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Enzymatic Regulation of Steroidogenesis and Nuclear Receptor Activation: Special Focus on Vitamin D and Sex Hormones
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Steroid P450)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzyme-catalyzed reactions are important to regulate steroidogenesis and nuclear receptor activation. The present investigation examines the role of steroid metabolism catalyzed by CYP7B1 for regulation of hormone receptor activation and the effects of vitamin D on enzymatic regulation of steroidogenesis.

The study reports data indicating that CYP7B1 can regulate estrogenic signaling by converting estrogens into inactive or less active metabolites. Similar results were obtained for CYP7B1-mediated metabolism of some androgen receptor ligands, indicating that CYP7B1 can be involved also in the regulation of androgenic signaling. CYP7B1 substrates and metabolites were found to exert androgenic effects in a cell line-specific manner. Furthermore, cell line differences were observed in the expression pattern for androgen receptor comodulators.

This thesis reports that 1α,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of CYP21A2 and CYP17A1 leading to suppressed production of aldosterone, dehydroepiandrosterone and androstenedione in adrenocortical cells. These are novel findings on vitamin D action.

A mechanism is reported for the vitamin D-mediated regulation of the CYP21A2 gene. Data indicate that vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) are key comodulators in this novel vitamin D receptor (VDR)-mediated mechanism. Furthermore, the results indicate that altered expression levels of VDIR and WSTF can shift the suppressing effect of vitamin D to a stimulatory effect. Also, epigenetic components were found to be involved in the effects of vitamin D on CYP21A2 transcriptional rate. In addition, a functional vitamin D response element was identified in the CYP21A2 promoter.

This study also reports that 1α,25-dihydroxyvitamin D3 affects sex hormone production in a tissue-specific way. Gene expression and enzyme activity of aromatase were found to be downregulated in cells derived from breast, but not in cells derived from prostate and adrenal cortex. The production of estradiol and dihydrotestosterone was altered in a tissue-selective manner following vitamin D treatment. These findings are of importance for the discussion on vitamin D as a potential anti-breast cancer agent.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 146
Keyword [en]
adrenal steroidogenesis, CYP7B1, vitamin D, calcitriol, enzymatic regulation, transcriptional regulation, CYP21A2, aromatase, sex hormone, estrogen, androgen, nuclear receptor
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-151740ISBN: 978-91-554-8093-6OAI: oai:DiVA.org:uu-151740DiVA: diva2:413207
Public defence
2011-06-10, Uppsala Biomedical Center, room C4:301, Husargatan 3, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Funder
Swedish Research Council
Available from: 2011-05-20 Created: 2011-04-16 Last updated: 2011-07-01Bibliographically approved
List of papers
1. Effects of CYP7B1-mediated catalysis on estrogen receptor activation.
Open this publication in new window or tab >>Effects of CYP7B1-mediated catalysis on estrogen receptor activation.
2010 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1801, no 9, 1090-1097 p.Article in journal (Refereed) Published
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.

Keyword
CYP7B1, estrogen, hydroxylation, steroid metabolism, ER-mediated response
National Category
Pharmaceutical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-100776 (URN)10.1016/j.bbalip.2010.05.011 (DOI)000280627800012 ()20553962 (PubMedID)
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2011-07-01Bibliographically approved
2. Effects of CYP7B1-related steroids on androgen receptor activation in different cell lines
Open this publication in new window or tab >>Effects of CYP7B1-related steroids on androgen receptor activation in different cell lines
2012 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1821, no 7, 973-979 p.Article in journal (Refereed) Published
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.

Keyword
Androgen, CYP7B1, Hydroxylation, AR-mediated response, Steroid metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-151739 (URN)10.1016/j.bbalip.2012.03.007 (DOI)000305359200004 ()
Available from: 2011-04-16 Created: 2011-04-16 Last updated: 2012-08-01Bibliographically approved
3. 1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells
Open this publication in new window or tab >>1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells
2010 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1801, no 9, 1056-1062 p.Article in journal (Refereed) Published
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.

Keyword
Steroidogenesis, Calcitriol, 1 alpha, 25-dihydroxyvitamin D-3, Regulation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-135611 (URN)10.1016/j.bbalip.2010.04.009 (DOI)000280627800008 ()20420936 (PubMedID)
Available from: 2010-12-07 Created: 2010-12-07 Last updated: 2011-07-22Bibliographically approved
4. 1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism
Open this publication in new window or tab >>1α,25-Dihydroxyvitamin D3 exerts tissue-specific effects on estrogen and androgen metabolism
2011 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1811, no 4, 263-270 p.Article in journal (Refereed) Published
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.

Keyword
Vitamin D, Calcitriol, Androgens, Estrogens, Breast cancer, Aromatase
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Biochemistry
Identifiers
urn:nbn:se:uu:diva-146842 (URN)10.1016/j.bbalip.2011.01.004 (DOI)000288876400005 ()21262387 (PubMedID)
Available from: 2011-02-21 Created: 2011-02-21 Last updated: 2011-07-01Bibliographically approved
5. Vitamin D-mediated regulation of CYP21A2 transcription – a novel mechanism for vitamin D action
Open this publication in new window or tab >>Vitamin D-mediated regulation of CYP21A2 transcription – a novel mechanism for vitamin D action
2012 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1820, no 10, 1553-1559 p.Article in journal (Refereed) Published
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.

Keyword
Steroidogenesis, Vitamin D, Calcitriol, CYP21A2, Steroid
National Category
Chemical Sciences
Research subject
Pharmaceutical Biochemistry
Identifiers
urn:nbn:se:uu:diva-151457 (URN)10.1016/j.bbagen.2012.04.017 (DOI)000307369700014 ()
Available from: 2011-04-12 Created: 2011-04-12 Last updated: 2012-10-09Bibliographically approved

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