“These experiments clearly demonstrate the existence of a fourth vitamin whose specific property, as far as we can tell at present, is to regulate the metabolism of the bones.” McCollum et al., Journal of Biological Chemistry, 1922

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Pettersson, H., Lundqvist, J. and Norlin, M.

Effects of CYP7B1-mediated catalysis on estrogen receptor ac-tivation. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1801:1090–1097 (2010)

II Lundqvist, J. and Norlin, M. Effects of CYP7B1-related steroids on hormone receptor activa-tion in different cell lines. Manuscript

III Lundqvist, J., Norlin, M. and Wikvall, K. 1 ,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1801:1056–1062 (2010)

IV Lundqvist, J., Norlin, M. and Wikvall, K. 1 ,25-Dihydroxyvitamin D3 exerts tissue-specific effects on es-trogen and androgen metabolism. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1811:263–270 (2011)

V Lundqvist, J., Norlin, M. and Wikvall, K. Vitamin D-mediated regulation of CYP21A2 transcription – a novel mechanism for vitamin D action. Submitted

Reprints were made with permission from the publisher.

Contents

Introduction...................................................................................................11 Production and action of steroid hormones ..............................................11

Adrenal steroidogenesis.......................................................................11 Sex hormone production......................................................................14 The role of steroid 7 -hydroxylation ..................................................15 Estrogen and androgen receptors.........................................................16 Sex hormone-dependent cancers .........................................................16

From vitamin D towards hormone D .......................................................17 Production, bioactivation and metabolism...........................................18 Mode of action.....................................................................................19 Vitamin D and adrenal steroidogenesis ...............................................20 Vitamin D and sex hormones ..............................................................20 Vitamin D and breast cancer................................................................21

Aims of the present investigation..................................................................22

Experimental procedures ..............................................................................23 Materials...................................................................................................23 Cell culture and treatment ........................................................................23 Analysis of steroid hormone secretion .....................................................24 Reverse transcriptase-polymerase chain reaction (RT-PCR) ...................24 Quantitative real time RT-PCR ................................................................25 Assays of enzyme activity........................................................................25 Analysis of nuclear receptor activation ....................................................26 Analysis of CYP21A2 promoter activity .................................................26 Gene silencing by small interfering RNA ................................................27 In silico analysis of gene promoters .........................................................27 Mutagenesis..............................................................................................27 Statistical analysis ....................................................................................27

Results and discussion ..................................................................................28 Effects of CYP7B1-related steroids on estrogen receptor activation .......28 Effects of CYP7B1-related steroids on androgen receptor activation......30

Discussion on the cell line differences ................................................31 Effects of 1 ,25-dihydroxyvitamin D3 on adrenal steroidogenesis .........33

Effects of vitamin D on gene expression .............................................34

Effects of vitamin D on hormone production and enzyme activity .....34 Discussion on adrenal bioactivation of vitamin D...............................35

Tissue-selective effects of vitamin D on sex hormone production ..........35 Vitamin D as a potential anti-breast cancer agent ...............................37 NCI-H295R cells as a model to study sex hormone production..........37 Discussion on putative mechanisms for tissue-selective regulation of aromatase .............................................................................................38

Mechanism for vitamin D-mediated effects on CYP21A2 ......................38 Effects of nuclear receptor and comodulator expression level ............39 Epigenetic mechanisms .......................................................................39 Identification of a functional VDRE....................................................40

Conclusions...................................................................................................41

Populärvetenskaplig sammanfattning ...........................................................42

Acknowledgements.......................................................................................44

References.....................................................................................................46

Abbreviations

3 -Adiol 5 -androstane-3 ,17 -diol 3 -Atriol 5 -androstane-3 ,7 ,17 -triol 3 -HSD 3 -hydroxysteroid dehydrogenase 5-Aza 5-aza-2’-deoxycytidine 7 -hydroxy-DHEA 7 -hydroxy-dehydroepiandrosterone 17 -HSD 17 -hydroxysteroid dehydrogenase Aene-diol 5-androstene-3 ,17 -diol Aene-triol 5-androstene-3 ,7 ,17 -triol AR androgen receptor ARE androgen response element bp base pair CAH congenital adrenal hyperplasia Calcidiol 25-hydroxyvitamin D3 Calcitriol 1 ,25-dihydroxyvitamin D3 CAR constitutive androstane receptor CYP cytochrome P450 DHEA dehydroepiandrosterone DHEA-S dehydroepiandrosterone sulfate DHT dihydrotestosterone DMSO dimethyl sulfoxide DNA deoxyribonucleic acid ELISA enzyme-linked immunosorbent assay ER estrogen receptor ER estrogen receptor ERE estrogen response element estradiol 17 -estradiol GAPDH glyceraldehyde-3-phosphate dehydrogenase HPLC high-performance liquid chromatography mRNA messenger ribonucleic acid NCoR-1 nuclear receptor co-repressor 1 nVDRE negative vitamin D responsive element POR P450 oxidoreductase pVDRE positive vitamin D responsive element RLU relative light units RT-PCR reverse transcriptase-polymerase chain reaction RXR retinoid X receptor

SF-1 steroidogenic factor-1 SMRT nuclear receptor co-repressor 2 SRA homo sapiens steroid receptor RNA activator SRC-1 human steroid receptor coactivator StAR steroidogenic acute regulatory protein SULT2A1 sulfotransferase 2A1 TBP TATA box binding protein TLC thin layer chromatography TSA trichostatin A UV ultraviolet VDIR VDR interacting repressor VDR vitamin D receptor VDRE vitamin D responsive element WSTF Williams syndrome transcription factor

11

Introduction

Production and action of steroid hormones All steroid hormones are synthesized from the common precursor choles-terol, which can be obtained from the diet or de novo synthesized from ace-tyl CoA. The production of steroid hormones is regulated via a number of enzymes of which a majority belongs to the cytochrome P450 (CYP) super-family. An overview of steroids and enzyme-catalyzed reactions of impor-tance for this thesis is shown in figures 1, 2 and 3.

Steroid hormones exert a wide range of physiological responses, includ-ing functions in the immune system, protein and carbohydrate metabolism, water and salt balance, reproductive system and development of sexual char-acteristics. Steroid hormones are synthesized in steroidogenic tissues such as the adrenal cortex, breast, ovaries, prostate and testis, either from cholesterol or from steroidogenic precursors secreted from other steroidogenic tissues. In the work presented in this thesis, enzymatic regulation of the production and action of steroid hormones has been investigated.

Adrenal steroidogenesis The adrenal cortex produces steroid hormones such as aldosterone, corticos-terone, cortisol, dehydroepiandrosterone (DHEA) and androstenedione. The adrenal steroidogenesis is quantitatively regulated by the transcription of CYP11A1 (cholesterol side-chain cleavage enzyme) and the activity of ster-oidogenic acute regulatory protein (StAR).

There are three adrenocortical zones, each with a distinct role in the pro-duction of steroid hormones; zona glomerulosa produces mineralocorticoids (e.g. aldosterone), zona fasciculata produces glucocorticoids (e.g. cortisol) and zona reticularis is the point of synthesis for adrenal androgens (e.g. DHEA). The qualitative regulation of adrenal steroidogenesis, determining which type of steroid that will be produced, is performed by the transcription and activity of CYP17A1. CYP17A1 catalyzes two different reactions, namely the 17 -hydroxylation and the 17,20-lyase reaction. The expression and activity of CYP17A1 differs between the three adrenal zones [1-3]. CYP17A1 is not expressed in zona glomerulosa leading to the produc-

12

Figure 1. Enzyme-catalyzed reactions in the adrenal steroidogenesis that are of im-portance for the work presented in this thesis.

tion of mineralocorticoids. In zona fasciculata, CYP17A1 is expressed and catalyzing 17 -hydroxylation, but not the 17,20-lyase activity, leading to glucocorticoid production. In zona reticularis, CYP17A1 catalyzes both 17 -hydroxylation and 17,20-lyase activities and adrenal androgens are therefore the main product of adrenal steroidogenesis in this adrenal zone [1]. Hence, in order to control the production of steroid hormones it is neces-sary to control the two activities of CYP17A1 separately. It is well-known that the manners of regulation for the two activities of CYP17A1 are princi-pally different. The 17 -hydroxylase activity is regulated via the expression

13

of the CYP17A1 gene, while the 17,20-lyase activity is regulated by post-transcriptional mechanisms [1-7]. The adrenal zone-specific activities of CYP17A1 are summarized in figure 2.

Three posttranscriptional mechanisms for regulation of 17,20-lyase activ-ity have been described; the abundance of P450 oxidoreductace (POR) [8, 9], allosteric action of cytochrome b5 [10] and serine phosphorylation of CYP17A1 [11-14]. Isolated 17,20-lyase deficiency is a rare condition first described by Zachmann et al. 1972 [15]. It has recently been reported that the 17,20-lyase deficiency could be a result of a mutated cytochrome b5 [16]. CYP17A1 deficiency, or impaired CYP17A1 activity due to altered posttranscriptional mechanisms, may lead to hypertension, hypokalemia and impaired development of sexual characteristics due to decreased production of adrenal androgens. Patients that are genetically male often present com-plete male pseudohermaphroditism while female patients may be infertile [17-19].

It has been reported that CYP17A1 strongly prefers 17 -hydroxypregnenolone over 17 -hydroxyprogesterone as a substrate for 17,20-lyase activity in humans [1, 20]. 17 -Hydroxyprogesterone may, however, be a substrate for CYP17A1 in other species [20].

CYP21A2 is a steroid 21-hydroxylase catalyzing the production of 11-deoxycorticosterone and 11-deoxycortisol, which are precursors for the pro-duction of corticosterone, aldosterone and cortisol. 21-Hydroxylase defi-ciency may lead to severe conditions such as congenital adrenal hyperplasia (CAH) and Addison´s disease [21]. CYP21A2 is exclusively expressed in the adrenal cortex [1]. Therefore, glucocorticoids and mineralocorticoids can not be synthesized in other tissues than the adrenal cortex.

14

Figure 2. Adrenal zone-specific activities of CYP17A1.

Sex hormone production Sex hormones are mainly produced in the gonads, breast and prostate. The sex hormones in these tissues are produced either by in situ synthesis from cholesterol or by enzyme catalyzed conversion of DHEA or androstenedione excreted to the circulation from the adrenal cortex.

The sex hormones are divided into two groups; androgens and estrogens. Androgens such as testosterone and 5 -dihydrotestosterone (DHT) are pro-duced via reactions catalyzed by 17 -hydroxysteroid dehydrogenase (17 -HSD) and 5 -reductase. Estrogens, such as 17 -estradiol (estradiol), are produced by aromatization of androgenic precursors, a reaction catalyzed by CYP19A1 (aromatase). Hence, the tissue-selective expression and activity of 5 -reductase and aromatase regulates the production of androgens and es-trogens [22-24]. Enzyme-catalyzed reactions in the sex hormone production of importance for this thesis are summarized in figure 3. For some of these steroids, it remains unclear if they act as estrogens or as androgens or if they are inactive metabolites [25]. To fully understand the estrogenic and/or an-drogenic signaling exerted by these steroids is of utmost importance in re-search on eg. breast and prostate carcinogenesis.

It is crucial to regulate the levels of sex hormones in order to achieve normal gonadal development. Abnormally high levels of estrogens and an-drogens are associated with increased risk for breast cancer and prostate cancer, respectively [23, 26-28].

15

Figure 3. Enzyme-catalyzed reactions in the sex hormone production.

The role of steroid 7 -hydroxylation The widely expressed steroid 7 -hydroxylase CYP7B1 is involved in the metabolism of a number of steroids reported to influence estrogenic and androgenic signaling. For instance, CYP7B1 converts DHEA, 5-androstene-3 ,17 -diol (Aene-diol) and 5 -androstane-3 ,17 -diol (3 -Adiol), to the corresponding 7 -hydroxy steroids [29-31]. Furthermore, it has recently been reported that 5 -androstane-3 ,17 -diol is a substrate for CYP7B1 [32]. The effect of this CYP7B1-catalyzed conversion of steroids on their ability to exert estrogenic and androgenic effects has been disputed. Some researchers have suggested that CYP7B1 substrates, but not products, may stimulate estrogen receptor (ER ), indicating that CYP7B1 activity could be a mechanism to decrease ER activity [33]. Other researchers have pro-posed that CYP7B1 products, but not substrates, can activate ER , suggest-ing that CYP7B1 activity would increase estrogenic signaling [34].

