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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. Bodin, Karl
    et al.
    Andersson, Ulla
    Rystedt, Eva
    Ellis, Eva
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Pikuleva, Irina
    Eggertsen, Gösta
    Björkhem, Ingemar
    Diczfalusy, Ulf
    Metabolism of 4 beta -hydroxycholesterol in humans2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 35, p. 31534-31540Article in journal (Refereed)
    Abstract [en]

    One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.

  • 3.
    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.

  • 4.
    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.

  • 5.
    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.

  • 6.
    Emanuelsson, Ida
    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.
    Protective effects of 27-and 24-hydroxycholesterol against staurosporine-induced cell death in undifferentiated neuroblastoma SH-SY5Y cells2012In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 525, no 1, p. 44-48Article in journal (Refereed)
    Abstract [en]

    Alterations in cholesterol metabolism have been linked to several neurodegenerative disorders, including Alzheimer's disease, multiple sclerosis and Parkinson's disease. Brain cholesterol is metabolized to the oxysterols 24-hydroxycholesterol and 27-hydroxycholesterol. Disturbed levels of these oxysterols are found in neurodegenerative conditions. In the current study we examined the effects of 27- and 24-hydroxycholesterol on viability of human neuroblastoma SH-SY5Y cells treated with staurosporine, a toxic substance that induces apoptosis. Analyses using MTT assay and measurement of lactate dehydrogenase release showed that presence of 27-hydroxycholesterol counteracted the toxic effects of staurosporine on these cells. Also, 27-hydroxycholesterol significantly decreased the staurosporine-mediated induction of caspase-3 and -7, known to be important in apoptotic events. 24-Hydroxycholesterol had similar effects on viability as 27-hydroxycholesterol in low concentrations, although in higher concentrations this oxysterol exacerbated the toxic effects of staurosporine. From these findings it may be concluded that effects of oxysterols on cellular viability are strongly dependent on the concentration and on the type of oxysterol. Previous studies on oxysterols have reported that these compounds are pro-apoptotic or trigger pathological changes that result in neurodegeneration. The present data indicate that, during some conditions, oxysterols may have neuroprotective effects.

  • 7.
    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.

  • 8.
    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.

  • 9.
    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.
    Effects of CYP7B1-related steroids on androgen receptor activation in different cell lines2012In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1821, no 7, p. 973-979Article in journal (Refereed)
    Abstract [en]

    The widely expressed steroid hydroxylase CYP7B1 is involved in metabolism of a number of steroids reported to influence estrogen and androgen signaling. Several studies by us and other investigators have linked this enzyme to effects on estrogen receptor activation. In a previous report we examined the effect of CYP7B1-mediated hormone metabolism for estrogen-mediated response in kidney-derived HEK293 cells. In the current study we used an androgen response element (ARE) reporter system to examine androgen-dependent response of some CYP7B1 substrates and CYP7B1-formed metabolites in several cell lines derived from different tissues. The results indicate significantly lower androgen receptor activation by CYP7B1-formed steroid metabolites than by the corresponding steroid substrates, suggesting that CYP7B1-mediated catalysis may decrease some androgenic responses. Thus, CYP7B1-dependent metabolism may be of importance not only for estrogenic signaling but also for androgenic. This finding, that CYP7B1 activity may be a regulator of androgenic signaling by converting AR ligands into less active metabolites, is also supported by real-time RT-PCR experiment where a CYP7B1 substrate, but not the corresponding product, was able to stimulate known androgen-sensitive genes. Furthermore, our data indicate that the effects of some steroids on hormone response element reporter systems are cell line-specific. For instance, despite transfection of the same reporter systems, 5-androstene-3β,17β-diol strongly activates an androgen-dependent response element in prostate cancer cells whereas it elicits only ER-dependent responses in kidney HEK293 cells. Potential roles of cell-specific metabolism or comodulator expression for the observed differences are discussed.

  • 10.
    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.

  • 11.
    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.

  • 12.
    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.

