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  • 1.
    Almokhtar, Mokhtar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ubhayasekera, S. J. Kumari A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Motor neuron-like NSC-34 cells as a new model for the study of vitamin D metabolism in the brain.2016In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 158, p. 178-188Article in journal (Refereed)
    Abstract [en]

    Vitamin D-3 is a pro-hormone, which is sequentially activated by 25- and 1 alpha-hydroxylation to form 25-hydroxyvitamin D-3 [25(OH)D-3] and 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)2D(3)], respectively. Subsequent inactivation is performed by 24-hydroxylation. These reactions are carried out by a series of CYP450 enzymes. The 25-hydroxylation involves mainly CYP2R1 and CYP27A1, whereas 1 alpha-hydroxylation and 24-hydroxylation are catalyzed by CYP27B1 and CYP24A1, respectively, and are tightly regulated to maintain adequate levels of the active vitamin D hormone, 1 alpha,25(OH)(2)D-3. Altered circulating vitamin D levels, in particular 25(OH)D-3, have been linked to several disorders of the nervous system, e.g., schizophrenia and Parkinson disease. However, little is known about the mechanisms of vitamin D actions in the neurons. In this study, we examined vitamin D metabolism and its regulation in a murine motor neuron-like hybrid cell line, NSC-34. We found that these cells express mRNAs for the four major CYP450 enzymes involved in vitamin D activation and inactivation, and vitamin D receptor (VDR) that mediates vitamin D actions. We also found high levels of CYP24A1-dependent 24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] production, that was inhibited by the well-known CYP enzyme inhibitor ketoconazole and by several inhibitors that are more specific for CYP24A1. Furthermore, CYP24A1 mRNA levels in NSC-34 cells were up-regulated by 1 alpha,25(OH)(2)D-3 and its synthetic analogs, EB1089 and tacalcitol. Our results suggest that NSC-34 cells could be a novel model for the studies of neuronal vitamin D metabolism and its mechanism of actions.

  • 2.
    Araya, Zufan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hosseinpour, Fardin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bodin, Karl
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1): a novel reaction by CYP27A12003In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1632, no 1-3, p. 40-47Article in journal (Refereed)
    Abstract [en]

    The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).

  • 3.
    Araya, Zufan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1.2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 372, no 2, p. 529-534Article in journal (Refereed)
    Abstract [en]

    The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Bj¨orkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5_-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

  • 4. Bjorkhem, I
    et al.
    Araya, Zufan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Rudling, M
    Angelin, B
    Einarsson, C
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver: no coordinate regulation of CYP7A1 and CYP27A1.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 30, p. 26804-26807Article in journal (Refereed)
    Abstract [en]

    It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7_-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis.

  • 5.
    Ellfolk, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gyllensten, Katarina
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Regulation of human vitamin D(3) 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells2009In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 75, no 6, p. 1392-1399Article in journal (Refereed)
    Abstract [en]

    In this study, we examined whether 1alpha,25-dihydroxyvitamin D(3) (calcitriol), phenobarbital, and the antiretroviral drug efavirenz, drugs used by patient groups with high incidence of low bone mineral density, could affect the 25-hydroxylase activity or expression of human 25-hydroxylases in dermal fibroblasts and prostate cancer LNCaP cells. Fibroblasts express the 25-hydroxylating enzymes CYP2R1 and CYP27A1. LNCaP cells were found to express two potential vitamin D 25-hydroxylases-CYP2R1 and CYP2J2. The presence in different cells of nuclear receptors vitamin D receptor (VDR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) was also determined. Phenobarbital suppressed the expression of CYP2R1 in fibroblasts and CYP2J2 in LNCaP cells. Efavirenz suppressed the expression of CYP2R1 in fibroblasts but not in LNCaP cells. CYP2J2 was slightly suppressed by efavirenz, whereas CYP27A1 was not affected by any of the two drugs. Calcitriol suppressed the expression of CYP2R1 in both fibroblasts and LNCaP cells but had no clear effect on the expression of either CYP2J2 or CYP27A1. The vitamin D(3) 25-hydroxylase activity in fibroblasts was suppressed by both calcitriol and efavirenz. In LNCaP cells, consumption of substrate (1alpha-hydroxyvitamin D(3)) was used as indicator of metabolism because no 1alpha,25-dihydroxyvitamin D(3) product could be determined. The amount of 1alpha-hydroxyvitamin D(3) remaining in cells treated with calcitriol was significantly increased. Taken together, 25-hydroxylation of vitamin D(3) was suppressed by calcitriol and drugs. The present study provides new information indicating that 25-hydroxylation of vitamin D(3) may be regulated. In addition, the current results may offer a possible explanation for the impaired bone health after treatment with certain drugs.