Conflicting data have been published regarding the effect of DHEA and 7 -hydroxy-DHEA on the androgen receptor. Mo et al. [35] have reported that DHEA, but not 7 -hydroxy-DHEA, exerts androgenic effects. Chen et al. [36] suggests that DHEA rather acts as an antagonist to the androgen receptor and Evaul et al. [37] have reported that DHEA has to be converted to androstenedione, a reaction catalyzed by 3 -hydroxysteroid dehydro-genase (3 -HSD), to gain androgenic effect. Aene-diol has been reported to

16

have androgenic effects by some researchers [38-40], while other researchers state that Aene-diol has to be converted to testosterone to gain androgenic properties [37]. In conclusion, the role of these CYP7B1 substrates and me-tabolites, and thereby the role of CYP7B1 activity, in estrogenic and andro-genic signaling is still not fully understood.

Estrogen and androgen receptors Estrogens and androgens exert most of their effects via nuclear receptors. Estrogenic effects are mediated by estrogen receptor (ER ) and estrogen receptor (ER ) while androgenic response is mediated by the androgen receptor (AR). These receptors are nuclear transcription factors that, when ligand activated, are able to regulate gene expression.

The two estrogen receptors are encoded by different genes and have sepa-rate expression patterns. ER is expressed in breast, liver, bone, brain, uro-genital tract and in the cardiovascular system while ER is expressed in breast, bone, urogenital tract, gastrointestinal tract, lung, cardiovascular sys-tem and in the brain [41]. Estrogens can either activate both ER and ER or be a specific ligand for one of the receptors. When ligand activated, the es-trogen receptors alter gene expression of estrogen sensitive genes by dimer formation (ER :ER , ER :ER or ER :ER ). The ligand activated receptor dimer binds to an estrogen receptor response element in the gene promoter and alters the transcriptional rate via recruitment of comodulators. Estrogens have also been found to exert rapid responses, which have been suggested to be mediated by membrane-bound receptors [42].

The androgen receptor is expressed in neuroendocrine and musculoskele-tal tissues and in the male urogenital system [43]. Ligand-activated androgen receptor alters gene expression of androgen-sensitive genes by a mechanism resembling the one for estrogen receptors. Ligand-activated androgen recep-tor forms a homodimer, binds to an androgen response element in the gene promoter and alters the transcriptional rate by recruitment of comodulators.

Sex hormone-dependent cancers Sex hormone signaling plays important roles in both breast and prostate car-cinogenesis.

A large majority of all breast cancers are classified as estrogen receptor positive, and most of these tumors rely on estrogens to proliferate [26]. For estrogen-dependent carcinomas arising in postmenopausal women, when the ovarian estrogen production is decreased, the local estrogen production in breast is crucial for the tumor development [44]. Therefore, regulation of estrogenic signaling and estrogen production has become key strategies in breast cancer treatment [23, 26]. The enzyme aromatase catalyzes the con-version of androgenic precursors to estrogens. The gene expression of aro-

17

matase is regulated by different promoters in a tissue-specific manner [24, 45]. The aromatase expression is higher in breast cancer tissue than in nor-mal breast tissue and the local estrogen levels in breast cancer tissue are higher than the circulating levels [46, 47]. Treatment with the antiestrogen tamoxifen has been first line endocrine therapy for more than thirty years, but today treatment with a third generation aromatase inhibitor like letrozole, anastrozole or exemestane is the recommended first line endocrine therapy for most postmenopausal women. However, treatment with both aromatase inhibitors and anti-estrogens are associated with risk of adverse effects (eg. osteoporosis) due to effects on peripheral estrogen metabolism [48, 49].

Androgenic signaling is important in a broad range of physiological proc-esses, including normal prostate development and prostate carcinogenesis. A majority of all prostate cancers are androgen dependent when diagnosed (ie. they rely on androgens to proliferate) [50]. Furthermore, cells in benign prostate hyperplasia rely on androgens to proliferate. Therefore, decreased androgen production and/or inhibited androgenic signaling have become key strategies in the treatment of prostate cancer and benign prostate hyperplasia [51] (eg. antiandrogens and 5 -reductase inhibitors). As an alternative strat-egy to decrease androgen production, a CYP17A1 inhibitor has undergone a phase III clinical trial as a treatment against castration-resistant prostate can-cer [52]. Furthermore, ER is expressed in the prostate and has been pro-posed to be involved in prostate growth inhibition [53, 54], although the role of estrogenic signaling in the regulation of prostate proliferation remains unclear.

From vitamin D towards hormone D Vitamin D was discovered in the early 20th century when it was described that rickets could be cured by sunlight or cod liver oil. This fat-soluble vita-min was the fourth vitamin described and therefore designated as vitamin D. Vitamin D was found to regulate intestinal absorption and renal reabsorption of calcium and bone metabolism. Later research has demonstrated that vita-min D can be obtained from the diet or be de novo synthesized from 7-dehydrocholesterol. Further, the active form of vitamin D, 1 ,25-dihydroxyvitamin D3, interacts with a receptor in the target cells, showing that vitamin D should be biochemically characterized as a hormone rather than as a vitamin [55].

During the last decades, the outlook on vitamin D has widened, from be-ing a vitamin solely involved in bone metabolism and calcium homeostasis, to being a multifunctional hormone known to affect a broad range of physio-logical processes. This includes effects on the immune system, brain and fetal development, insulin secretion, cancer, apoptosis, cell proliferation and differentiation as well as the cardiovascular system via the vitamin D recep-

18

tor (VDR) [55-58]. The vitamin D receptor is widely expressed and it has been suggested that 1 ,25-dihydroxyvitamin D3 may have other roles yet undiscovered [57, 59, 60]. It has recently been suggested that 1 ,25-dihydroxyvitamin D3 may affect the expression of up to 200 genes in hu-mans [61].

Production, bioactivation and metabolism There are two forms of vitamin D, vitamin D2 (ergocalciferol) which is syn-thesized in plants, yeast and fungi and vitamin D3 (cholecalciferol) which is synthesized in animals. Vitamin D3 is synthesized in the skin from 7-dehydrocholesterol upon exposure to UV-B radiation. Vitamin D3 is then bioactivated in two subsequent steps to gain the biologically active form of vitamin D (figure 4). In the first step, vitamin D3 is 25-hydroxylated to 25-hydroxyvitamin D3 (calcidiol). 25-Hydroxylation of vitamin D is a reaction that can be catalyzed by the mitochondrial CYP27A1 and the microsomal CYP3A4, CYP2R1 and CYP2J2 in humans. 25-Hydroxylation of vitamin D is mainly performed in the liver and calcidiol is then excreted into the circu-lation. Calcidiol is converted to 1 ,25-dihydroxyvitamin D3 (calcitriol) by 1 -hydroxylation, mainly performed in the kidneys. The principal human 1 -hydroxylase for 25-hydroxyvitamin D3 is CYP27B1. The 1 ,25-dihydroxyvitamin D3 produced is excreted into the circulation and acts as a hormone [55, 57, 58, 62-64] .

Extrarenal 1 -hydroxylation of 25-hydroxyvitamin D3 has been reported for a wide range of tissues, including colon, brain, mammary tissue, breast, pancreatic islets, parathyroid glands, placenta, prostate and keratinocytes [57, 62]. These findings indicate that 1 ,25-dihydroxyvitamin D3 may be produced locally and act in an intracrine or paracrine fashion. It may be speculated that the local concentration of 1 ,25-dihydroxyvitamin D3 in these tissues could be higher than the circulating levels.

The normal serum level of calcidiol is 50-100 nM and for calcitriol 50-125 pM [57]. Calcitriol is the most potent form of vitamin D even though calcidiol can exert some biological effects as well. The circulating levels of 1 ,25-dihydroxyvitamin D3 is tightly regulated via a feed-back mechanism where 1 ,25-dihydroxyvitamin D3 downregulates the expression of CYP27B1 and upregulates the expression of CYP24A1 [63].

Both calcidiol and calcitriol is metabolized by CYP24A1 to the less active compounds 24,25-dihydroxyvitamin D3 and 1 ,24,25-trihydroxyvitamin D3 respectively [65]. It has recently been reported that CYP11A1 can catalyze the production of 20-hydroxyvitamin D3 from vitamin D3 and the production of 1 ,20-dihydroxyvitamin D3 from 1 -hydroxyvitamin D3 [66-70]. Both these metabolites have been reported to exert biological effects on cell dif-ferentiation and gene expression in a way resembling the one of 1 ,25-

19

dihydroxyvitamin D3 [66, 67]. The physiological role, if any, of this CY11A1-mediated metabolism of vitamin D remains to be clarified.

Figure 4. Bioactivation of vitamin D3 to its hormonally active form, 1 , 25-dihydroxyvitamin D3.

Mode of action The bioactivated form of vitamin D alters the gene expression of a large number of genes. 1 ,25-Dihydroxyvitamin D3 may increase the expression of certain genes while it decreases the expression of other genes. Both these effects of 1 ,25-dihydroxyvitamin D3 are mediated by activation of the vi-tamin D receptor (VDR) which forms a heterodimer with the retinoid X re-ceptor (RXR). Extensive research has been conducted to understand the mechanism for 1 ,25-dihydroxyvitamin D3-mediated induction of gene ex-pression. The mechanism is now well known and based on a direct interac-tion between the ligand-activated VDR-RXR complex and a vitamin D re-sponsive element (VDRE) in the gene promoter. These positive VDRE (pVDRE) consist of a hexameric direct repeat of the consensus sequence 5´-RGKTCA (R=A or G, K=G or T) [71]. The two half sites are separated by a three nucleotide spacer. The ligand-activated VDR-RXR complex interacts with the pVDRE and acts as a transcription factor to increase the transcrip-tional rate by recruiting coactivators [57].

The mechanism for vitamin D-mediated downregulation of gene expres-sion, on the other hand, has in large part remained unclear [72]. Studies on the mechanism for 1 ,25-dihydroxyvitamin D3-mediated downregulation of gene expression have been performed for only a few genes. The proposed mechanism for those genes includes recruitment of corepressors such as VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) as well as epigenetic mechanisms such as histone deacetyla-tion and DNA methylation [73-77]. The negative vitamin D responsive ele-ments (nVDRE) that have been described only vaguely resembles the pVDRE described [77]. It has been suggested that the mechanism for vita-min D-mediated downregulation of gene expression does not include a direct

20

interaction between the ligand-activated VDR-RXR complex and the nVDRE, but rather an interaction between nVDRE and VDIR [73].

Vitamin D has also been reported to exert rapid effects only seconds or minutes after treatment. Due to the quick response, it has been suggested that these effects are non-genomic and mediated by membrane-bound receptors [78].

Vitamin D and adrenal steroidogenesis There are a few early reports in the literature suggesting a connection be-tween vitamin D and the adrenal steroidogenesis [79, 80]. De Toni et al. [79] reported in 1959 several clinical cases describing children with rickets hav-ing changes in urinary 17-ketosteroid levels. The authors suggested that the disturbance in steroid metabolism may have some bearing on the action of vitamin D and that it exists some relationship between such metabolic dis-turbance and the sensitivity or resistance of organisms to antirachitic vita-mins. In a study with puppies, Guseinova and collaborators found a decrease in the secretion of 17-ketosteroids after rachitogenic treatment [80]. In a more recent publication, a case has been reported where a woman with os-teomalacia had elevated serum levels of aldosterone and low levels of 25-hydroxyvitamin D [81]. The condition was normalized after 24 months of treatment with vitamin D. Chatterjee and collaborators [82, 83] have re-ported that ligand-activated VDR upregulates the expression of sulfotrans-ferase 2A1 (SULT2A1), an enzyme that catalyzes the conversion of dehy-droepiandrosterone (DHEA) to dehydroepiandrosterone-sulfate (DHEA-S).

Vitamin D and sex hormones The production of sex hormones is regulated by multiple enzymes (figure 3). Vitamin D has been reported to affect the expression and activity of several of these enzymes. Wang and Tuohimaa [84] have reported that 1 ,25-dihydroxyvitamin D3 upregulates the mRNA level for 17 -hydroxysteroid dehydrogenases (17 -HSD) type 2, 4 and 5 in cell lines derived from human prostate. In keratinocytes, Hughes et al. [85] have reported that 1 ,25-dihydroxyvitamin D3 stimulates the expression of 17 -HSD type 1 and 2.