  • 13.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP7B1 (cytochrome P450, family 7, subfamily B, polypeptide 1)2009In: Atlas of Genetics and Cytogenetics in Oncology and Haematology, E-ISSN 1768-3262, Vol. AprilArticle in journal (Refereed)
  • 14.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Effects of vitamin D in the nervous system: Special focus on interaction with steroid hormone signalling and a possible role in the treatment of brain cancer2020In: Journal of neuroendocrinology, ISSN 0953-8194, E-ISSN 1365-2826, Vol. 32, no 1, article id e12799Article, review/survey (Refereed)
    Abstract [en]

    The active vitamin D hormone, 1,25-dihydroxyvitamin D-3, exerts many physiological actions in the body, including effects on the nervous system. Studies of steroidogenesis in cells of the nervous system and elsewhere not only indicate that 1,25-dihydroxyvitamin D-3 affects steroidogenic pathways but also suggest varying responses in different cell types. For example, 1,25-dihydroxyvitamin D-3 stimulates the expression of aromatase in human glioma but not in human neuroblastoma cells or rat astrocytes. However, in astrocytes, 1,25-dihydroxyvitamin D-3 suppresses 3 beta-hydroxysteroid dehydrogenase and steroid 17-hydroxylase/lyase. Other studies indicate cross-talk between vitamin D signalling and signalling of oestrogens, progesterone or glucocorticoids. Reported data indicate synergistic effects of combinations of 1,25-dihydroxyvitamin D-3 and other steroid hormones on neuroinflammation, neurite outgrowth and neuroprotection. Also, dysregulation of steroid pathways affecting brain cells is found in vitamin D deficiency. Thus, several studies suggest that active vitamin D may affect steroid hormone synthesis and/or signalling in the nervous system, although the potential mechanisms for these responses remain unclear. 1,25-Dihydroxyvitamin D-3 suppresses proliferation in several cell types and is therefore of interest in cancer treatment. Also, epidemiological studies associate vitamin D levels with cancer risk or outcomes. Reported data on tumours of the nervous system are mainly on glioma, a common type of brain cancer. Expression of the vitamin D receptor in glioma tumours is associated with improved survival. Several studies show that 1,25-dihydroxyvitamin D-3 and vitamin D analogues (synthetic vitamin D-like compounds) suppress proliferation and migration in human vitamin D receptor-expressing glioma cell lines. Studies on mechanisms for actions of 1,25-dihydroxyvitamin D-3 or its analogues indicate regulation of cell cycle proteins and senescence markers. These compounds also show synergism in combination with other cancer therapies treating glioma. From the data available, vitamin D analogues emerge as interesting candidates for the future improved treatment of human glioma and possibly also other cancers of the nervous system.

  • 15.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Regulation of cellular steroid levels with special focus on oxysterol and estrogen metabolism2008In: Future Lipidology, ISSN 1746-0875, Vol. 3, no 3, p. 337-346Article, review/survey (Refereed)
    Abstract [en]

    Steroids play essential roles in a number of physiological systems. However, disturbed functions or abnormal levels of steroids can result in serious health problems. The cellular levels of steroids are regulated by enzymes and other factors that control the formation and elimination of these compounds. Recent findings on steroid action and metabolism have identified novel links between two groups of steroids, oxysterols and estrogens. A better understanding of cellular processes related to these steroids is of interest for developing future therapies in several conditions, including endocrine tumors and hyperlipidemia.