  • 6.
    Ellfolk, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Isolation and properties of the CYP2D25 promoter: Transcriptional regulation by vitamin D3 metabolites2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 345, no 2, p. 568-572Article in journal (Refereed)
    Abstract [en]

    Previous studies have suggested that hepatic production of 25-hydroxyvitamin D3 may be suppressed by 1α,25-dihydroxyvitamin D3. However, the molecular details of these observations have not been clarified. In the current study, the 5´-flanking DNA sequence of CYP2D25, a porcine microsomal vitamin D 25-hydroxylase, was isolated and analyzed. The CYP2D25 promoter contains a putative vitamin D response element (VDRE). The promoter activity was markedly suppressed by 1α,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in presence of vitamin D receptor (VDR). The data suggest that VDR-mediated inhibition of 25-hydroxylase(s) by vitamin D3 metabolites at the transcriptional level may play an important role in the regulation of 25-hydroxyvitamin D3 production in liver and other tissues.

  • 7.
    Emanuelsson, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Almokhtar, Mokhtar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gröndbladh, Alfhild
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nylander, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Svensson, Anne-Lie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Svenningsen, Åsa Fex
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Expression and regulation of CYP17A1 and 3β-hydroxysteroid dehydrogenase in cells of the nervous system: potential effects of vitamin D on brain steroidogenesis2018In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 113, p. 46-55Article in journal (Refereed)
    Abstract [en]

    Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3b-hydroxysteroid dehydrogenase (3b-HSD). 3b-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3b-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3b-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3b-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3b-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3b-HSD, particularly on the transcriptional level, may play a role in the nervous system.

  • 8.
    Emanuelsson, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Friman, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells2018In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544Article in journal (Refereed)
    Abstract [en]

    The active form of vitamin D (1,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 M. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.

  • 9. Hansson, Magnus
    et al.
    Wikvall, Kjell
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Babiker, Amir
    Regulation of sterol 27-hydroxylase in human monocyte-derived macrophages: up-regulation by transforming growth factor beta1.2005In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1687, no 1-3, p. 44-51Article in journal (Refereed)
  • 10.
    Hosseinpour, Fardin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ellfolk, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Phenobarbital suppresses vitamin D3 25-hydroxylase expression: A potential new mechanism for drug-induced osteomalacia2007In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 357, no 3, p. 603-607Article in journal (Refereed)
    Abstract [en]

    Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D3 due to decreased 25-hydroxylation of vitamin D3 in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D3 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.

  • 11.
    Hosseinpour, Fardin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ibranovic, Ines
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    25-Hydroxylation of vitamin D3 in primary cultures of pig hepatocytes:evidence for a role of both CYP2D25 and CYP27A12003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 303, no 3, p. 877-883Article in journal (Refereed)
    Abstract [en]

    There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).

