UVB-induced CYP24 induction suggesting the possibility of a paracrine or autocrine role for the photoproduced 1,25(OH)2D3. In conclusion, preconfluent CaCo-2 cells and THP-1 macrophages are able to induce vitamin D activity upon UVB irradiation and hence combine all parts of the vitamin D photoendocrine system, a characteristic which is therefore not keratinocyte specific.     

Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes

Human keratinocytes are fully competent cells of the vitamin D (VD) hormone system. They have the capacity to generate VD, to convert it to hormonally active 1alpha,25(OH)(2)D(3) and subsequently, to metabolize the hormone by self-induced CYP24. These reactions generate a cascade of highly transient products and, eventually terminate biologic activity. To elucidate regulatory principles in the VD cascade in more detail, we made use of novel selective CYP24 inhibitors, recently synthesized by our group. Here, we describe the effects of VID400 and SDZ 89-443 on the metabolism of 20 nM (3)H-25(OH)D(3) in human keratinocytes, analyzed by sensitive HPLC methods. First, we present evidence that freshly generated 1alpha,25(OH)(2)D(3) does not down-regulate 1alpha-hydroxylation, as commonly assumed. The transient time course of 1alpha,25(OH)(2)D(3), could be explained by its fast 24-hydroxylation to polar products, undetectable by usual HPLC-analysis of organic extracts. On inhibition of CYP24, 1alpha-hydroxylation continued throughout extended periods, indicating its constitutive nature. Asking whether 1alpha,25(OH)(2)D(3) derived metabolites [1alpha,25(OH)(2)-3epi-D(3), 1alpha,24(R),25(OH)(3)D(3), 1alpha,25(OH)(2)-24oxo-D(3), 1alpha,23(S),25(OH)(3)-24-oxo-D(3) and calcitroic acid] would regulate 1alpha-hydroxylase, we pre-treated cells with 20 nM of these metabolites for 5 h and 24 h. Subsequent incubation with (3)H-25(OH)D(3) demonstrated that neither metabolite substantially impaired 1alpha-hydroxylase, while all of them transiently induced CYP24 activity. Analyzing the effects of VID400 on the kinetics of (3)H-25(OH)D(3), we showed that 1alpha-hydroxylation rather than 24-hydroxylation was rate-limiting in the C-24 oxidation pathway - again suggesting constitutive expression of 1alpha-hydroxylase. CYP24 inhibitors effectively increased the levels and lifetime of all transient 1alpha-hydroxylated metabolites, especially of 1alpha,25(OH)(2)-3epi-D(3) that became the predominant lipid soluble metabolite. Highly increased levels of 1alpha,23(S),25(OH)(3)-24-oxo-D(3), the metabolite preceding side chain cleavage, indicated involvement of CYP24 also in the terminal step of the cascade. Besides using inhibitors of CYP24 as tools to explore mechanisms in the VD cascade, they also appear to be valuable to discover the intrinsic biologic functions of distinct metabolites.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.