  • 16.
    Norlin, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Chiang, John Y. L.
    Transcriptional regulation of human oxysterol 7alpha-hydroxylase by sterol response element binding protein2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 316, no 1, p. 158-164Article in journal (Refereed)
    Abstract [en]

    Oxysterol 7alpha-hydroxylase (CYP7B1) metabolizes oxysterols, potent regulators of lipid homeostasis. Very little is known about transcriptional regulation of human CYP7B1. The present results indicate that sterol response element binding protein (SREBP), a family of oxysterol-responsive transcription factors that stimulates cholesterol synthesis, may be an important regulator of CYP7B1. SREBP suppressed a human CYP7B1 luciferase reporter gene in several cell lines, most markedly in rat hepatoma McA-RH7777 cells. An SREBP-1-responsive region was mapped to a GC-rich sequence in the proximal CYP7B1 promoter, containing binding sites for the basal transcriptional activator Sp1. Mutagenesis of this sequence abolished SREBP-1-mediated suppression. Data indicated that SREBP does not bind this sequence but affects the gene indirectly, probably via interaction with Sp1. Our findings indicate that CYP7B1 transcription is controlled by SREBP and reveal a link between oxysterol-sensitive regulators and oxysterol metabolism. We propose that CYP7B1 is important for regulating cellular sterol content and protects against oxysterol-mediated toxicity.

  • 17.
    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.

  • 18.
    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.

  • 19.
    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.

  • 20.
    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)
  • 21.
    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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  • 22.
    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.

  • 23.
    Olsson, Frida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sarri, Niki
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Papadopoulos, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lennartsson, 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.
    Effects of 1a,25-dihydroxyvitamin D-3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells2022In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 31, article id 101313Article in journal (Refereed)
    Abstract [en]

    The active hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D-3, is reported to have 1000s of biological targets. The growth-suppressive properties of 1 alpha,25-dihydroxyvitamin D-3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1 alpha,25-dihydroxyvitamin D-3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indi-cated suppression of p70-S6 kinase levels by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G cells, whereas the levels of PLC gamma, a target for phospholipid signaling, was slightly increased. Activation of STAT3, an important regulator of malignancy, was suppressed by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1 alpha,24-dihydroxyvitamin D-3), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1 alpha,25-dihydroxyvitamin D-3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumor-igenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.

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  • 24.
    Pettersson, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Holmberg, Lisa
    Axelson, Magnus
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    CYP7B1-mediated metabolism of dehydroepiandrosterone and 5alpha-androstane-3beta,17beta-diol--potential role(s) for estrogen signaling2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 8, p. 1778-1789Article in journal (Refereed)
    Abstract [en]

    CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3 beta-Adiol may play an important role for estrogen receptor beta-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor beta activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3 beta-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3 beta-Adiol (a CYP7B1 substrate) and 7 alpha-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor beta activation. The data obtained indicated that 3 beta-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor beta activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3 beta-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the K-cat/K-m values were in the same order of magnitude. A high dehydroepiandrosterone/3 beta-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3 beta-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3 beta-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3 beta-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor beta signaling.

  • 25.
    Pettersson, Hanna
    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.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Effects of CYP7B1-mediated catalysis on estrogen receptor activation.2010In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1801, no 9, p. 1090-1097Article in journal (Refereed)
    Abstract [en]

    Most of the many biological effects of estrogens are mediated via the estrogen receptors ER alpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ER beta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3 beta,17 beta-diol (Aene-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7 alpha-hydroxy-DHEA, previously suggested to affect ER beta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3 beta-Adiol. CYP7B1-mediated metabolism of 3 beta-Adiol has been proposed to influence ER beta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ER alpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7 alpha-hydroxymetabolite will result in loss of action.

  • 26.
    Pettersson, Hanna
    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.
    Oliw, Ernst
    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.
    CYP7B1-mediated metabolism of 5 alfa-androstane-3 alfa,17 beta-diol (3 alfa-Adiol): A novel pathway for potential regulation of the cellular levels of androgens and neurosteroids2009In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1791, no 12, p. 1206-1215Article in journal (Refereed)
    Abstract [en]

    The current study presents data indicating that 5alfa-androstane-3alfa,17beta-diol (3alfa-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3alfa-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABAA) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3a-hydroxylated steroids. 3alfa-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. Also, recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3alfa-Adiol hydroxylation. In addition, 3alfa-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3alfa-Adiol which may impact intracellular levels of dihydrotestosterone and GABAA-modulating steroids.

  • 27.
    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.

