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Expression and regulation of steroid metabolizing enzymes in cells of the nervous and skeletal systems: Special focus on vitamin D metabolism
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Little is known about the mechanisms of vitamin D actions in the brain and bone. In this study, the metabolism of vitamin D and its regulation in various cell cultures of the nervous and skeletal systems were examined.

Human osteosarcoma Saos-2 cells, human primary osteoblasts (hOB) and murine motor neuron-like NSC-34 cells were found to express mRNA for all enzymes required in vitamin D3 metabolism as well as the vitamin D receptor (VDR) that mediates vitamin D actions. Also, production of 24,25-dihydroxyvitamin D3 was found in these cells. Studies on vitamin D metabolism in NSC-34 cells and in primary neuron-enriched cells from rat cerebral cortex indicate formation of a previously unknown major metabolite formed from 25-hydroxyvitamin D3. Evaluation of the NSC-34 cells suggests that this cell line could be a novel model for studies of neuronal vitamin D metabolism and its regulation by endogenous and exogenous compounds.

Treatment with glucocorticoids down regulated mRNA expression for the CYP24A1 gene in Saos-2 and hOB cells. Additionally, the glucocorticoid prednisolone showed suppression of CYP24A1-mediated metabolism and CYP24A1 promoter activity in Saos-2 cells. In NSC-34 cells, CYP24A1 mRNA levels were up-regulated by prednisolone, 1α,25-dihydroxyvitamin D3 and its synthetic analogues, EB1089 and tacalcitol. Formation of an endogenous glucocorticoid, 11-deoxycortisol, was observed in Saos-2 cells. Effects of glucocorticoids on the vitamin D system in bone cells may contribute to the adverse side effects in long-term treatment with glucocorticoids. Also, there may be a correlation between the administration of corticosteroids and adverse effects in the CNS.

Expression and effects of vitamin D on steroidogenic enzymes were studied in primary neuron-enriched rat cortex cells, primary rat astrocytes and human neuroblastoma SH-SY5Y cells. These different cell cultures all expressed CYP17A1, whereas only astrocytes expressed 3β-hydroxysteroid dehydrogenase (3β-HSD). 1α,25-Dihydroxyvitamin D3 suppressed mRNA levels and enzyme activity of CYP17A1 in SH-SY5Y cells and astrocytes. 1α,25-Dihydroxyvitamin D3 suppressed enzyme activity and mRNA levels of 3β-HSD in astrocytes. The results suggest that vitamin D-mediated regulation of CYP17A1 and 3β-HSD may play a role in the nervous system.

The results presented here contribute to our understanding of vitamin D metabolism and effects of glucocorticoids in the brain and bone.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 242
National Category
Chemical Sciences Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-333920ISBN: 978-91-513-0154-9 (print)OAI: oai:DiVA.org:uu-333920DiVA, id: diva2:1158259
Public defence
2018-01-18, B7:101a, BMC, Husargatan 3, Uppsala, 13:15 (Swedish)
Opponent
Supervisors
Available from: 2017-12-21 Created: 2017-11-19 Last updated: 2018-03-08
List of papers
1. Motor neuron-like NSC-34 cells as a new model for the study of vitamin D metabolism in the brain.
Open this publication in new window or tab >>Motor neuron-like NSC-34 cells as a new model for the study of vitamin D metabolism in the brain.
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2016 (English)In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 158, p. 178-188Article in journal (Refereed) Published
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.