It has been shown that 1 ,25-dihydroxyvitamin D3 alters the aromatase activity in placental cells [86, 87], prostate cells [88] and osteoblasts [89, 90]. Kinuta et al. [91] have reported that vitamin D receptor null mutant mice have a decreased aromatase activity in the ovary, testis and epididymis. Recently, it was reported that 1 ,25-dihydroxyvitamin D3 alters the gene expression of aromatase in a tissue-selective manner [92]. In breast cancer cell lines, vitamin D treatment resulted in decreased aromatase gene expres-sion, while the same treatment increased the aromatase gene expression in osteosarcoma cell lines. 1 ,25-Dihydroxyvitamin D3 has therefore been

21

proposed to be a tissue-selective aromatase modulator [92]. Furthermore, 1 ,25-dihydroxyvitamin D3 and analogs have been reported to alter sex hor-mone signaling in breast cancer cells by suppressing the expression of estro-gen receptor [93-96]. It has also been reported that vitamin D deficiency alters reproductive functions in both male and female rats, indicating that vitamin D may affect sex hormone signaling [97, 98].

Vitamin D and breast cancer It is well-known that 1 ,25-dihydroxyvitamin D3 exerts anti-proliferative and pro-differentiating effects and has therefore been proposed to be of po-tential use as an anti-cancer agent [99-102]. Epidemiological studies have revealed relationships between solar UV-B exposure, which is needed for vitamin D3 production, and decreased incidence and mortality in breast can-cer. Low 25-hydroxyvitamin D3 serum level has been found to be correlated to breast cancer risk [100, 103]. 1 ,25-Dihydroxyvitamin D3 has also been reported to affect apoptosis and expression of tumor suppressor genes [100, 103]. As discussed above, it has been reported that 1 ,25-dihydroxyvitamin D3 may alter the estrogenic signaling by suppression of gene expression for aromatase [92] and estrogen receptor [93-96]. These effects are of impor-tance for the discussion of vitamin D as an anti-cancer agent against breast cancer.

22

Aims of the present investigation

The overall aim of the present investigation was to study the enzymatic regu-lation of steroidogenesis and nuclear receptor activation with a special focus on the effects of vitamin D on steroidogenesis and the effects of metabolism of steroids for their ability to activate estrogen and androgen receptors.

The specific aims were:

To study the role of CYP7B1-mediated catalysis for nuclear receptor activation

To study the effects of 1 ,25-dihydroxyvitamin D3 on adrenal ster-oid hormone production and expression and activity of key adreno-cortical enzymes

To study the effects of 1 ,25-dihydroxyvitamin D3 on enzymes of importance for estrogen and androgen metabolism

To study the mechanism for 1 ,25-dihydroxyvitamin D3-mediated effects on CYP21A2

23

Experimental procedures

Materials 1 ,25-Dihydroxyvitamin D3 was obtained from Solvay, Duphar, The Nether-lands. CYP21A2 promoter-luciferase reporter constructs were a kind gift from professor Walter L. Miller, University of California, San Francisco. Expression vectors for VDIR and WSTF were kind gifts from professor Shi-geaki Kato, University of Tokyo. The full-length human vitamin D receptor (VDR) expression vector was a gift from Dr. Leonard Freedman, the Merck Research Laboratories, West Point. The expression vector for the human retinoid X receptor (RXR) was a kind gift from Dr. Ronald M. Evans, How-ard Hughes Medical Institute, The Salk Institute for Biological Studies, San Diego. The human ER and ER expression vectors and the ERE (estrogen response element) luciferase reporter vector were generous gifts from Dr. P. Chambon, Institut de génétique et de biologie moléculaire et cellulaire, Strasbourg, and Dr. K. Arcaro, University of Massachusetts, respectively. The human AR expression vector and the ARE (androgen response element) luciferase reporter vector were generous gifts from Dr. A. Brinkmann and Dr. J. Trapman, Erasmus Medical Centre, Rotterdam. The vitamin D recep-tor antagonist TEI-9647 was a generous gift from Teijin Pharma, Tokyo. NCI-H295R cells and Y-1 cells were a kind gift from professor Ingvar Brandt and Caco-2 cells from professor Per Artursson, both at Uppsala uni-versity. All other chemicals were of analytical grade and purchased from various commercial sources.

Cell culture and treatment Human adrenocortical carcinoma NCI-H295R cells (ATCC CRL-2128) were cultured as a monolayer in Dulbecco´s modified Eagle´s medium/Nutrient Mixture F-12 Ham (Sigma) supplemented with 1% ITS Plus premix (BD Biosciences), 2.5% NuSerum (VWR), 1% L-glutamine (Gibco) and 1% an-tibiotic/antimycotic (Gibco). The cells were subcultured once a week and the medium was changed two to three times a week.

Human breast adenocarcinoma MCF-7 cells (ATCC HTB-22), human embryonic kidney HEK-293 cells (ATCC CRL-1573) and human epithelial colorectal adenocarcinoma Caco-2 cells (ATCC HTB-37) were cultured as a

24

monolayer in Dulbecco´s modified Eagle´s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics/antimycotics (Gibco). MCF-7 cells were subcultured once a week and the medium was changed once a week. Caco-2 cells were subcultured twice a week. HEK-293 cells were subcultured twice a week.

Mouse adrenocortical Y-1 cells (ATCC CCL-79) were cultured as a monolayer in F-12K medium (ATCC) supplemented with 15% horse serum (ATCC) and 2.5% fetal bovine serum (Gibco) and 1% antibiotic/antimycotic (Gibco). Cells were subcultured once a week and the medium was changed every 48 hour.

Human prostate adenocarcinoma LNCaP cells (ATCC CRL-1740) and human prostate carcinoma DU-145 cells (ATCC HTB-81) were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic/antimycotic (Gibco). The cells were subcultured twice a week.

All cells were cultured in a humidified environment at 37°C with 5% CO2. Cells were treated with different hormones or other substances dis-solved in ethanol or dimethyl sulfoxide (DMSO). The control group was treated with the same amount of vehicle (ethanol and/or DMSO).

Analysis of steroid hormone secretion In order to analyze the steroid hormone secretion, the cell culture medium from treated cells was collected and the hormone levels were analyzed using enzyme-linked immunosorbent assay (ELISA). Secretion of aldosterone, corticosterone, cortisol, androstenedione, DHEA, DHEA-S, estradiol, testos-terone and dihydrotestosterone was measured. The ELISA kits were pur-chased from Demeditech Diagnostics GmbH, Germany. The absorbance was measured at 450 nm, using a Polarstar Optima (BMG Labtech) plate reader. The analyses were performed in accordance with the manufacturer’s recom-mendations.

Reverse transcriptase-polymerase chain reaction (RT-PCR) Expression of CYP11A1, CYP17A1, CYP21A2, 3 -HSD, CYP11B1, CYP11B2, VDR, VDR interacting repressor (VDIR), Williams syndrome transcription factor (WSTF), 5 -reductase, aromatase, Hem45, AR, nuclear receptor co-repressor 1 (NCoR-1), nuclear receptor co-repressor 2 (SMRT), human steroid receptor coactivator (SRC-1) and homo sapiens steroid recep-tor RNA activator (SRA) mRNA in different cell lines were measured by RT-PCR. RNA was isolated using RNeasy Mini kit (Qiagen) and reversed tran-

25

scribed to cDNA by Reverse Transcription System (Promega). The PCR reac-tion was performed with the AmpliTaq Gold system (Applied Biosystems).

Quantitative real time RT-PCR Relative expression level of CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, 3 -HSD, VDIR, 5 -reductase, aromatase, Hem45 and NCoR-1 mRNA in different cell lines were measured by real time RT-PCR. RNA was isolated using RNeasy Mini kit (Qiagen) and reversed transcribed to cDNA by Reverse Transcription System (Promega).

The real time RT-PCR analysis was performed with Power SYBR Green Master Mix (Applied Biosystems) or iQ SYBR Green Supermix (Bio-Rad) using an iQ5 Real-Time PCR Detection System (Bio-Rad). All experiments were conducted in accordance with the manufacturer’s recommendations. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or TATA binding protein (TBP) was used as endogenous control. All real time RT-PCR data were normalized to the endogenous control. The relative mRNA expression was calculated with the standard curve method and expressed as fold change compared to the control group. Non-template wells were used as negative control. Melt curve analysis was performed routinely.

Assays of enzyme activity The enzyme activity of CYP21A2 and CYP17A1 was measured in NCI-H295R cells. The CYP21A2 activity was measured by addition of [3H]-17 -hydroxyprogesterone which was 21-hydroxylated to 11-deoxycortisol. The substrate and metabolite was separated using thin layer chromatography with trichloroethane:acetone 70:30 as mobile phase. The silica gel plates were then analyzed on a LB2723 Berthold scanner.

In order to measure the 17 -hydroxylation activity of CYP17A1, cells were pretreated with the CYP11B1 inhibitor metyrapone (Sigma). 11-deoxycorticosterone was then added and metabolized to 11-deoxycortisol. The substrate and metabolite was separated in a reversed phase high per-formance liquid chromatography system on a 125 × 4 mm LiChrosphere RP 18 column (5 m; Merck) with acetonitril:water 40:60 as mobile phase and monitored with an UV detector.

The 17,20-lyase activity of CYP17A1 was measured by analyzing the conversion of 17 -hydroxypregnenolone into DHEA in cell culture, while inhibiting 3 -HSD activity with trilostane. The amount of DHEA secreted was determined using ELISA, as described above.

The aromatase enzyme activity was determined in MCF-7 cells, LNCaP cells and NCI-H295R cells, by measuring the conversion of testosterone to

26

estradiol while preventing the 5 -reductase-catalyzed conversion of testos-terone to dihydrotestosterone with finasteride. Testosterone was added to the cell culture and the production of estradiol was measured using ELISA, as described above.

Analysis of nuclear receptor activation Cells were transiently transfected with a nuclear receptor-responsive luciferase reporter vector (AR-responsive or ER-responsive) together with expression vectors containing cDNA for either the androgen receptor (AR), the estrogen receptor (ER ) or the estrogen receptor (ER ). All cells were cotransfected with a pCMV -galactosidase plasmid (in order to stan-dardize for transfection efficiency). The transfection was carried out using Lipofectamine (Invitrogen) (NCI-H295R cells, HEK-293 cells, DU-145 cells and LNCaP cells) in accordance with the manufacturer’s recommendations or calcium co-precipitation (HEK-293 cells) as previously described [104]. The AR-responsive vector contains an androgen response element (ARE) coupled to luciferase. The ER-responsive vector contains an estrogen re-sponse element (ERE) coupled to luciferase. Transfected cells were treated for 24-40 hours with different steroids dissolved in ethanol and the levels of androgen-dependent or estrogen-dependent luciferase activity were meas-ured as previously described [104, 105] and compared with the luciferase levels in cells treated with the same volume of ethanol. Luciferase activity is expressed as relative light units (RLU) divided by -galactosidase activity. Data are expressed as fold change compared to control standard deviation.

Analysis of CYP21A2 promoter activity Cells were transiently transfected with luciferase reporter constructs contain-ing different lengths of the CYP21A2 promoter, spanning from -9027 to +13, coupled to the luciferase gene [106]. In all experiments, pCMV -galactosidase plasmid was cotransfected, in order to standardize for transfec-tion efficiency. Cells were transfected using Lipofectamine 2000 (Invitro-gen) in accordance with the manufacturer’s recommendations. In some ex-periments, the cells were cotransfected with expression vectors for human vitamin D receptor (VDR), human retinoid X receptor (RXR), human VDIR and/or human WSTF.

Following transfection, the cells were treated for 24 hours with different substances dissolved in ethanol or dimethyl sulfoxide. Control samples were treated with the same amount of ethanol and/or dimethyl sulfoxide.

After the treatment, cells were lysed and luciferase and -galactosidase activities were measured as previously described [104, 105]. Luciferase re-

27

porter activity is expressed as relative light units (RLU) divided by -galactosidase activity. Reporter activity is presented as fold change com-pared to the control group standard deviation.

Gene silencing by small interfering RNA Transfection of siRNA was used to silence the expression of VDIR in NCI-H295R cells. Transfection was carried out with DharmaFECT transfection reagent 1 (Thermo Scientific) in accordance with the manufacturer’s rec-ommendations. As siRNA, the siGENOME SMARTpool M-009384-00-0005 (Thermo Scientific) was used. Following gene silencing, the effect of 1 ,25-dihydroxyvitamin D3 on the mRNA level of CYP21A2 was deter-mined, using real-time RT-PCR. The effectiveness of siRNA silencing was controlled by measuring VDIR mRNA levels using real time RT-PCR. The VDIR mRNA was decreased by 70% following siRNA transfection.

In silico analysis of gene promoters The CYP21A2 promoter (9 kb upstream) and different aromatase promoto-ers were in silico-analyzed for putative VDREs. The promoter sequences were manually searched for consensus sequences for VDRE described by Matilainen et al. [71] and for previously described negative VDREs [77].