  • 28.
    Raykova, Doroteya
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kermpatsou, Despoina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Malmqvist, Tony
    Atlas Antibodies AB, Bromma, Sweden..
    Harrison, Philip J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rubin Sander, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stiller, Christiane
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leino, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ricardo, Sara
    Univ Porto, Fac Med, Porto, Portugal.;Univ Porto, Differentiat & Canc Grp, Inst Mol Pathol & Immunol, Inst Res & Innovat Hlth I3S,Univ Porto Ipatimu, Porto, Portugal.;Univ Inst Hlth Sci IUCS, Dept Sci, CESPU, Gandra, Portugal..
    Klemm, Anna H
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    David, Leonor
    Univ Porto, Fac Med, Porto, Portugal.;Univ Porto, Differentiat & Canc Grp, Inst Mol Pathol & Immunol, Inst Res & Innovat Hlth I3S,Univ Porto Ipatimu, Porto, Portugal..
    Spjuth, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vemuri, Kalyani
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Sundqvist, Anders
    Uppsala University, Science for Life Laboratory, SciLifeLab. 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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Klaesson, Axel
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kampf, Caroline
    Atlas Antibodies AB, Bromma, Sweden..
    Söderberg, Ola
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A method for Boolean analysis of protein interactions at a molecular level2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 4755Article in journal (Refereed)
    Abstract [en]

    Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

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    FULLTEXT01
  • 29.
    Rosén, Jacob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Stattin, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Uppsala Clinical Research Center (UCR). Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Lipcsey, Miklós
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care, Hedenstierna laboratory.
    Frithiof, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care, Hedenstierna laboratory.
    Malinovschi, Andrei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Lung- allergy- and sleep research.
    Hultström, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    ECG pathology and its association with death in critically ill COVID-19 patients, a cohort study.2021In: PLOS ONE, E-ISSN 1932-6203, Vol. 16, no 12, article id e0261315Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: We investigated the prevalence of ECG abnormalities and their association with mortality, organ dysfunction and cardiac biomarkers in a cohort of COVID-19 patients admitted to the intensive care unit (ICU).

    METHODS: This cohort study included patients with COVID-19 admitted to the ICU of a tertiary hospital in Sweden. ECG, clinical data and laboratory findings during ICU stay were extracted from medical records and ECGs obtained near ICU admission were reviewed by two independent physicians.

    RESULTS: Eighty patients had an acceptable ECG near ICU-admission. In the entire cohort 30-day mortality was 28%. Compared to patients with normal ECG, among whom 30-day mortality was 16%, patients with ECG fulfilling criteria for prior myocardial infarction had higher mortality, 63%, odds ratio (OR) 9.61 (95% confidence interval (CI) 2.02-55.6) adjusted for Simplified Acute Physiology Score 3 and patients with ST-T abnormalities had 50% mortality and OR 6.05 (95% CI 1.82-21.3) in univariable analysis. Both prior myocardial infarction pattern and ST-T pathology were associated with need for vasoactive treatment and higher peak plasma levels of troponin-I, NT-pro-BNP (N-terminal pro-Brain Natriuretic Peptide), and lactate during ICU stay compared to patients with normal ECG.

    CONCLUSION: ECG with prior myocardial infarction pattern or acute ST-T pathology at ICU admission is associated with death, need for vasoactive treatment and higher levels of biomarkers of cardiac damage and strain in severely ill COVID-19 patients, and should alert clinicians to a poor prognosis.

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    fulltext
  • 30.
    Svenningsen, Åsa Fex
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Wicher, Grzegorz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. 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.
    Pettersson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Corell, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism2011In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 58, no 6, p. 620-624Article in journal (Refereed)
    Abstract [en]

    The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1-mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor β, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism.