National Category
Chemical Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-271604 (URN)10.1016/j.jsbmb.2015.12.010 (DOI)000372690200018 ()26704532 (PubMedID)
Funder
Swedish Research Council, 621-2008-3562, 621-2011-4423
Available from: 2016-01-11 Created: 2016-01-11 Last updated: 2017-12-01Bibliographically approved
2. Effects of glucocorticoids on vitamin D3 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts
Open this publication in new window or tab >>Effects of glucocorticoids on vitamin D3 24-hydroxylase (CYP24A1) in Saos-2 cells and primary human osteoblasts
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Vitamin D is essential for bone function and deficiency in active vitamin D hormone can lead to rickets, osteomalacia or osteoporosis. Long-term treatment with glucocorticoids is known to result in osteoporosis and a substantially increased risk of fractures. Although the actions of vitamin D and glucocorticoids play important roles for bone function and in the development of osteoporosis, much remains unclear regarding the effects of these compounds in cells of the bone. In the current study, the human osteosarcoma Saos-2 cell line and primary human osteoblast-like cells were found to express mRNA for the vitamin D receptor as well as both activating and deactivating enzymes in vitamin D3 metabolism. These bone cells exhibited the CYP24A1-mediated 24-hydroxylation, involved in deactivation of the active vitamin D3 form. However, bioactivating vitamin D3 hydroxylase activities could not be detected in either of these cells. Several commonly used therapeutic glucocorticoids, including prednisolone, down regulated mRNA expression for the CYP24A1 gene as well as the CYP24A1-mediated 24-hydroxylase activity in both Saos-2 and primary human osteoblast-like cells. Prednisolone had the strongest suppressive effect on CYP24A1 expression. Results from experiments with a human CYP24A1 promoter-luciferase reporter gene in Saos-2 cells, co-transfected with the glucocorticoid receptor, showed that treatment with prednisolone significantly suppresses the CYP24A1 promoter activity. Thus, the results of the present study showed suppression by glucocorticoids on expression of CYP24A1 mRNA, CYP24A1-mediated metabolism and CYP24A1 promoter activity in human osteoblast-like cells. As part of the present investigation we examined if glucocorticoids are formed locally in Saos-2 cells. The experiments indicate formation of 11-deoxycortisol, a steroid that has glucocorticoid activity and is able to bind to the glucocorticoid receptor.

Our data showing suppression by glucocorticoids on CYP24A1 expression in human osteoblast-like cells suggest a previously unknown mechanism for effects of glucocorticoids in human bone, where these compounds may act by increasing the normal levels of active vitamin D.

National Category
Chemical Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-333914 (URN)
Available from: 2017-11-18 Created: 2017-11-18 Last updated: 2017-11-19
3. Expression and regulation of CYP17A1 and 3β-hydroxysteroid dehydrogenase in cells of the nervous system: potential effects of vitamin D on brain steroidogenesis
Open this publication in new window or tab >>Expression and regulation of CYP17A1 and 3β-hydroxysteroid dehydrogenase in cells of the nervous system: potential effects of vitamin D on brain steroidogenesis
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2018 (English)In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 113, p. 46-55Article in journal (Refereed) Published
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.

Keywords
vitamin D, brain, metabolism, neurons, astrocytes, neurosteroids
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-333916 (URN)10.1016/j.neuint.2017.11.007 (DOI)000428495900005 ()29162485 (PubMedID)
Available from: 2017-11-18 Created: 2017-11-18 Last updated: 2018-07-19Bibliographically approved
4. Vitamin D metabolism in the nervous system: potential effects of glucocorticoids
Open this publication in new window or tab >>Vitamin D metabolism in the nervous system: potential effects of glucocorticoids
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Several studies have reported that neuronal function is influenced by vitamin D and it has been proposed that low serum 25-hydroxyvitamin D3 levels may be a risk factor for several brain disorders. However, little is known about the activation and metabolism as well as mechanisms of action of vitamin D in the neurons. In a previous study, we reported that the mouse motor neuron-like hybrid cell line NSC-34 expresses mRNA for CYP24A1 as well as the CYP24A1-mediated enzyme activity (Almokhtar et al., 2016). In contrast, the present results show that neither mRNA expression nor enzymatic activities of vitamin D3 metabolizing CYP enzymes could be detected in primary neuron-enriched cells from rat embryonic cortex. However, the levels of 25-hydroxyvitamin D3 incubated with cultured primary cells decreased substantially, indicating metabolism of this substrate. NSC-34 cells were found to produce, besides 24,25-dihydroxyvitamin D3, a major as yet unknown 25-hydroxyvitamin D3 metabolite in addition. The current results indicate drug-mediated regulation of vitamin D3 metabolism in cells of the nervous system. The results showing effects of prednisolone on expression of CYP24A1 mRNA, CYP24A1-mediated hydroxylase activity and 25-hydroxvitamin D3 consumption in NSC-34 cells are all consistent with an increased 25-hydroxyvitamin D3 metabolism. The results indicate that therapeutic treatment with glucocorticoids may lead to a potential decrease in active forms of vitamin D3 in brain cells. It might be speculated that administration of corticosteroids leading to reported CNS adverse effects may at least in part be due to effects of glucocorticoids on vitamin D3 metabolism.

National Category
Chemical Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-333917 (URN)
Available from: 2017-11-18 Created: 2017-11-18 Last updated: 2017-11-19

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