Mutagenesis Mutagenesis was used to produce deletion mutants where putative nVDREs in the CYP21A2-promoter luciferase constructs had been deleted. Quick-Change II XL Site-Directed Mutagenesis Kit (Agilent Technologies) was used in accordance with the manufacturer’s recommendations. Primers were purchased from Thermo Fisher Scientific GmbH, Germany.

Statistical analysis Analysis of statistical significance was performed using either ANOVA or Student’s t-test in Microsoft Excel. P-values <0.05 were considered statisti-cally significant.

28

Results and discussion

Effects of CYP7B1-related steroids on estrogen receptor activation In paper I, we have examined the role of CYP7B1-mediated catalysis in estrogenic signaling. This research has been conducted by investigating the estrogenic effects exerted by some CYP7B1 substrates and metabolites.

The enzyme CYP7B1 catalyzes 7 -hydroxylation of a number of ster-oids. The chemical structures of some CYP7B1 substrates and metabolites of importance for this study are shown in figure 5. The aim of paper I was to examine the effect of this 7 -hydroxylation on the steroids´ ability to stimu-late estrogen receptor (ER ) and estrogen receptor (ER ). Previously, conflicting data have been published regarding the effect of CYP7B1-mediated metabolism on estrogenic signaling where some researchers have reported that 7 -hydroxylation abolishes the estrogenic effect of steroids [33] while other have reported that 7 -hydroxylated products are more po-tent estrogens than the corresponding CYP7B1 substrates [34].

In the present study, we examined the estrogenic effects of several CYP7B1-related steroids using an estrogen response element (ERE) luciferase reporter system transiently transfected into human embryonic kid-ney HEK-293 cells. Our results showed a significant stimulation of both estrogen receptor and estrogen receptor by the CYP7B1 substrates Aene-diol and 3 -Adiol while DHEA did not stimulate any of the estrogen recep-tors. To study the effects of CYP7B1-mediated steroid metabolism on a ster-oid´s ability to activate the estrogen receptor , we treated transfected cells with the corresponding 7 -hydroxylated products (Aene-triol, 3 -Atriol, 7 -hydroxy-DHEA). Neither of these steroids were able to activate estrogen receptor . Furthermore, we found that Aene-diol, but not Aene-triol, could activate estrogen receptor . These results indicate that CYP7B1-mediated 7 -hydroxylation of Aene-diol and 3 -Adiol may alter the estrogenic signal-ing by metabolizing ER and ER ligands to inactive metabolites.

The mRNA level of HEM45, a gene known to be stimulated by estrogens, was markedly upregulated by Aene-diol while the CYP7B1-formed metabo-lite exerted significantly less effect on HEM45 gene expression, lending further support to the conclusion that 7 -hydroxylation abolishes the estro-genic effect of Aene-diol. CYP7B1-mediated metabolism of 3 -Adiol has been proposed to influence ER -mediated growth suppression [33]. Our

29

results indicate that Aene-diol, a steroid reported to be present in higher in-traprostatic concentrations than 3 -Adiol [107], 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 and ER and that CYP7B1-mediated conversion of Aene-diol into a 7 -hydroxymetabolite will result in loss of action. These findings do not support the suggestion by Martin et al. [34] that CYP7B1-catalyzed 7 -hydroxylation increases estrogenic signaling. In conclusion, data presented in this paper show that CYP7B1 may act as a regulator of estrogenic signal-ing by metabolizing estrogenic steroids to inactive metabolites. This paper also reports findings indicating that Aene-diol may play a role in estrogenic signaling in the prostate.

To understand the estrogenic and/or androgenic effects of these steroids, and the role of CYP7B1-mediated catalysis, is of importance to understand normal prostate development as well as prostate carcinogenesis. Further-more, information regarding the regulation of estrogenic and androgenic signaling can be used in the development of new treatment strategies against prostate cancer.

30

Figure 5. Some CYP7B1 substrates and metabolites.

Effects of CYP7B1-related steroids on androgen receptor activation In paper II, we have investigated the role of CYP7B1 in androgenic signal-ing. Androgenic signaling is important in normal prostate development as well as in prostate carcinogenesis. Since a majority of all prostate cancers rely on androgens to proliferate, it is of importance to investigate the mecha-nisms that regulate the levels of androgens in the prostate. CYP7B1 is a widely expressed steroid hydroxylase involved in the metabolism of a num-ber of steroids reported to influence estrogen and androgen signaling. The effect of CYP7B1-mediated metabolism on a steroid´s ability to exert effect on sex hormone receptors has been disputed.

In this study, we have examined whether the CYP7B1 substrates DHEA and Aene-diol and their corresponding 7 -hydroxy products, formed in a reaction catalyzed by CYP7B1, could trigger an androgenic response in an

31

androgen-dependent luciferase reporter system. These experiments have been conducted in different cell lines derived from human embryonic kid-ney, human adrenal cortex and human prostate. The results indicate signifi-cantly lower androgen receptor activation by CYP7B1-formed metabolites than by the corresponding substrate. Thus, CYP7B1-mediated metabolism may be of importance in the regulation of androgenic signaling, by convert-ing AR ligands into less active metabolites. Furthermore, we found cell line-specific differences in the steroids´ ability to trigger the androgen-dependent response (figure 6). These differences between cell lines could be a result of cell line differences in expression of comodulators of importance for the androgen receptor or by cell line-specific metabolism of the steroids during the assay.

Discussion on the cell line differences The studied steroids were able to trigger an AR-dependent response in a cell-line specific manner. In human embryonic kidney HEK-293 cells, dihydro-testosterone stimulated the AR-dependent reporter system but neither treat-ment with CYP7B1 substrates DHEA or Aene-diol nor CYP7B1 metabolites 7 -hydroxy-DHEA or Aene-triol resulted in an AR-dependent response.

In human adrenocortical carcinoma NCI-H295R cells, dihydrotestoster-one and the CYP7B1 substrates DHEA and Aene-diol acted as androgens while the corresponding 7 -hydroxylated steroids 7 -hydroxy-DHEA and Aene-triol did not. Furthermore, we studied the androgenic effects of these steroids in two cell lines derived from human prostate; LNCaP and DU-145. In DU-145 cells, both CYP7B1 substrates and metabolites stimulated the AR-dependent reporter system, but the effect of 7 -hydroxy-DHEA and Aene-triol was significantly lower than the effect of the corresponding CYP7B1 substrates. In LNCaP cells, on the other hand, Aene-diol acted as an androgen, while DHEA, 7 -hydroxy-DHEA and Aene-triol were unable to stimulate the reporter system. This indicates that the cellular milieu may vary between LNCaP cells and DU-145 cells in a manner that is of impor-tance for androgenic signaling.

To investigate the observed cell line differences in androgenic signaling, we studied cell line differences in AR comodulator expression and putative cell-specific metabolism of DHEA and Aene-diol. Over 170 potential AR comodulators have been reported [43, 108] and it has been proposed that the expression levels of these coactivators and corepressors could alter the an-drogenic signaling. Overexpression of multiple AR coactivators has been reported to be associated with prostate carcinogenesis and prostate cancer progression [43]. Tissue-selective expression of AR comodulators has also been described, indicating that the expression level of comodulators could be important in the regulation of tissue-specific androgenic signaling [108]. Furthermore, animal knockout models, as well as studies where different

32

comodulators have been overexpressed, demonstrate that the expression level of comodulators could be of importance for AR´s ability to trigger an-drogenic response [108]. Therefore, we studied whether the expression of four AR comodulators differed between the four cell lines, using RT-PCR. Such a cell line specificity in comodulator expression could be a possible mechanism for the observed differences in androgenic signaling. We found that all four cell lines expressed the comodulators NCoR-1, SRC-1, SRA and SMRT. However, the result from the RT-PCR experiments indicated that the expression level of NCoR-1 differed between the cell lines. In order to study this in detail, the expression level of NCoR-1 mRNA was measured using real-time RT-PCR. We found that the expression level of the comodulator NCoR-1 differed markedly between the studied cell lines, where NCoR-1 mRNA level in LNCaP >> HEK-293 > NCI-H295R DU-145. It has been reported by Miyamoto et al. [38] that overexpression of the AR comodulator ARA70 altered the capacity of Aene-diol to trigger an AR dependent re-sponse. The observed differences in NCoR-1 expression between the cell lines studied in our paper could play a role for the observed differences in androgenic signaling. Further research is, however, needed to fully under-stand the role of comodulator expression in cell line-specific regulation of androgenic signaling.

Another possible mechanism for the observed cell line differences in an-drogenic signaling is cell-specific metabolism of the added steroid, during the assay. To investigate this, we studied different aspects of expression and activity of the enzyme 3 -HSD. Evaul et al. [37] have suggested that both DHEA and Aene-diol have to be converted to androstenedione and testoster-one, respectively, to gain androgenic effect, reactions catalyzed by 3 -HSD. That suggestion indicates that cell line-specific expression of 3 -HSD could be an explanation for the cell line differences that we have observed. How-ever, we found that all four studied cell lines did express 3 -HSD. Further-more, we found that both DHEA and Aene-diol exert androgenic effects in DU-145 cells even when 3 -HSD is inhibited by trilostane. In summary, these findings do not support the suggestion that cell line differences in 3 -HSD expression could explain the observed differences in androgenic signal-ing between cell lines nor that 3 -HSD-catalyzed conversion is required to obtain androgenic effect of DHEA and Aene-diol.

In conclusion, data presented in paper II indicate that CYP7B1 may be a regulator of androgenic response, at least in some tissues, by converting AR ligands into less active metabolites. Further research has to be conducted to examine the mechanism and role for the observed tissue selectivity in andro-gen receptor activation.

33

0

5

10

15

20

25

30

HEK-293 NCI-H295R LNCaP DU-145

Rep

orte

r act

ivity

- f

old

chan

ge v

s E

tOH

Aene-diolAene-triol

*

*

*

*

Figure 6. Effects of CYP7B1 substrate Aene-diol and CYP7B1 metabolite Aene-triol on AR-mediated response in HEK-293 cells, NCI-H295R cells, LNCaP cells and DU-145 cells. Cells were transiently transfected with AR-responsive luciferase reporter system and an expression vector containing cDNA för AR. The cells were then treated with 1 M of either Aene-diol or Aene-triol. *Statistically significant response compared to negative control (p<0.05).

Effects of 1 ,25-dihydroxyvitamin D3 on adrenal steroidogenesis Previous clinical studies have reported a connection between vitamin D and steroidogenesis but the characteristics of this relationship have remained uninvestigated. The aim of the study presented in paper III was to investigate the possible effects of vitamin D on steroidogenesis at molecular level. The active form of vitamin D, 1 ,25-dihydroxyvitamin D3, is a multifunctional hormone known to affect a broad range of physiological processes, mainly by altering gene expression. Human adrenocortical carcinoma NCI-H295R cells, a widely used model for human adrenal cortex [109-113], were cul-tured and treated with 1 ,25-dihydroxyvitamin D3. The amount of hormone produced was then measured using enzyme-linked immunosorbent assay (ELISA), the mRNA levels of steroidogenic enzymes were determined using real time RT-PCR and the enzyme activities of key enzymes were measured using thin layer chromatography, high-performance liquid chromatography or ELISA.

Following 1 ,25-dihydroxyvitamin D3 treatment, hormone production, mRNA levels and enzyme activities were altered for key components in the

34

adrenal steroidogenesis. In summary, this paper reports previously unknown biological effects of vitamin D on the adrenal steroidogenesis. The effects of 1 ,25-dihydroxyvitamin D3 are summarized in figure 7.

Figure 7. Summary of effects of 1 ,25-dihydroxyvitamin D3 on adrenal steroido-genesis. + represents increased gene expression and/or enzyme activity while - represents decreased hormone production, gene expression and/or enzyme activity.

Effects of vitamin D on gene expression The mRNA levels of three key enzymes in the adrenal steroidogenesis, CYP11A1, CYP17A1 and CYP21A2 were found to be altered by 1 ,25-dihydroxyvitamin D3 treatment. CYP11A1 and CYP17A1 mRNA levels were upregulated after vitamin D treatment, while CYP21A2 mRNA level was suppressed by the same treatment. No significant changes were ob-served in the mRNA levels of CYP11B1, CYP11B2 and 3 HSD.

Effects of vitamin D on hormone production and enzyme activity We found that 1 ,25-dihydroxyvitamin D3 treatment decreased the produc-tion of corticosterone, androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), while the production of aldosterone and cor-tisol was unaltered. Furthermore, we measured the enzyme activity of CYP21A2 and CYP17A1 by adding a known amount of substrate to the cell culture and measuring the turnover of substrate to product. In resemblance with the effects on mRNA level, CYP21A2 enzyme activity was suppressed

35

by 1 ,25-dihydroxyvitamin D3. CYP17A1 catalyzes two separate reactions, the 17 -hydroxylation and the 17,20-lyase reaction. We found that treatment with vitamin D resulted in increased 17 -hydroxylation activity of CYP17A1, but in decreased 17,20-lyase activity of CYP17A1.