  • 31.
    Tang, Wanjin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Eggertsen, Gösta
    Chiang, John Y.L.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Estrogen-mediated regulation of CYP7B1: a possible role for controlling DHEA levels in human tissues2006In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 100, no 1-3, p. 42-51Article in journal (Refereed)
    Abstract [en]

    The current study examines regulation of CYP7B1, a DHEA 7alpha-hydroxylase, by sex hormones. Transfection with estrogen receptor alpha and treatment with 17beta-estradiol in human embryonic kidney 293 cells significantly increased CYP7B1 catalytic activity and mRNA, and stimulated a human CYP7B1 reporter gene. Transfection with estrogen receptor beta showed similar but less significant effects. In the absence of receptors, 17beta-estradiol suppressed CYP7B1 activity, suggesting that estrogenic effects may be different in cells not expressing receptors. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. DHEA secreted by fetal adrenals is an essential precursor for placental estrogen formation. Since CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway, estrogen receptor-mediated up-regulation of CYP7B1 should lead to less DHEA available for sex hormone synthesis and may help to maintain normal levels of estrogens and androgens in human tissues, especially during fetal development. Regulation by estrogens may also be of importance in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function.

  • 32.
    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.
    Regulation of steroid hydroxylase CYP7B1 by androgens and estrogens in prostate cancer LNCaP cells2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 344, no 2, p. 540-546Article in journal (Refereed)
    Abstract [en]

    The present study reports effects of androgens and estrogens on human CYP7B1 transcription in prostate cancer LNCaP cells. Studies with rodents have suggested a role for the CYP7B1 enzyme in balancing cellular hormone levels important for prostate growth. Little is, however, known about the regulation of human CYP7B1. The current study showed strong suppression of a human CYP7B1 luciferase reporter gene by dihydrotestosterone (DHT) in prostate cancer LNCaP cells. Also, DHT and overexpression of androgen receptor (AR) suppressed CYP7B1 promoter activity and CYP7B1-mediated catalysis in kidney-derived HEK293 cells. Effects on CYP7B1 transcription were observed also by estrogen receptors (ER). The effects appeared different for different estrogens. CYP7B1 was stimulated by synthetic ER agonists but suppressed by 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol in LNCaP cells. Our data indicate an important role for CYP7B1 in balancing prostate hormone levels in human cells. In particular, the data suggest that androgens may control intraprostatic levels of estrogen via regulation of CYP7B1-mediated metabolism.

  • 33.
    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.
    Regulation of steroid hydroxylase CYP7B1 by androgens and estrogens in prostate cancer LNCaP cells2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 344, no 2, p. 540-546Article in journal (Refereed)
    Abstract [en]

    The present study reports effects of androgens and estrogens on human CYP7B1 transcription in prostate cancer LNCaP cells. Studies with rodents have suggested a role for the CYP7B1 enzyme in balancing cellular hormone levels important for prostate growth. Little is, however, known about the regulation of human CYP7B1. The current study showed strong suppression of a human CYP7B1 luciferase reporter gene by dihydrotestosterone (DHT) in prostate cancer LNCaP cells. Also, DHT and overexpression of androgen receptor (AR) suppressed CYP7B1 promoter activity and CYP7B1-mediated catalysis in kidney-derived HEK293 cells. Effects on CYP7B1 transcription were observed also by estrogen receptors (ER). The effects appeared different for different estrogens. CYP7B1 was stimulated by synthetic ER agonists but suppressed by 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol in LNCaP cells. Our data indicate an important role for CYP7B1 in balancing prostate hormone levels in human cells. In particular, the data suggest that androgens may control intraprostatic levels of estrogen via regulation of CYP7B1-mediated metabolism.

  • 34.
    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.

  • 35.
    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.