  • 12.
    Hosseinpour, Fardin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Porcine microsomal vitamin D-3 25-hydroxylase (CYP2D25): Catalytic properties, tissue distribution, and comparison with human CYP2D62000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 44, p. 34650-34655Article in journal (Refereed)
    Abstract [en]

    The metabolic activation of the prohormone vitamin D(3) requires a 25-hydroxylation that has been reported to be catalyzed by both mitochondrial CYP27A and a microsomal vitamin D(3) 25-hydroxylase in the liver. CYP27A has been extensively studied, but its role as a physiologically important vitamin D(3) 25-hydroxylase has been questioned. The present paper reports that the microsomal vitamin D(3) 25-hydroxylase, purified from pig liver, converted vitamin D(3) into 25-hydroxyvitamin D(3) in substrate concentrations which are within the physiological range (apparent K(m) = 0.1 microm). The enzyme 25-hydroxylated vitamin D(3), 1 alpha-hydroxyvitamin D(3) and vitamin D(2) and also converted tolterodine, a substrate for human CYP2D6, into its 5-hydroxymethyl metabolite. Tolterodine inhibited the microsomal 25-hydroxylation, whereas quinidine, an inhibitor of CYP2D6, did not markedly inhibit the reaction. The primary structure of the microsomal vitamin D(3) 25-hydroxylase, designated CYP2D25, shows 77% identity with that of human CYP2D6. Northern blot and reverse transcription-polymerase chain reaction experiments revealed that CYP2D25 mRNA is expressed in higher levels in liver than in kidney and in small amounts in adrenals, brain, heart, intestine, lung, muscle, spleen, and thymus. Experiments with human liver microsomes and recombinantly expressed CYP2D6 strongly indicate that the microsomal 25-hydroxylation of vitamin D(3) in human liver is catalyzed by an enzyme different from CYP2D6.

  • 13.
    Lundell, Kerstin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hansson, Ronnie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cloning and expression of a pig liver taurochenodeoxycholic acid 6α-hydroxylase (CYP4A21): a novel member of the CYP4A subfamily2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 13, p. 9606-9612Article in journal (Refereed)
    Abstract [en]

    A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6 alpha -hydroxylase. This enzyme catalyzes a 6 alpha -hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6 alpha -hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6 alpha -hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.

  • 14.
    Lundell, Kerstin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gene structure of pig sterol 12alpha-hydroxylase (CYP8B1) and expression in fetal liver: comparison with expression of taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21)2003In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1634, no 3, p. 86-96Article in journal (Refereed)
    Abstract [en]

    Cholic acid is the major trihydroxy bile acid formed in most mammals. The domestic pig (Sus scrofa) is an exception. The bile of adult pig is devoid of cholic acid whereas hyocholic acid is found in amounts equal to that of cholic acid in humans. The pathway leading to formation of hyocholic acid is believed to be species-specific and to have evolved in the pig to compensate for a nonexistent or deficient cholic acid biosynthesis. However, a high level of cholic acid has recently been found in the bile of fetal pig. Here we describe that a gene encoding the key enzyme in cholic acid biosynthesis, the sterol 12alpha-hydroxylase (CYP8B1), is in fact present in the pig genome. The deduced amino acid sequence shows 81% identity to the human and rabbit orthologues. CYP8B1 mRNA is expressed at significant levels in fetal pig liver. Both CYP8B1 and the key enzyme in hyocholic acid formation, taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21), were found to be expressed in pig liver in a developmental-dependent but opposite fashion.

  • 15.
    Lundell, Kerstin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Species-specific and age-dependent bile acid composition: Aspects on CYP8B and CYP4A subfamilies in bile acid biosynthesis2008In: Current drug metabolism, ISSN 1389-2002, E-ISSN 1875-5453, Vol. 9, no 4, p. 323-331Article, review/survey (Refereed)
    Abstract [en]

    The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12 alpha or 6 alpha position, respectively, has a profound effect on the hydrophilic-hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12 alpha-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6 alpha-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6 alpha-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.

  • 16.
    Lundqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells2010In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1801, no 9, p. 1056-1062Article in journal (Refereed)
    Abstract [en]

    The current study presents data indicating that 1 alpha,25-dihydroxyvitamin D-3 affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1 alpha,25-dihydroxyvitamin D-3 suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1 alpha,25-dihydroxyvitamin D-3 on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1 alpha,25-dihydroxyvitamin D3. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally the 17 alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1 alpha,25-dihydroxyvitamin D-3-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1 alpha,25-dihydroxyvitamin D-3-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

  • 17.
    Lundqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism2011In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1811, no 4, p. 263-270Article in journal (Refereed)
    Abstract [en]

    It is well-known that 1α,25-dihydroxyvitamin D(3) and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D(3) has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D(3) on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D(3) treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D(3) exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D(3) stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D(3) exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D(3) as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.