It is well-known that the manners of regulation for the two activities of CYP17A1 are principally different. The 17 -hydroxylase activity is regu-lated via the expression of the CYP17A1 gene, while the 17,20-lyase activity is regulated by posttranscriptional mechanisms [1, 4-7]. Three posttranscrip-tional mechanism for regulation of 17,20-lyase activity have been described; the abundance of P450 oxidoreductase (POR) [8, 9], allosteric action of cy-tochrome b5 [10] and serine phosphorylation of CYP17A1 [11-14]. The discrepancy between the 1 ,25-dihydroxyvitamin D3-mediated increase in expression of CYP17A1 mRNA and the suppression of 17,20-lyase activity could be a result of posttranscriptional mechanisms affected by 1 ,25-dihydroxyvitamin D3.

Discussion on adrenal bioactivation of vitamin D Extrarenal production of active vitamin D metabolites has been reported for multiple organs. Slominski and collaborators [66-70] have reported that the adrenal enzyme CYP11A1 catalyzes the conversion of vitamin D3 to 20-hydroxyvitamin D3 and the conversion of 1 -hydroxyvitamin D3 to 1 ,20-dihydroxyvitamin D3. 1 -hydroxyvitamin D3 is an exogenous substance used to treat hypovitaminosis D caused by insufficient 1 -hydroxylase ca-pacity. Both 20-hydroxyvitamin D3 and 1 ,20-dihydroxyvitamin D3 have been reported to exert effects resembling those of 1 ,25-dihydroxyvitamin D3 [66, 67]. Due to the high expression of CYP11A1 in the adrenal cortex, it may be speculated that 20-hydroxyvitamin D3 and 1 ,20-dihydroxyvitamin D3 are produced in the adrenal cortex. However, it remains to be investigated whether these vitamin D metabolites alters the adrenal steroidogenesis in the same manner as 1 ,25-dihydroxyvitamin D3.

Tissue-selective effects of vitamin D on sex hormone production The aim of paper IV was to examine the effects of 1 ,25-dihydroxyvitamin D3 on estrogen and androgen metabolism in different cell lines of human origin derived from adrenal cortex, breast and prostate. The effects of 1 ,25-dihydroxyvitamin D3 on endogenous production of estradiol, testosterone and dihydrotestosterone as well as the aromatase enzyme activity and the mRNA expression of aromatase and 5 -reductase were determined. The effects are summarized in table 1.

36

Interestingly, we found that 1 ,25-dihydroxyvitamin D3 exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were de-creased, while production of androgens was markedly increased. In adreno-cortical NCI-H295R cells, 1 ,25-dihydroxyvitamin D3 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. Furthermore, we studied the effects of 1 ,25-dihydroxyvitamin D3 on three different aroma-tase promoters, and found that the transcriptional rate of these promoters were affected by 1 ,25-dihydroxyvitamin D3 in a cell line-specific manner.

This study reports data important for the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism. Further-more, this paper reports important knowledge about 1 ,25-dihydroxyvitamin D3 as an interesting substance for further research in the field of breast can-cer prevention and treatment.

Table 1. Effects of 1 ,25-dihydroxyvitamin D3 on estrogen and androgen metabo-lism, summary of results reported in paper IV. represents increased; represents decreased; - represents unaltered.

Cell line Effect of treatment with 1 ,25-dihydroxyvitamin D3

Aromatase mRNA level MCF-7 LNCaP NCI-H295R Aromatase enzyme activity MCF-7 LNCaP - NCI-H295R Estradiol production MCF-7 LNCaP - NCI-H295R /- Dihydrotestosterone production MCF-7 LNCaP NCI-H295R Expression of promoter specific aromatase transcripts

MCF-7 (p I.3, p I.4 and p II)

LNCaP No statistically significant alterations

NCI-H295R (p I.3 and p I.4), (p II)

37

Vitamin D as a potential anti-breast cancer agent 1 ,25-Dihydroxyvitamin D3 and vitamin D analogs exert anti-proliferative and pro-differentiating effects on cells in vitro and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Epide-miological studies have revealed relationships between solar UV-B expo-sure, which is needed for vitamin D3 production, and decreased incidence and mortality in breast cancer as well as between 25-hydroxyvitamin D3 serum levels and breast cancer risk [103]. 1 ,25-Dihydroxyvitamin D3 has also been reported to affect apoptosis and expression of tumor suppressor genes [100, 103].

To regulate estrogen production and signaling are key strategies in breast cancer treatment, since a large part of all breast cancers depends on estro-gens to proliferate. To regulate the activity of aromatase, that catalyzes the conversion of testosterone to estradiol, is therefore of utmost importance in breast cancer treatment. It has recently been reported that 1 ,25-dihydroxyvitamin D3 alters the gene expression and enzyme activity of aro-matase in a tissue-specific manner [92]. The results presented in paper IV add knowledge showing that aromatase promoter activity, expression and enzyme activity are regulated by 1 ,25-dihydroxyvitamin D3 in a tissue-selective manner. This lends further support to the hypothesis that the bioac-tivated form of vitamin D acts as a selective aromatase modulator [92]. Fur-thermore, the suppressing effect of 1 ,25-dihydroxyvitamin D3 on the aro-matase enzyme activity is abolished by cotreatment with a VDR inhibitor, showing that the effect is mediated by VDR.

Treatment with both aromatase inhibitors and anti-estrogens are associ-ated with risk of adverse effects due to effects on peripheral estrogen me-tabolism (eg. osteoporosis) [48, 49]. The vitamin D-mediated tissue-selective regulation of aromatase expression and activity is therefore an interesting strategy to affect estrogen levels in breast without effects on the peripheral estrogen metabolism. Hence, vitamin D or analogs are interesting substances in further research on breast cancer treatment and prevention.

NCI-H295R cells as a model to study sex hormone production The NCI-H295R cell line has been proposed as a screening tool to study the effects of endocrine disruptors on estrogen and androgen production and has undergone a validation program supported by the Organization for Economic Cooperation and Development (OECD) [114-117]. Therefore, we studied whether this cell line reacted to 1 ,25-dihydroxyvitamin D3 treatment in the same way as cell lines derived from important endocrine target tissues. As shown in table 1, adrenocortical NCI-H295R cells respond to 1 ,25-dihydroxyvitamin D3 treatment in a different way than breast cancer MCF-7 cells and prostate cancer LNCaP cells. The observed differences between

38

NCI-H295R cells and cells derived from important endocrine target tissues, and the molecular basis for these differences, need to be addressed and clari-fied if NCI-H295R cells should be used as a model for estrogen and andro-gen metabolism.

Discussion on putative mechanisms for tissue-selective regulation of aromatase The mechanism for the vitamin D-mediated tissue-selective regulation of aromatase remains to be clarified. Aromatase uses different promoters in different tissues and a negative vitamin D response element has been pro-posed in the aromatase promoter I.3 [92]. Therefore, it has been suggested that the cell line differences in vitamin D-mediated alterations in aromatase expression could be a result of tissue-selective use of aromatase promoter. However, our finding that 1 ,25-dihydroxyvitamin D3 upregulates the pro-moter I.3-specific transcript in NCI-H295R cells but downregulates the same promoter-specific transcript in MCF-7 cells does not support this suggested mechanism. It may be speculated that the observed tissue-differences could be a result of varying expression of comodulators in the different cell lines. Further research is needed to elucidate the mechanism for this tissue-selective effect of 1 ,25-dihydroxyvitamin D3.

Mechanism for vitamin D-mediated effects on CYP21A2 In Paper III, we reported that 1 ,25-dihydroxyvitamin D3 decreases mRNA level and enzyme activity of the steroid 21-hydroxylase CYP21A2. In paper V, we have studied the mechanism for the 1 ,25-dihydroxyvitamin D3-mediated effects on CYP21A2. In this paper, the effect of 1 ,25-dihydroxyvitamin D3 on the promoter activity of CYP21A2 has been studied in a luciferase reporter assay system in cell lines derived from human adre-nal cortex, human colon and mouse adrenal cortex.

1 ,25-Dihydroxyvitamin D3 was found to alter the promoter activity of CYP21A2 via a vitamin D receptor (VDR)-mediated mechanism. We report that the mechanism for 1 ,25-dihydroxyvitamin D3-mediated suppression of CYP21A2 includes the key comodulators VDR interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF) as well as epigenetic mechanisms such as histone deacetylation and DNA methylation.

In conclusion, this paper reports a mechanism for the effects of 1 ,25-dihydroxyvitamin D3 on CYP21A2. This is a previously unknown regulatory pathway for CYP21A2 transcription. Furthermore, we report data indicating a novel mechanism of vitamin D action, showing that the expression level of

39

key comodulators may shift a suppressing effect of vitamin D to a stimula-tory effect.

Effects of nuclear receptor and comodulator expression level We found that altered expression levels of nuclear receptors VDR and RXR as well as the expression levels of comodulators VDIR and WSTF influ-enced the effects of 1 ,25-dihydroxyvitamin D3 on the promoter activity of CYP21A2. When the CYP21A2 promoter-luciferase reporter system was treated with 1 ,25-dihydroxyvitamin D3, the promoter activity was de-creased in all cell lines studied. Surprisingly, when VDR and RXR was overexpressed, the effect of vitamin D shifted from a suppressing effect on the promoter activity to a stimulatory effect. Thus, overexpression of VDR and RXR reversed the previously observed effect of vitamin D on CYP21A2 promoter activity from suppression to stimulation. Consequently, the expres-sion level of the nuclear receptors VDR and RXR are crucial for the effects of 1 ,25-dihydroxyvitamin D3 on CYP21A2 promoter activity.

Although the mechanism for vitamin D-mediated suppression of gene ex-pression remains unclear, it has been reported that VDIR and WSTF act as key comodulators in vitamin D-mediated transcriptional repression of CYP27B1 and the parathyroid hormone gene. Both these comodulators were overexpressed in NCI-H295R cells in order to examine if they play a role in the vitamin D-mediated effects on CYP21A2 transcription. When both VDR/RXR and VDIR/WSTF were overexpressed, 1 ,25-dihydroxyvitamin D3 suppressed the transcriptional rate of CYP21A2 by approximately 50%, which is in the same order of magnitude as the suppression of CYP21A2 mRNA level and enzyme activity that were reported in paper III. The sup-pressing effect of 1 ,25-dihydroxyvitamin D3 on CYP21A2 gene expression was abolished when the VDIR expression was silenced using siRNA. This result lends further support to the suggestion that VDIR is a key comodulator in vitamin D-mediated repression of gene expression.

Our results show that both VDIR and WSTF play important roles in the 1 ,25-dihydroxyvitamin D3-mediated suppression of CYP21A2 and that altered expression levels of these comodulators may shift a suppressing ef-fect of 1 ,25-dihydroxyvitamin D3 to a stimulatory effect.

Epigenetic mechanisms It has been proposed that epigenetic mechanisms, such as DNA demethyla-tion and histone deacetylation, may affect 1 ,25-dihydroxyvitamin D3-mediated suppression of transcription [75]. Furthermore, epigenetic compo-nents in expression control have been reported for multiple cytochrome P450 enzymes [118, 119]. To examine whether these mechanisms are involved in the 1 ,25-dihydroxyvitamin D3-mediated effects on CYP21A2 transcription,

40

NCI-H295R cells transfected with the CYP21A2 promoter-luciferase con-struct and expression vectors for VDR and RXR, were treated with 1 ,25-dihydroxyvitamin D3 and the methyltransferase inhibitor 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A.

Treatment with 1 ,25-dihydroxyvitamin D3 alone increased the promoter activity of CYP21A2. However, the 1 ,25-dihydroxyvitamin D3-mediated increase in promoter activity was significantly decreased when the cells were co-treated with a methyltransferase inhibitor and/or a histone deacetylase inhibitor. In a separate experiment, we showed that the 1 ,25-dihydroxyvitamin D3-mediated effect on CYP21A2 mRNA level was altered when the cells were cotreated with 5-aza-2'-deoxycytidine and trichostatin A.

Our finding that the effect of vitamin D on CYP21A2 transcriptional rate was altered when cells were cotreated with a histone deacetylase inhibitor and/or a DNA methyltransferase inhibitor, indicate that epigenetic modifica-tions are involved in the effects of vitamin D on CYP21A2. This indicates that the complex formed by nuclear receptors and comodulators that medi-ates the effect of vitamin D on CYP21A2 also may include factors that func-tion as histone deacetylases and DNA methyltransferases.