  • 36.
    Tang, Wanjin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Pettersson, Hanna
    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.
    Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling2008In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 112, no 1-3, p. 63-73Article in journal (Refereed)
    Abstract [en]

    The steroid hydroxylase CYP7B1 metabolizes neurosteroids, cholesterol derivatives, and estrogen receptor (ER) ligands. Previous studies identified CYP7B1 as a target for regulation by estrogen. The present study examines the mechanism for estrogen-mediated regulation of the human CYP7B1 gene promoter. Treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), abolished ER-mediated up-regulation of a CYP7B1 promoter-luciferase reporter in HepG2 cells, whereas overexpression of PI3K or Akt significantly increased estrogenic up-regulation of CYP7B1. Overexpression of dominant-negative mutant Akt abolished ER-mediated stimulation of CYP7B1 in HepG2 cells. Data indicated no binding of ER to CYP7B1 promoter sequences, suggesting that ER interacts with the PI3K/Akt pathway without binding to the gene. At low ER levels, overexpression of Akt suppressed CYP7B1 promoter activity, suggesting that its effect on CYP7B1 is different when estrogens are absent. In HEK293 cells, CYP7B1 transcription was much less affected by Akt, indicating that the mechanism for up-regulation of CYP7B1 is different in different cell types. Other experiments indicated that MAPK signalling may affect basal CYP7B1 levels. The current results, indicating that regulation of CYP7B1 by ER can be mediated via the PI3K/Akt signal pathway, a regulatory pathway important for cellular survival and growth, suggest an important role for CYP7B1 in cellular growth, particularly in connection with estrogenic signalling.

  • 37.
    Tevell, Annica
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry. Analytisk Farmaceutisk kemi.
    Lennernäs, Hans
    Department of Pharmacy.
    Jönsson, Mats
    Norlin, Maria
    Department of Pharmaceutical Biosciences. Farm biokemi.
    Lennernäs, Bo
    Bondesson, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Medicinal Chemistry. Analytisk Farmaceutisk Kemi.
    Hedeland, Mikael
    Flutamide metabolism in four different species in vitro and identification of flutamide metabolites in human patient urine by high performance liquid chromatography/tandem mass spectrometry.2006In: Drug Metab Dispos, ISSN 0090-9556, Vol. 34, no 6, p. 984-92Article in journal (Refereed)
  • 38.
    Wicher, Grzegorz
    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.
    Estrogen-mediated regulation of steroid metabolism in rat glial cells; effects on neurosteroid levels via regulation of CYP7B1-mediated catalysis2015In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 145, p. 21-27Article in journal (Refereed)
    Abstract [en]

    Many neuroactive steroids, including dehydroepiandrosterone (DHEA), pregnenolone, 27-hydroxycholesterol and 17 beta-estradiol, are known to affect development and function of the brain and nervous system. These and other steroids can undergo tissue and/or cell-specific enzymatic conversions into steroid metabolites. Carefully regulated production of steroids with various physiological effects is important for cells of the nervous system. Astrocytes express many steroidogenic enzymes and are considered important producers of brain steroids. The quantitative roles of different pathways for steroid metabolism in rat astrocytes are not clear. In the current study we examined effects of estrogens on steroid metabolism catalyzed by CYP7B1 and other enzymes in primary cultures of rat astrocytes. The CYP7B1 enzyme, which has been linked to neurodegenerative disease, is involved in the metabolism of several important neurosteroids. In the present study, we found that 7 alpha-hydroxylation, performed by CYP7B1, is the quantitatively most important pathway for DHEA metabolism in rat astrocytes. In addition, our present experiments on catalytic steroid conversions revealed that estrogens significantly suppress the CYP7B1-catalyzed metabolism of not only DHEA but also of pregnenolone and 27-hydroxycholesterol in rat astrocytes. These novel findings point to a regulatory mechanism for control of the cellular levels of these neurosteroids via CYP7B1. Our hypothesis that estrogens can regulate neurosteroid levels via this enzymatic reaction was supported by experiments using ELISA to assay levels of DHEA and pregnenolone in the presence or absence of estrogen. Furthermore, the present results show that estrogen suppresses CYP7B1-catalyzed 7 alpha-hydroxylation also in primary cultures of rat Schwann cells, indicating that regulation by estrogen via this enzyme may be of relevance in both the CNS and the PNS. 

  • 39.
    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.

  • 40.
    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.

1 - 40 of 40
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