  • 18.
    Lundqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Vitamin D-mediated regulation of CYP21A2 transcription – a novel mechanism for vitamin D action2012In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1820, no 10, p. 1553-1559Article in journal (Refereed)
    Abstract [en]

    Background

    1α,25-Dihydroxyvitamin D3 has recently been reported to decrease expression and activity of CYP21A2. In this paper, we have studied the mechanisms for the 1α,25-dihydroxyvitamin D3-mediated effect on CYP21A2 transcriptional rate.

    Methods

    We have studied the effects of 1α,25-dihydroxyvitamin D3 using luciferase reporter constructs containing different lengths of the CYP21A2 promoter. These constructs were transfected into cell lines derived from human and mouse adrenal cortex. The mechanism for the effects of vitamin D on the CYP21A2 promoter was studied using chromatin immunoprecipitation assay, mutagenesis and gene silencing by siRNA.

    Results

    1α,25-Dihydroxyvitamin D3 was found to alter the promoter activity via a VDR-mediated mechanism, including the comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). The involvement of comodulator VDIR was confirmed by gene silencing. We identified a vitamin D response element in the CYP21A2 promoter. Interaction between this novel response element and VDR, WSTF and VDIR was shown by chromatin immunoprecipitation assay. When this sequence was deleted, the effect of 1α,25-dihydroxyvitamin D3 was abolished, indicating that this sequence in the CYP21A2 promoter functions as a vitamin D response element. Interestingly, an altered balance between nuclear receptors and comodulators reversed the suppressing effect of vitamin D to a stimulatory effect.

    General significance

    This paper reports data important for the understanding of the mechanisms for vitamin D-mediated suppression of gene expression as well as for the vitamin D-mediated effects on CYP21A2. We report a novel mechanism for effects of 1α,25-dihydroxyvitamin D3.

  • 19. Nebert, Daniel W.
    et al.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Miller, Walter L.
    Human cytochromes P450 in health and disease2013In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 368, no 1612, p. 20120431-Article, review/survey (Refereed)
    Abstract [en]

    There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases-confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development.

  • 20.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andersson, Ulla
    Björkhem, Ingemar
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 44, p. 34046-34053Article in journal (Refereed)
    Abstract [en]

    A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.

  • 21.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lundqvist, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ellfolk, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hellström Pigg, Maritta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Gustafsson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Drug-mediated gene regulation of vitamin D3 metabolism in primary human dermal fibroblasts2017In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 120, no 1, p. 59-63Article in journal (Refereed)
    Abstract [en]

    Vitamin D metabolism was studied in primary human dermal fibroblasts with focus on drug-mediated gene regulation related to adverse side effects of antiretroviral drugs used in HIV therapy. The fibroblasts expressed mRNA for cytochrome P450 (CYP) enzymes catalysing bioactivating (CYP2R1, CYP27A1 and CYP27B1) and catabolic reactions (CYP24A1). The cells produced both 25-hydroxyvitamin D3 and 1a,25-dihydroxyvitamin D3. The results demonstrate that primary dermal fibroblasts have an active vitamin D3 metabolising system. High incidence of low bone mineral density is a concern for HIV-infected patients treated with antiretroviral drugs. Osteomalacia and severe vitamin D deficiency have been reported. We investigated whether drug-mediated gene regulation could be a possible mechanism behind these adverse drug effects. Fibroblasts were treated with different drugs used in HIV therapy and the 1a,25-dihydroxyvitamin D3 levels and relative mRNA-levels for crucial enzymes were determined. Efavirenz, stavudine and ritonavir significantly downregulated the bioactivating CYP2R1 and upregulated the catabolic CYP24A1. The drugs reduced bioactivating enzyme activities and cellular levels of 1a,25-dihydroxyvitamin D3. The current results indicate that effects on gene expression may lead to disturbed vitamin D-metabolism and decreased cellular levels of active vitamin D3. The data are consistent with the impaired bone health in patients treated with certain antiretroviral drugs.