Identification of a functional VDRE NCI-H295R cells were transfected with progressive deletion constructs of the CYP21A2 promoter and expression vectors containing cDNA for VDR and RXR. When transfected cells were treated with 1 ,25-dihydroxyvitamin D3, a 9 kb long promoter construct was markedly upregulated by the treat-ment. The other deletion constructs were not affected to the same extent. This result indicates that the promoter sequence most important for 1 ,25-dihydroxyvitamin D3-mediated effects on CYP21A2 transcription is located in the region between -5.6 kb and -9 kb.

An in silico analysis of the CYP21A2 promoter revealed putative vitamin D response elements, based on sequence similarity to previously described response elements for vitamin D. We found that when one of the putative response elements was deleted using mutagenesis, the effect of 1 ,25-dihydroxyvitamin D3 was abolished. We conclude from this, that the se-quence CACCTG located at -8205 to -8210 in the CYP21A2 promoter (fig-ure 8) is important for the effects of 1 ,25-dihydroxyvitamin D3 on the pro-moter activity and that it might function as a VDRE.

-8214ggaCACCTGggc-8202 Figure 8. The functional VDRE identified in the CYP21A2 promoter.

41

Conclusions

This thesis reports previously unknown biological effects of vitamin D on steroidogenesis and new data on the role of steroid metabolism for regulation of sex hormone signaling. Based on the results presented in this thesis, it can be concluded that:

Estrogenic signaling may be altered by CYP7B1-mediated metabo-lism of ER and ER ligands to less active metabolites

Androgenic signaling may be altered by CYP7B1-mediated metabo-lism of AR ligands to less active metabolites in a cell line-specific manner

The hormone production in human adrenocortical NCI-H295R cells is affected by 1 ,25-dihydroxyvitamin D3 via alteration of CYP17A1 and CYP21A2 mRNA levels and enzyme activities

The metabolism of androgens and estrogens is altered by 1 ,25-dihydroxyvitamin D3 in a cell line-specific manner

The transcriptional rate of CYP21A2 is repressed by 1 ,25-dihydroxyvitamin D3. The effect is mediated by nuclear receptors VDR and RXR and comodulators VDIR and WSTF via a functional VDRE in the CYP21A2 promoter

42

Populärvetenskaplig sammanfattning

Enzymer är ämnen som påskyndar kemiska reaktioner. De kemiska reaktio-nerna i en viss vävnad, till exempel tillverkningen av hormoner, kan styras genom att reglera mängden av ett eller flera enzymer som finns i den vävna-den. I denna avhandling studeras hur dessa enzymsystem kan regleras för att styra tillverkningen av binjurehormoner och könshormoner.

Könshormoner kan vara antingen östrogener eller androgener, vilka har olika effekter i cellerna. Att förstå hur tillverkning, effekt och nedbrytning av könshormoner sker är viktigt ur flera aspekter, till exempel för att få ytterli-gare kunskap om vad som orsakar bröstcancer och prostatacancer samt hur nya behandlingsstrategier för dessa sjukdomar kan utvecklas. I kroppen till-verkas flera könshormon-liknande ämnen, för vilka det fortfarande är oklart om de fungerar som östrogener eller som androgener. I den här avhandlingen studeras om dessa steroider kan fungera som östrogener, som androgener eller som både östrogener och androgener. Jag har funnit att steroiden 5-androsten-3 ,17 -diol kan fungera både som östrogen och som androgen, i vart fall i vissa vävnader. Ytterligare forskning behövs för att klarlägga var-för 5-androsten-3 ,17 -diol fungerar som androgen i vissa vävnadstyper men inte i andra.

Flera steroider, bland annat 5-androsten-3 ,17 -diol, kan omvandlas till andra steroider med hjälp av enzymet CYP7B1. Jag har studerat om dessa CYP7B1-bildade steroider kan fungera som östrogener eller androgener. Mina resultat visar att de CYP7B1-bildade steroiderna har mycket sämre östrogen effekt och androgen effekt än utgångsmaterialet. Att reglera hur aktivt enzymet CYP7B1 är, kan alltså vara ett sätt för cellen att reglera hur mycket östrogener och androgener som ska finnas i cellen. Att förstå hur kroppen reglerar effekten av östrogener och androgener är viktigt för att kunna utveckla nya läkemedel mot könshormonberoende cancer.

Vitamin D är ett fettlösligt vitamin som bildas i huden då den bestrålas av solljus. Vitamin D kan även tas upp från kosten. I kroppen aktiveras vitamin D i två steg till ett ämne, 1 ,25-dihydroxyvitamin D3, som har många olika effekter. Det har länge varit känt att vitamin D är viktigt för skelettet, men under de senaste årtiondena har det rapporterats att vitamin D också påverkar många andra fysiologiska processer. Brist på vitamin D har kunnat kopplas till ökad risk att drabbas av olika cancerformer och autoimmuna sjukdomar. Vitamin D anses också påverka immunförsvaret.

43

I denna avhandling har jag studerat hur vitamin D påverkar tillverkningen av binjurehormoner och könshormoner. Binjurehormoner som aldosteron, kortisol och dehydroepiandrosteron är viktiga för att reglera immunförsvaret, nedbrytningen av kolhydrater och proteiner samt salt- och vätskebalansen. Jag fann att vitamin D påverkar tillverkningen av aldosteron och dehydroe-piandrosterone, genom att påverka mängden och aktiviteten av enzymerna CYP21A2 och CYP17A1. Dessa enzymer har nyckelroller i regleringen av tillverkningen av binjurehormoner.

Jag har även studerat hur vitamin D påverkar tillverkningen av könshor-moner. Mina resultat visar att vitamin D påverkar tillverkningen av östroge-ner på olika sätt i olika vävnadstyper. I celler från bröstvävnad minskade tillverkningen av östrogener då cellerna behandlades med vitamin D. I celler från binjurevävnad och prostatavävnad hade vitamin D motsatt effekt och tillverkningen av östrogener ökade eller förblev oförändrad. Att vitamin D påverkar östrogenproduktionen på detta sätt är ett viktigt fynd som kan an-vändas för att i framtiden utveckla vitamin D-liknande läkemedel mot bröst-cancer.

Avslutningsvis har jag i detalj undersökt mekanismen för de effekter av vitamin D på enzymet CYP21A2 som redovisas ovan. Jag fann att vitamin D utövar sin effekt på CYP21A2 genom att tillsammans med sin receptor binda till den reglerande sekvensen för den gen som kodar för CYP21A2. Jag fann även att ytterligare två ämnen, VDIR och WSTF, är helt nödvändiga för att vitamin D ska kunna utöva den korrekta effekten på CYP21A2.

44

Acknowledgements

The work presented in this thesis was carried out at the Division of Bio-chemistry, Department of Pharmaceutical Biosciences, Faculty of Pharmacy, Uppsala University, Sweden. The Swedish Academy of Pharmaceutical Sci-ences, Apotekare C D Carlssons stiftelse, Elisabet och Alfred Ahlqvists stiftelse, Anna Maria Lundins stipendiefond, The Federation of European Biochemical Societies and The Biochemical Society are gratefully acknowl-edged for generous travel grants. This work was financially supported by the Swedish Research Council Medicine. I would like to express my sincere gratitude to Professor Kjell Wikvall and associate professor Maria Norlin for providing excellent supervision and supportive leadership, for sharing your vast knowledge and experiences in biochemical research and for being open-minded to my ideas. Drs. Maria Ellfolk and Hanna Pettersson for pleasant company and good times both in Uppsala and abroad and for introducing me to all aspects of being a Ph D student. Present and former members of the division of biochemistry: professor Matti Lang, Dr. Ronnie Hansson, Ida Emanuelsson, Dr. Wanjin Tang and Dr. Kyle Christian for pleasant company and nice discussions. I am especially grateful to former staff member Angela Lannerbro for excellent technical advices and for keeping the laboratory in good shape. Agneta Bergström, for providing outstanding administrative support. Magnus Jansson, for computer support. Professor Agneta Oskarsson, Swedish University of Agricultural Sciences, and professor Ernst Oliw, Uppsala University, for valuable comments and suggestions at my half time seminar.

45

Professor Per Artursson and professor Ingvar Brandt, both at Uppsala Uni-versity, for their kind gifts of Caco-2 cells, Y-1 cells and NCI-H295R cells respectively, to professor Walter L. Miller, at University of California, San Francisco, for his kind gift of CYP21A2-promoter-luciferase reporter con-structs, to professor Shigeaki Kato, at University of Tokyo, for his kind gift of expression vectors for VDIR and WSTF and to Teijin Pharma, Tokyo, for the generous gift of the VDR antagonist TEI-9647.

My parents, Gunilla and Bernt, for always supporting me and believing in me. Alf, for patiently listening to five years of endless discussions about vitamin D and the adrenal cortex.

46

References

[1] W. Miller, Steroidogenic Enzymes, Endocr Dev. 13 (2008) 1-18. [2] W. Miller, Androgen synthesis in adrenarche, Reviews in Endocrine

& Metabolic Disorders 10 (2009) 3-17. [3] W.E. Rainey, Y. Nakamura, Regulation of the adrenal androgen

biosynthesis, The Journal of Steroid Biochemistry and Molecular Biology 108 (2008) 281-286.

[4] A. Nguyen, A. Conley, Adrenal androgens in humans and nonhuman primates: production, zonation and regulation, Endocr Dev. 13 (2008) 33-54.

[5] W.L. Miller, N. Huang, V. Agrawal, K.M. Giacomini, Genetic varia-tion in human P450 oxidoreductase, Molecular and Cellular Endo-crinology 300 (2009) 180-184.

[6] W.L. Miller, R.J. Auchus, D.H. Geller, The regulation of 17,20 lyase activity, Steroids 62 (1997) 133-142.

[7] P.F. Hall, Cytochrome P-450 C21scc: One enzyme with two actions: Hydroxylase and lyase, The Journal of Steroid Biochemistry and Molecular Biology 40 (1991) 527-532.

[8] D. Lin, S.M. Black, Y. Nagahama, W.L. Miller, Steroid 17 alpha-hydroxylase and 17,20-lyase activities of P450c17: contributions of serine106 and P450 reductase, Endocrinology 132 (1993) 2498-2506.

[9] K. Yanagibashi, P.F. Hall, Role of electron transport in the regula-tion of the lyase activity of C21 side-chain cleavage P-450 from por-cine adrenal and testicular microsomes, Journal of Biological Chem-istry 261 (1986) 8429-8433.

[10] R.J. Auchus, T.C. Lee, W.L. Miller, Cytochrome b 5 Augments the 17,20-Lyase Activity of Human P450c17 without Direct Electron Transfer, Journal of Biological Chemistry 273 (1998) 3158-3165.

[11] A.V. Pandey, S.H. Mellon, W.L. Miller, Protein Phosphatase 2A and Phosphoprotein SET Regulate Androgen Production by P450c17, Journal of Biological Chemistry 278 (2003) 2837-2844.

[12] A.V. Pandey, W.L. Miller, Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17, Journal of Biological Chemistry 280 (2005) 13265-13271.

[13] L.H. Zhang, H. Rodriguez, S. Ohno, W.L. Miller, Serine phosphory-lation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome, Proceedings of the National Academy of Sciences of the United States of America 92 (1995) 10619-10623.

47

[14] Y.-H. Wang, M.K. Tee, W.L. Miller, Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Pro-tein Kinase A, Endocrinology 151 (2010) 1677-1684.

[15] M. Zachmann, J. Vollmin, W. Hamilton, A. Prader, Steroid 17, 20-desmolase deficiency: a new cause fo male pseudohermaphroditism, Clinical Endocrinology 1 (1972) 369-385.

[16] R.C. Kok, M.A. Timmerman, K.P. Wolffenbuttel, S.L.S. Drop, F.H. de Jong, Isolated 17,20-Lyase Deficiency due to the Cytochrome b5 Mutation W27X, J Clin Endocrinol Metab 95 (2010) 994-999.

[17] M.I. New, Inborn errors of adrenal steroidogenesis, Molecular and Cellular Endocrinology 211 (2003) 75-84.

[18] W. Miller, Disorders of androgen synthesis from cholesterol to dehydroepiandrosterone, Med Princ Pract. 14 (2005) 58-68.

[19] C. Flück, A. Pandey, Clinical and biochemical consequences of p450 oxidoreductase deficiency, Endocr Dev. 20 (2011) 63-79.

[20] A.A. Gilep, T.A. Sushko, S.A. Usanov, At the crossroads of steroid hormone biosynthesis: The role, substrate specificity and evolution-ary development of CYP17, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1814 (2011) 200-209.