  • 22.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Pettersson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Androgen receptor-mediated regulation of the anti-atherogenic enzyme CYP27A1 involves the JNK/c-jun pathway2011In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 506, no 2, p. 236-241Article in journal (Refereed)
    Abstract [en]

    CYP27A1, an enzyme with several important roles in cholesterol homeostasis and vitamin D3 metabolism, has been ascribed anti-atherogenic properties. This study addresses an important problem regarding how this enzyme, involved in cholesterol metabolism in the liver and peripheral tissues, is regulated. Our results identify the human CYP27A1 gene as a new target for the JNK/c-jun pathway. Initial experiments showed that an inhibitor of c-Jun N-terminal kinase (JNK) downregulated basal CYP27A1 promoter activity whereas overexpression of JNK slightly enhanced promoter activity. Androgen receptor (AR)-mediated upregulation of mRNA levels and endogenous enzyme activity was recently reported. In the present study, the AR antagonist nilutamide blocked the androgen induction of CYP27A1. The present data revealed that inhibition of the JNK/c-jun pathway abolishes the AR-mediated effect on CYP27A1 transcription and enzyme activity, whereas overexpression of JNK markedly increased androgenic upregulation of CYP27A1. In conclusion, the current results indicate involvement of the JNK/c-jun pathway in AR-mediated upregulation of human CYP27A1. The link to JNK signaling is interesting since inflammatory processes may upregulate CYP27A1 to clear cholesterol from peripheral tissues.

  • 23.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Toll, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bjorkhem, Ingemar
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    24-Hydroxycholesterol is a substrate for hepatic cholesterol 7 alpha-hydroxylase (CYP7A)2000In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 41, no 10, p. 1629-1639Article in journal (Refereed)
    Abstract [en]

    (24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.

  • 24.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    von Bahr, Sara
    Björkhem, Ingemar
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation2003In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 44, no 8, p. 1515-1522Article in journal (Refereed)
    Abstract [en]

    The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.

  • 25.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1)2010In: Atlas of Genetics and Cytogenetics in Oncology and Haematology, E-ISSN 1768-3262Article in journal (Refereed)
  • 26.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Enzymatic activation in vitamin D signaling: Past, present and future2023In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 742, article id 109639Article, review/survey (Refereed)
    Abstract [en]

    Vitamin D signaling is important in regulating calcium homeostasis essential for bone health but also displays other functions in cells of several tissues. Disturbed vitamin D signaling is linked to a large number of diseases. The multiple cytochrome P450 (CYP) enzymes catalyzing the different hydroxylations in bioactivation of vitamin D3 are crucial for vitamin D signaling and function. This review is focused on the progress achieved in identification of the bioactivating enzymes and their genes in production of 1α,25-dihydroxyvitamin D3 and other active metabolites. Results obtained on species- and tissue-specific expression, catalytic reactions, substrate specificity, enzyme kinetics, and consequences of gene mutations are evaluated. Matters of incomplete understanding regarding the physiological roles of some vitamin D hydroxylases are critically discussed and the authors will give their view of the importance of each enzyme for vitamin D signaling. Roles of different vitamin D receptors and an alternative bioactivation pathway, leading to 20-hydroxylated vitamin D3 metabolites, are also discussed. Considerable progress has been achieved in knowledge of the vitamin D3 bioactivating enzymes. Nevertheless, several intriguing areas deserve further attention to understand the pleiotropic and diverse activities elicited by vitamin D signaling and the mechanisms of enzymatic activation necessary for vitamin D-induced responses.