[21] N. Krone, W. Arlt, Genetics of congenital adrenal hyperplasia, Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 181-192.

[22] S.J. Ellem, G.P. Risbridger, Aromatase and regulating the estro-gen:androgen ratio in the prostate gland, The Journal of Steroid Bio-chemistry and Molecular Biology 118 (2010) 246-251.

[23] G.P. Risbridger, I.D. Davis, S.N. Birrell, W.D. Tilley, Breast and prostate cancer: more similar than different, Nat Rev Cancer 10 (2010) 205-212.

[24] E.R. Simpson, C. Clyne, G. Rubin, W.C. Boon, K. Robertson, K. Britt, C. Speed, M. Jones, Aromatase - a brief overview, Annual Re-view of Physiology 64 (2002) 93-127.

[25] H. Lardy, A. Marwah, P. Marwah, L. Gerald, C19-5-ene Steroids in Nature, Vitamins & Hormones 71 (2005) 263-299.

[26] S. Ali, R.C. Coombes, Endocrine-responsive breast cancer and strategies for combating resistance, Nat Rev Cancer 2 (2002) 101-112.

[27] H.I. Scher, C.L. Sawyers, Biology of Progressive, Castration-Resistant Prostate Cancer: Directed Therapies Targeting the Andro-gen-Receptor Signaling Axis, Journal of Clinical Oncology 23 (2005) 8253-8261.

[28] S. Balk, K. Knudsen, AR, the cell cycle, and prostate cancer, Nucl Recept Signal. 6 (2008) e001.

[29] A.R. Stiles, J.G. McDonald, D.R. Bauman, D.W. Russell, CYP7B1: One Cytochrome P450, Two Human Genetic Diseases, and Multiple Physiological Functions, Journal of Biological Chemistry 284 (2009) 28485-28489.

48

[30] D.L. Auci, C.L. Reading, J.M. Frincke, 7-Hydroxy androstene ster-oids and a novel synthetic analogue with reduced side effects as a potential agent to treat autoimmune diseases, Autoimmunity Re-views 8 (2009) 369-372.

[31] R. Lathe, Steroid and sterol 7-hydroxylation: ancient pathways, Steroids 67 (2002) 967-977.

[32] H. Pettersson, J. Lundqvist, E. Oliw, M. Norlin, CYP7B1-mediated metabolism of 5 -androstane-3 ,17 -diol (3 -Adiol): A novel pathway for potential regulation of the cellular levels of androgens and neurosteroids, Biochimica et Biophysica Acta (BBA) - Molecu-lar and Cell Biology of Lipids 1791 (2009) 1206-15.

[33] Z. Weihua, R. Lathe, M. Warner, J.-A. Gustafsson, An endocrine pathway in the prostate, ER , AR, 5 -androstane-3 ,17 -diol, and CYP7B1, regulates prostate growth, PNAS 99 (2002) 13589-13594.

[34] C. Martin, M. Ross, K.E. Chapman, R. Andrew, P. Bollina, J.R. Seckl, F.K. Habib, CYP7B Generates a Selective Estrogen Receptor

Agonist in Human Prostate, J Clin Endocrinol Metab 89 (2004) 2928-2935.

[35] Q. Mo, S.-f. Lu, N.G. Simon, Dehydroepiandrosterone and its me-tabolites: Differential effects on androgen receptor trafficking and transcriptional activity, The Journal of Steroid Biochemistry and Molecular Biology 99 (2006) 50-58.

[36] F. Chen, K. Knecht, E. Birzin, J. Fisher, H. Wilkinson, M. Mojena, C.T. Moreno, A. Schmidt, S.-i. Harada, L.P. Freedman, A.A. Reszka, Direct Agonist/Antagonist Functions of Dehydroepiandros-terone, Endocrinology 146 (2005) 4568-4576.

[37] K. Evaul, R. Li, M. Papari-Zareei, R.J. Auchus, N. Sharifi, 3 -Hydroxysteroid Dehydrogenase Is a Possible Pharmacological Tar-get in the Treatment of Castration-Resistant Prostate Cancer, Endo-crinology 151 (2010) 3514-3520.

[38] H. Miyamoto, S. Yeh, H. Lardy, E. Messing, C. Chang, 5-Androstenediol is a natural hormone with androgenic activity in hu-man prostate cancer cells, Proc Natl Acad Sci U S A. 95 (1998) 11083-8.

[39] P. Marwah, A. Marwah, H.A. Lardy, H. Miyamoto, C. Chang, C19-Steroids as androgen receptor modulators: Design, discovery, and structure-activity relationship of new steroidal androgen receptor an-tagonists, Bioorganic & Medicinal Chemistry 14 (2006) 5933-5947.

[40] A. Mizokami, E. Koh, H. Fujita, Y. Maeda, M. Egawa, K. Koshida, S. Honma, E.T. Keller, M. Namiki, The Adrenal Androgen Andros-tenediol Is Present in Prostate Cancer Tissue after Androgen Depri-vation Therapy and Activates Mutated Androgen Receptor, Cancer Research 64 (2004) 765-771.

[41] S. Nilsson, J.A. Gustafsson, Estrogen Receptors: Therapies Targeted to Receptor Subtypes, Clin Pharmacol Ther 89 (2011) 44-55.

49

[42] S. Lokuge, B.N. Frey, J.A. Foster, C.N. Soares, M. Steiner, The rapid effects of estrogen: a mini-review, Behavioural Pharmacology 21 (2010) 465-472.

[43] S. Koochekpour, Androgen receptor signaling and mutations in prostate cancer, Asian Journal of Andrology 12 (2010) 639-657.

[44] S. Hironobu, M. Yasuhiro, N. Shuji, S. Takashi, In situ estrogen production and its regulation in human breast carcinoma: From en-docrinology to intracrinology, Pathology International 59 (2009) 777-789.

[45] S.E. Bulun, Z. Lin, H. Zhao, M. Lu, S. Amin, S. Reierstad, D. Chen, Regulation of Aromatase Expression in Breast Cancer Tissue, An-nals of the New York Academy of Sciences 1155 (2009) 121-131.

[46] S. Chen, Aromatase and breast cancer, Frontiers in Bioscience 3 (1998) d922-d933.

[47] W.R. Miller, T.J. Anderson, W.J.L. Jack, Relationship between tu-mour aromatase activity, tumour characteristics and response to therapy, The Journal of Steroid Biochemistry and Molecular Biology 37 (1990) 1055-1059.

[48] G. Mazziotti, E. Canalis, A. Giustina, Drug-induced Osteoporosis: Mechanisms and Clinical Implications, The American Journal of Medicine 123 (2010) 877-884.

[49] Q. Khan, B. Kimler, C. Fabian, The Relationship Between Vitamin D and Breast Cancer Incidence and Natural History, Current Oncol-ogy Reports 12 (2010) 136-142.

[50] N. Ahmad, R. Kumar, Steroid hormone receptors in cancer devel-opment: A target for cancer therapeutics, Cancer Letters 300 (2011) 1-9.

[51] E.J. Folkerd, M. Dowsett, Influence of Sex Hormones on Cancer Progression, Journal of Clinical Oncology 28 (2010) 4038-4044.

[52] M. Salem, J. Garcia, Abiraterone Acetate, A Novel Adrenal Inhibi-tor in Metastatic Castration-Resistant Prostate Cancer, Current On-cology Reports 13 (2011) 92-96.

[53] I. Leav, K.-M. Lau, J.Y. Adams, J.E. McNeal, M.-E. Taplin, J. Wang, H. Singh, S.-M. Ho, Comparative Studies of the Estrogen Receptors and and the Androgen Receptor in Normal Human Prostate Glands, Dysplasia, and in Primary and Metastatic Carci-noma, The American Journal of Pathology 159 (2001) 79-92.

[54] S. Signoretti, M. Loda, Estrogen Receptor in Prostate Cancer: Brake Pedal or Accelerator? The American Journal of Pathology 159 (2001) 13-16.

[55] L.A. Plum, H.F. DeLuca, Vitamin D, disease and therapeutic oppor-tunities, Nat Rev Drug Discov 9 (2010) 941-955.

[56] A.S. Dusso, A.J. Brown, E. Slatopolsky, Vitamin D, Am J Physiol Renal Physiol 289 (2005) F8-F28.

[57] A.W. Norman, From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health, Am J Clin Nutr 88 (2008) 491S-499S.

50

[58] M.F. Holick, Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease, Am J Clin Nutr 80 (2004) 1678S-1688S.

[59] G. Jones, S.A. Strugnell, H.F. DeLuca, Current Understanding of the Molecular Actions of Vitamin D, Physiol. Rev. 78 (1998) 1193-1231.

[60] H.F. DeLuca, Overview of general physiologic features and func-tions of vitamin D, Am J Clin Nutr 80 (2004) 1689S-1696S.

[61] H.A. Ramagopalan SV, Berlanga AJ, Maugeri NJ, Lincoln MR, Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton SM, Wat-son CT, Morahan JM, Giovannoni G, Ponting CP, Ebers GC, Knight JC., A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution., Genome Res. 20 (2010) 1352-1360.

[62] M.F. Holick, Vitamin D: A millenium perspective, Journal of Cellu-lar Biochemistry 88 (2003) 296-307.

[63] D.E. Prosser, G. Jones, Enzymes involved in the activation and inac-tivation of vitamin D, Trends in Biochemical Sciences 29 (2004) 664-673.

[64] M. Karlgren, S.-i. Miura, M. Ingelman-Sundberg, Novel extra-hepatic cytochrome P450s, Toxicology and Applied Pharmacology 207 (2005) 57-61.

[65] K.K. Deeb, D.L. Trump, C.S. Johnson, Vitamin D signalling path-ways in cancer: potential for anticancer therapeutics, Nat Rev Can-cer 7 (2007) 684-700.

[66] B. Zbytek, Z. Janjetovic, R. Tuckey, M. Zmijewski, T. Sweatman, E. Jones, M. Nguyen, A. Slominski, 20-Hydroxyvitamin D3, a product of vitamin D3 hydroxylation by cytochrome P450scc, stimulates keratinocyte differentiation., J Invest Dermatol 128 (2008) 2271-2280.

[67] R.C. Tuckey, Z. Janjetovic, W. Li, M.N. Nguyen, M.A. Zmijewski, J. Zjawiony, A. Slominski, Metabolism of 1 -hydroxyvitamin D3 by cytochrome P450scc to biologically active 1 ,20-dihydroxyvitamin D3, The Journal of Steroid Biochemistry and Mo-lecular Biology 112 (2008) 213-219.

[68] A. Slominski, I. Semak, J. Wortsman, J. Zjawiony, W. Li, B. Zbytek, R.C. Tuckey, An alternative pathway of vitamin D2 metabo-lism, FEBS Journal 273 (2006) 2891-2901.

[69] A. Slominski, I. Semak, J. Zjawiony, J. Wortsman, M.N. Gandy, J. Li, B. Zbytek, W. Li, R.C. Tuckey, Enzymatic Metabolism of Ergos-terol by Cytochrome P450scc to Biologically Active 17 ,24-Dihydroxyergosterol, Chemistry & Biology 12 (2005) 931-939.

[70] A. Slominski, I. Semak, J. Zjawiony, J. Wortsman, W. Li, A. Szczesniewski, R.C. Tuckey, The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism, FEBS Journal 272 (2005) 4080-4090.

51

[71] M. Matilainen, M. Malinen, K. Saavalainen, C. Carlberg, Regulation of multiple insulin-like growth factor binding protein genes by 1 ,25-dihydroxyvitamin D3, Nucl. Acids Res. 33 (2005) 5521-5532.

[72] J.W. Pike, M.B. Meyer, The Vitamin D Receptor: New Paradigms for the Regulation of Gene Expression by 1,25-Dihydroxyvitamin D3, Endocrinology & Metabolism Clinics of North America 39 (2010) 255-269.

[73] R. Fujiki, M.-S. Kim, Y. Sasaki, K. Yoshimura, H. Kitagawa, S. Kato, Ligand-induced transrepression by VDR through association of WSTF with acetylated histones, EMBO J 24 (2005) 3881-3894.

[74] A. Murayama, M. Kim, J. Yanagisawa, K. Takeyama, S. Kato, Transrepression by a liganded nuclear receptor via a bHLH activator through co-regulator switching, EMBO Journal 23 (2004) 1598-608.

[75] M. Kim, T. Kondo, I. Takada, M.-Y. Youn, Y. Yamamoto, S. Taka-hashi, T. Matsumoto, S. Fujiyama, Y. Shirode, I. Yamaoka, H. Kita-gawa, K.-I. Takeyama, H. Shibuya, F. Ohtake, S. Kato, DNA de-methylation in hormone-induced transcriptional derepression, Na-ture 461 (2009) 1007-1012.