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  • 27.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Enzymes in the conversion of cholesterol into bile acids2007In: Current molecular medicine, ISSN 1566-5240, E-ISSN 1875-5666, Vol. 7, no 2, p. 199-218Article, review/survey (Refereed)
    Abstract [en]

    This article aims to give an overview on the characterization, properties and regulation of enzymes, particularly the cytochrome (CYP) P450 enzymes, in the formation of bile acids from cholesterol. Bile acids are biologically active molecules that promote absorption of dietary lipids in the intestine and stimulate biliary excretion of cholesterol. Bile acids and oxysterols, formed from cholesterol, act as ligands to nuclear receptors regulating the expression of important genes in cholesterol homeostasis. Thus, the bioactivation of cholesterol into bile acids is crucial for regulation of cholesterol homeostasis. The primary human bile acids, cholic acid and chenodeoxycholic acid, are formed from cholesterol via several pathways involving many different enzymes. Many of these enzymes are cytochrome P450 (CYP) enzymes, introducing a hydroxyl group in the molecule. The “classic” pathway of bile acid formation starts with a 7α-hydroxylation of cholesterol by CYP7A1 in the liver. The “acidic” pathway starts with a hepatic or extrahepatic 27-hydroxylation by CYP27A1. There also exist some quantitatively minor pathways which may be of importance under certain conditions. Formation of cholic acid requires insertion of a 12α-hydroxyl group performed by CYP8B1. Oxysterols are precursors to bile acids, participate in cholesterol transport and are known to affect the expression of several genes in cholesterol homeostasis. Enzymes with capacity to form and metabolize oxysterols are present in liver and extrahepatic tissues. The enzymes, nuclear receptors and transcription factors involved in bile acid biosynthesis are potential pharmaceutical targets for the development of new drugs to control hypercholesterolemia and to prevent atherosclerosis and other diseases related to disturbed cholesterol homeostasis. The review will also discuss some inborn errors of bile acid biosynthesis and the recently acquired knowledge on the genetic defects underlying these diseases.

  • 28.
    Pettersson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Andersson, Ulla
    Avd. för klinisk kemi, KI, Huddinge, Sverige.
    Pikuleva, Irina
    Department of clinical chemistry and toxicology, University of Texas medical branch, Galveston, USA.
    Björkhem, Ingemar
    Avd. för klinisk kemi. KI, Huddinge, Sverige.
    Misharin, Alexander Yu
    Inst. of biomedical chemisrty, Russian academy of medical sciences, Moscow, Russia.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Metabolism of a novel side chain modified Delta 8(14)-15-ketosterol, a potential cholesterol lowering drug: 28-hydroxylation by CYP27A12008In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1781, no 8, p. 383-390Article in journal (Refereed)
    Abstract [en]

    The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.

  • 29.
    Tang, Wanjin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Glucocorticoid receptor-mediated upregulation of human CYP27A1, a potential anti-atherogenic enzyme2008In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1781, no 11-12, p. 718-723Article in journal (Refereed)
    Abstract [en]

    Sterol 27-hydroxylase (CYP27A1) is required for the hepatic conversion of cholesterol into bile acids and for production of 27-hydroxycholesterol which affects cholesterol homeostasis in several ways. Dexamethasone increases hepatic bile acid biosynthesis and CYP27A1-mediated enzyme activity in HepG2 cells. This study examines the mechanism of the dexamethasone-induced effect on the human CYP27A1 promoter. Dexamethasone treatment of HepG2 cells overexpressed with glucocorticoid receptor alpha (GRalpha) increased the CYP27A1 promoter activity more than four-fold as compared with untreated cells. The GR-antagonist mifepristone almost completely abolished the dexamethasone-induced effect on the promoter activity. Progressive deletion analysis of the CYP27A1 promoter indicated that sequences involved in GR-mediated induction by dexamethasone are present in a region between -1094 and -792. Several putative GRE sites could be found in this region and EMSA experiments revealed that two of these could bind GR. Site-directed mutagenesis of GR-binding sequences in the CYP27A1 promoter identified a GRE at -824/-819 important for GR-mediated regulation of the transcriptional activity. Endogenous and pharmacological glucocorticoids may have a strong impact on several aspects of cholesterol homeostasis and other processes related to CYP27A1-mediated metabolism. The glucocorticoid-mediated induction of human CYP27A1 transcription is of particular interest due to the anti-atherogenic properties ascribed to this enzyme.