[76] M. Kim, R. Fujiki, H. Kitagawa, S. Kato, 1 ,25(OH)2D3-induced DNA methylation suppresses the human CYP27B1 gene, Molecular and Cellular Endocrinology 265-266 (2007) 168-173.

[77] M. Kim, R. Fujiki, A. Murayama, H. Kitagawa, K. Yamaoka, Y. Yamamoto, M. Mihara, K.-i. Takeyama, S. Kato, 1 ,25(OH)2D3-Induced Transrepression by Vitamin D Receptor through E-Box-Type Elements in the Human Parathyroid Hormone Gene Promoter, Mol Endocrinol 21 (2007) 334-342.

[78] A.W. Norman, W.H. Okamura, J.E. Bishop, H.L. Henry, Update on biological actions of 1 ,25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3, Molecular and Cellular Endocrinology 197 (2002) 1-13.

[79] E. de Toni, S. Nordio, The Relationship between Calcium-Phosphorus Metabolism, the `Krebs Cycle' and Steroid Metabolism, Archives of Disease in Childhood 34 (1959) 371-382.

[80] A.O. Natanson, A.V. Chuvaev, The effect of a rachitogenic regimen on the function of the rat adrenal cortex, Voprosy pitaniia 29 (1970) 46-47.

[81] H. Taylor, E. Elbadawy, Renal tubular acidosis type 2 with Fan-coni's syndrome, osteomalacia, osteoporosis, and secondary hyper-aldosteronism in an adult consequent to vitamin D and calcium defi-ciency: effect of vitamin D and calcium citrate therapy, Endocrine practice 12 (2006) 559-567.

[82] B. Chatterjee, I. Echchgadda, C. Seog Song, Vitamin D Receptor Regulation of the Steroid/Bile Acid Sulfotransferase SULT2A1, Methods in Enzymology Volume 400 (2005) 165-191.

[83] I. Echchgadda, C.S. Song, A.K. Roy, B. Chatterjee, Dehydroepian-drosterone Sulfotransferase Is a Target for Transcriptional Induction by the Vitamin D Receptor, Mol Pharmacol 65 (2004) 720-729.

52

[84] J.-H. Wang, P. Tuohimaa, Regulation of 17 -hydroxysteroid dehy-drogenase type 2, type 4 and type 5 by calcitriol, LXR agonist and 5 -dihydrotestosterone in human prostate cancer cells, J Steroid Biochem Mol Biol 107 (2007) 100-105.

[85] S.V. Hughes, E. Robinson, R. Bland, H.M. Lewis, P.M. Stewart, M. Hewison, 1,25-Dihydroxyvitamin D3 Regulates Estrogen Metabo-lism in Cultured Keratinocytes, Endocrinology 138 (1997) 3711-3718.

[86] D. Barrera, E. Avila, G. Hernández, A. Halhali, B. Biruete, F. Lar-rea, L. Díaz, Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol, The Journal of Steroid Biochemistry and Molecular Biology 103 (2007) 529-532.

[87] T. Sun, Y. Zhao, D.J. Mangelsdorf, E.R. Simpson, Characterization of a Region Upstream of Exon I.1 of the Human CYP19 (Aroma-tase) Gene That Mediates Regulation by Retinoids in Human Chori-ocarcinoma Cells, Endocrinology 139 (1998) 1684-1691.

[88] Y.-R. Lou, T. Murtola, P. Tuohimaa, Regulation of aromatase and 5 -reductase by 25-hydroxyvitamin D3, 1 ,25-dihydroxyvitamin D3, dexamethasone and progesterone in prostate cancer cells, The Journal of Steroid Biochemistry and Molecular Biology 94 (2005) 151-157.

[89] R. Takayanagi, K. Goto, S. Suzuki, S. Tanaka, S. Shimoda, H. Nawata, Dehydroepiandrosterone (DHEA) as a possible source for estrogen formation in bone cells: correlation between bone mineral density and serum DHEA-sulfate concentration in postmenopausal women, and the presence of aromatase to be enhanced by 1,25-dihydroxyvitamin D3 in human osteoblasts, Mech Ageing Dev 123 (2002) 1107-1114.

[90] S. Tanaka, M. Haji, R. Takayanagi, S. Tanaka, Y. Sugioka, H. Nawata, 1,25-Dihydroxyvitamin D3 enhances the enzymatic activity and expression of the messenger ribonucleic acid for aromatase cy-tochrome P450 synergistically with dexamethasone depending on the vitamin D receptor level in cultured human osteoblasts, Endocri-nology 137 (1996) 1860-1869.

[91] K. Kinuta, H. Tanaka, T. Moriwake, K. Aya, S. Kato, Y. Seino, Vi-tamin D Is an Important Factor in Estrogen Biosynthesis of Both Female and Male Gonads, Endocrinology 141 (2000) 1317-1324.

[92] A.V. Krishnan, S. Swami, L. Peng, J. Wang, J. Moreno, D. Feldman, Tissue-Selective Regulation of Aromatase Expression by Calcitriol: Implications for Breast Cancer Therapy, Endocrinology 151 (2010) 32-42.

[93] S. Swami, A.V. Krishnan, D. Feldman, 1 ,25-Dihydroxyvitamin D3 Down-Regulates Estrogen Receptor Abundance and Suppresses Es-trogen Actions in MCF-7 Human Breast Cancer Cells, Clinical Can-cer Research 6 (2000) 3371-3379.

[94] M. Simboli-Campbell, C.J. Narvaez, K. VanWeelden, M. Tennis-wood, J. Welsh, Comparative effects of 1,25(OH)2D3 and EB1089

53

on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells, Breast Cancer Research and Treatment 42 (1997) 31-41.

[95] S.Y. James, A.G. Mackay, L. Binderup, K.W. Colston, Effects of a new synthetic vitamin D analogue, EB1089, on the oestrogen-responsive growth of human breast cancer cells, J Endocrinol 141 (1994) 555-563.

[96] A. Stoica, M. Saceda, A. Fakhro, H.B. Solomon, B.D. Fenster, M.B. Martin, Regulation of estrogen receptor- gene expression by 1,25-dihydroxyvitamin D in MCF-7 cells, Journal of Cellular Biochemis-try 75 (1999) 640-651.

[97] B.P. Halloran, H.F. Deluca, Effect of Vitamin D Deficiency on Fer-tility and Reproductive Capacity in the Female Rat, J. Nutr. 110 (1980) 1573-1580.

[98] G.G. Kwiecinski, G.I. Petrie, H.F. DeLuca, Vitamin D is Necessary for Reproductive Functions of the Male Rat, J. Nutr. 119 (1989) 741-744.

[99] G.L. Christensen, J.S. Jepsen, C.K. Fog, I.J. Christensen, A.E. Lyk-kesfeldt, Sequential Versus Combined Treatment of Human Breast Cancer Cells with Antiestrogens and the Vitamin D Analogue EB1089 and Evaluation of Predictive Markers for Vitamin D Treat-ment, Breast Cancer Research and Treatment 85 (2004) 53-63.

[100] A.V. Krishnan, S. Swami, D. Feldman, Vitamin D and breast cancer: Inhibition of estrogen synthesis and signaling, The Journal of Ster-oid Biochemistry and Molecular Biology 121 (2010) 343-348.

[101] S.S. Larsen, I. Heiberg, A.E. Lykkesfeldt, Anti-oestrogen resistant human breast cancer cell lines are more sensitive towards treatment with the vitamin D analogue EB1089 than parent MCF-7 cells, Br J Cancer 84 (2001) 686-690.

[102] J. Welsh, Targets of Vitamin D Receptor Signaling in the Mammary Gland, Journal of Bone and Mineral Research 22 (2007) V86-V90.

[103] B.A. Ingraham, B. Bragdon, A. Nohe, Molecular basis of the poten-tial of vitamin D to prevent cancer, Current Medical Research and Opinion 24 (2008) 139-149.

[104] H. Pettersson, L. Holmberg, M. Axelson, M. Norlin, CYP7B1-mediated metabolism of dehydroepiandrosterone and 5 -androstane-3 ,17 -diol – potential role(s) for estrogen signaling, FEBS Journal 275 (2008) 1778-1789.

[105] W. Tang, G. Eggertsen, J.Y.L. Chiang, M. Norlin, Estrogen-mediated regulation of CYP7B1: A possible role for controlling DHEA levels in human tissues, The Journal of Steroid Biochemistry and Molecular Biology 100 (2006) 42-51.

[106] S.D. Wijesuriya, G. Zhang, A. Dardis, W.L. Miller, Transcriptional Regulatory Elements of the Human Gene for Cytochrome P450c21 (Steroid 21-Hydroxylase) Lie within Intron 35 of the Linked C4B Gene, Journal of Biological Chemistry 274 (1999) 38097-38106.

54

[107] K.-D. Voigt, W. Bartsch, Intratissular androgens in benign prostatic hyperplasia and prostatic cancer, Journal of Steroid Biochemistry 25 (1986) 749-757.

[108] H.V. Heemers, D.J. Tindall, Androgen Receptor (AR) Coregulators: A Diversity of Functions Converging on and Regulating the AR Transcriptional Complex, Endocr Rev 28 (2007) 778-808.

[109] A.F. Gazdar, H.K. Oie, C.H. Shackleton, T.R. Chen, T.J. Triche, C.E. Myers, G.P. Chrousos, M.F. Brennan, C.A. Stein, R.V. La Rocca, Establishment and Characterization of a Human Adrenocor-tical Carcinoma Cell Line That Expresses Multiple Pathways of Steroid Biosynthesis, Cancer Res 50 (1990) 5488-5496.

[110] A. Oskarsson, E. Ullerås, K.E. Plant, J.P. Hinson, P.S. Goldfarb, Steroidogenic gene expression in H295R cells and the human adre-nal gland: adrenotoxic effects of lindane in vitro, J Appl Toxicol 26 (2006) 484-492.

[111] W.E. Rainey, I.M. Bird, J.I. Mason, The NCI-H295 cell line: a pluripotent model for human adrenocortical studies, Mol Cell Endo-crinol 100 (1994) 45-50.

[112] W.E. Rainey, I.M. Bird, C. Sawetawan, N.A. Hanley, J.L. McCarthy, E.A. McGee, R. Wester, J.I. Mason, Regulation of hu-man adrenal carcinoma cell (NCI-H295) production of C19 steroids, J Clin Endocrinol Metab 77 (1993) 731-737.

[113] W.E. Rainey, K. Saner, B.P. Schimmer, Adrenocortical cell lines, Mol Cell Endocrinol 228 (2004) 23-38.

[114] T. Gracia, K. Hilscherova, P.D. Jones, J.L. Newsted, X. Zhang, M. Hecker, E.B. Higley, J.T. Sanderson, R.M.K. Yu, R.S.S. Wu, J.P. Giesy, The H295R system for evaluation of endocrine-disrupting ef-fects, Ecotoxicology and Environmental Safety 65 (2006) 293-305.

[115] M. Hecker, H. Hollert, R. Cooper, A.-M. Vinggaard, Y. Akahori, M. Murphy, C. Nellemann, E. Higley, J. Newsted, R. Wu, P. Lam, J. Laskey, A. Buckalew, S. Grund, M. Nakai, G. Timm, J. Giesy, The OECD validation program of the H295R steroidogenesis assay for the identification of In vitro inhibitors and inducers of testosterone and estradiol production. Phase 2: Inter-laboratory pre-validation studies, Environmental Science and Pollution Research 14 (2007) 23-30.

[116] M. Hecker, J.L. Newsted, M.B. Murphy, E.B. Higley, P.D. Jones, R. Wu, J.P. Giesy, Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: Hormone produc-tion, Toxicology and Applied Pharmacology 217 (2006) 114-24.

[117] E. Higley, J. Newsted, X. Zhang, J. Giesy, M. Hecker, Assessment of chemical effects on aromatase activity using the H295R cell line, Environmental Science and Pollution Research 17 (2010) 1137-1148.

[118] A. Gomez, M. Ingelman-Sundberg, Epigenetic and microRNA-dependent control of cytochrome P450 expression: a gap between DNA and protein, Pharmacogenomics 10 (2009) 1067-1076.

55

[119] C. Rodriguez-Antona, A. Gomez, M. Karlgren, S. Sim, M. Ingel-man-Sundberg, Molecular genetics and epigenetics of the cyto-chrome P450 gene family and its relevance for cancer risk and treatment, Human Genetics 127 (2010) 1-17.