  • 30.
    Tang, Wanjin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Regulation of human CYP27A1 by estrogens and androgens in HepG2 and prostate cells2007In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 462, no 1, p. 13-20Article in journal (Refereed)
    Abstract [en]

    The regulation of the human CYP27A1 gene by estrogens and androgens was studied in human liver-derived HepG2 and prostate cells. Our results show that the promoter activity, enzymatic activity and mRNA levels of CYP27A1 in HepG2 cells are downregulated by estrogen in presence of ERα or ERβ. Similar effects by estrogen were found in RWPE-1 prostate cells. In contrast, estrogen markedly upregulated the transcriptional activity of CYP27A1 in LNCaP prostate cancer cells. 5α-Dihydrotestosterone and androgen receptor upregulated the transcriptional activity of CYP27A1 in HepG2 cells. Progressive deletion experiments indicate that the ERβ-mediated effects in HepG2 and LNCaP cells are conferred to the same region (−451/+42) whereas ERα-mediated effects on this promoter are more complex. The results indicate that the stimulating effect of androgen in HepG2 cells is conferred to a region upstream from –792 in the CYP27A1 promoter. In summary, we have identified the human CYP27A1 gene as a target for estrogens and androgens. The results imply that expression of CYP27A1 may be affected by endogenous sex hormones and pharmacological compounds with estrogenic or androgenic effects.

  • 31.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cytochrome P450 enzymes in the bioactivation of vitamin D to its hormonal form (review)2001In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 7, no 2, p. 201-209Article, review/survey (Refereed)
    Abstract [en]

    The formation of 1alpha,25-dihydroxyvitamin D3 requires a 25-hydroxylation followed by a 1alpha-hydroxylation catalyzed by cytochrome P450 (CYP) enzymes in liver and kidney. The aim of this review is to give a brief summary of our research on the cytochrome P450 enzymes catalyzing the 25-hydroxylation and 1alpha-hydroxylation and to discuss the results in relation to other published literature on these enzymes. Two hepatic P450 enzymes catalyzing 25-hydroxylation of vitamin D3 exist in mammalian liver - one mitochondrial and one microsomal. The mitochondrial vitamin D3 25-hydroxylase is apparently identical with CYP27A, an obligatory enzyme in bile acid biosynthesis in liver. The microsomal 25-hydroxylase has been purified to apparent homogeneity from pig liver. The enzyme catalyzed 25-hydroxylation of vitamin D3, 1alpha-hydroxyvitamin D3, vitamin D2 and 1alpha-hydroxyvitamin D2. A cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase has been isolated in this laboratory. The primary structure of vitamin D3 25-hydroxylase shows 70-80% identity with members of the CYP2D subfamily and has been designated CYP2D25. Three different 1alpha-hydroxylating cytochromes P450 in kidney, i.e. CYP27A, CYP27B and a microsomal 1alpha-hydroxylase, have been described. Mitochondrial cytochrome P450, catalyzing 1alpha-hydroxylation and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3, was partially purified from pig kidney. Purification and inhibition experiments as well as experiments with a monoclonal antibody against CYP27A indicated that one single enzyme catalyzes both 1alpha- and 27-hydroxylation. Treatment of rats with a single i.v. dose of 1alpha,25-dihydroxyvitamin D3 resulted in a marked suppression of CYP27A mRNA levels in kidney. The results suggest a role for CYP27A as a renal mitochondrial 1alpha-hydroxylase. Subsequently, several research groups reported the isolation of cDNA encoding mouse, rat and human kidney 25-hydroxyvitamin D3 1alpha-hydroxylase. The amino acid sequences deduced from these cDNA clones were similar but differed from that of CYP27A. This 1alpha-hydroxylase constitutes a new CYP27 subfamily, CYP27B. The expression of CYP27B was found to be influenced by vitamin D status and parathyroid hormone. Mutations in the CYP27B gene have been identified in patients with pseudovitamin D-deficiency rickets. A microsomal P450 catalyzing 1alpha-hydroxylation of 25-hydroxyvitamin D3 has been purified to apparent homogeneity from pig kidney. This finding demonstrate the presence of a microsomal 1alpha-hydroxylase in addition to the mitochondrial 1alpha-hydroxylases in kidney. The relative importance and regulation of the different renal 1alpha-hydroxylases in the bioactivation of vitamin D3 under normal and pathological conditions will be subject for future studies.

  • 32.
    Wikvall, Kjell
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ellfolk, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lundqvist, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Regulation of CYP enzymes in steroidogenesis with particular focus on cholesterol metabolism and vitamin D-related processes2009In: Proceedings of the 16th International Conference on Cytochrome P450, Nago, Okinawa, Japan, June 21-25 / [ed] Hirofumi Shoun and Hideo Ohkawa, Bologna: Medimond , 2009, p. 177-182Conference paper (Refereed)
    Abstract [en]

    CYP27A1 is an enzyme essential for cholesterol homeostasis and is considered an anti-atherogenic enzyme. CYP27A1 is required in bile acid biosynthesis and participates in oxysterol formation, cholesterol transport and cholesterol elimination (Fig 1). Considering these important functions, mechanisms for regulation of the human CYP27A1 gene are of great interest (1). CYP27A1 is also a vitamin D 25-hydroxylase, catalyzing the first step in the bioactivation of vitamin D into the multifunctional hormone 1,25-dihydroxyvitamin D. Fig 2 shows the bioactivation of vitamin D. Multiple vitamin D 25-hydroxylases exist in humans. In addition to CYP27A1, other known vitamin D 25-hydroxylases are CYP2R1, CYP2J2 and CYP3A4 (2). The active 1,25-dihydroxyvitamin D is a calcium-regulating hormone. Newly discovered functions of this hormone are regulation of cell growth, regulation of immune function and regulation of blood pressure and insulin production. In the current presentation, we focus on our recent studies on the regulation of CYP27A1 and the CYP enzymes catalyzing the first step in the bioactivation of vitamin D.

  • 33.
    Zayny, Ahmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Almokhtar, Mokhtar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ljunggren, Östen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrinology and mineral metabolism.
    Ubhayasekera, Kumari
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Kibar, Pinar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Effects of glucocorticoids on vitamin D3-metabolizing 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts2019In: Molecular and Cellular Endocrinology, ISSN 0303-7207, E-ISSN 1872-8057, Vol. 496, article id 110525Article in journal (Refereed)
    Abstract [en]

    Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to bone disorders. Long-term treatment with glucocorticoids results in osteoporosis and increased risk of fractures. Much remains unclear regarding the effects of these compounds in bone cells. In the current study, human osteosarcoma Saos-2 cells and primary human osteoblasts were found to express mRNA for the vitamin D receptor as well as activating and deactivating enzymes in vitamin D-3 metabolism. These bone cells exhibited CYP24A1-mediated 24-hydroxylation which is essential for deactivation of the active vitamin form. However, bioactivating vitamin D-3 hydroxylase activities could not be detected in either of these cells. Several glucocorticoids, including prednisolone, down regulated CYP24A1 mRNA and CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblasts. Also, prednisolone significantly suppressed a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells co-transfected with the glucocorticoid receptor. Thus, the results of the present study show suppression by glucocorticoids on CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of this study we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid with glucocorticoid activity, which can bind the glucocorticoid receptor. Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblasts suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may interfere with regulation of active vitamin D levels.

  • 34.
    Zufan, Araya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hormonal regulation of the human sterol 27-hydroxylase gene (CYP27A1)2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 372, no Part 2, p. 529-534Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence data reported in this paper will appear in EMBL Nucleotide Sequence Database under the accession number AJ 544720. The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D(3). Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Bj örkhem (1997) J. Biol. Chem. 272, 26253-26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5'-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2-3-fold, whereas thyroxine (T(4)) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T(4) and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position -1094 bp. The putative response elements for dexamethasone were mapped to positions between -792 and -1095 bp. The -451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T(4) and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

1 - 34 of 34
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