“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 activation.
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 activation 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 estrogen 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
3 -Atriol
3 -HSD
5-Aza
7 -hydroxy-DHEA
17 -HSD
Aene-diol
Aene-triol
AR
ARE
bp
CAH
Calcidiol
Calcitriol
CAR
CYP
DHEA
DHEA-S
DHT
DMSO
DNA
ELISA
ER
ER
ERE
estradiol
GAPDH
HPLC
mRNA
NCoR-1
nVDRE
POR
pVDRE
RLU
RT-PCR
RXR
5 -androstane-3 ,17 -diol
5 -androstane-3 ,7 ,17 -triol
3 -hydroxysteroid dehydrogenase
5-aza-2’-deoxycytidine
7 -hydroxy-dehydroepiandrosterone
17 -hydroxysteroid dehydrogenase
5-androstene-3 ,17 -diol
5-androstene-3 ,7 ,17 -triol
androgen receptor
androgen response element
base pair
congenital adrenal hyperplasia
25-hydroxyvitamin D3
1 ,25-dihydroxyvitamin D3
constitutive androstane receptor
cytochrome P450
dehydroepiandrosterone
dehydroepiandrosterone sulfate
dihydrotestosterone
dimethyl sulfoxide
deoxyribonucleic acid
enzyme-linked immunosorbent assay
estrogen receptor
estrogen receptor
estrogen response element
17 -estradiol
glyceraldehyde-3-phosphate dehydrogenase
high-performance liquid chromatography
messenger ribonucleic acid
nuclear receptor co-repressor 1
negative vitamin D responsive element
P450 oxidoreductase
positive vitamin D responsive element
relative light units
reverse transcriptase-polymerase chain reaction
retinoid X receptor
SF-1
SMRT
SRA
SRC-1
StAR
SULT2A1
TBP
TLC
TSA
UV
VDIR
VDR
VDRE
WSTF
steroidogenic factor-1
nuclear receptor co-repressor 2
homo sapiens steroid receptor RNA activator
human steroid receptor coactivator
steroidogenic acute regulatory protein
sulfotransferase 2A1
TATA box binding protein
thin layer chromatography
trichostatin A
ultraviolet
VDR interacting repressor
vitamin D receptor
vitamin D responsive element
Williams syndrome transcription factor
Introduction
Production and action of steroid hormones
All steroid hormones are synthesized from the common precursor cholesterol, which can be obtained from the diet or de novo synthesized from acetyl CoA. The production of steroid hormones is regulated via a number of
enzymes of which a majority belongs to the cytochrome P450 (CYP) superfamily. An overview of steroids and enzyme-catalyzed reactions of importance for this thesis is shown in figures 1, 2 and 3.
Steroid hormones exert a wide range of physiological responses, including functions in the immune system, protein and carbohydrate metabolism,
water and salt balance, reproductive system and development of sexual characteristics. 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, corticosterone, 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 steroidogenic acute regulatory protein (StAR).
There are three adrenocortical zones, each with a distinct role in the production 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-
11
Figure 1. Enzyme-catalyzed reactions in the adrenal steroidogenesis that are of importance 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 necessary to control the two activities of CYP17A1 separately. It is well-known
that the manners of regulation for the two activities of CYP17A1 are principally different. The 17 -hydroxylase activity is regulated via the expression
12
of the CYP17A1 gene, while the 17,20-lyase activity is regulated by posttranscriptional mechanisms [1-7]. The adrenal zone-specific activities of
CYP17A1 are summarized in figure 2.
Three posttranscriptional mechanisms for regulation of 17,20-lyase activity 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 complete 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 11deoxycorticosterone and 11-deoxycortisol, which are precursors for the production of corticosterone, aldosterone and cortisol. 21-Hydroxylase deficiency 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.
13
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 produced 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 estrogens [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 androgenic signaling exerted by these steroids is of utmost importance in research 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 androgens are associated with increased risk for breast cancer and prostate
cancer, respectively [23, 26-28].
14
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-androstene3 ,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 proposed that CYP7B1 products, but not substrates, can activate ER , suggesting 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 dehydrogenase (3 -HSD), to gain androgenic effect. Aene-diol has been reported to
15
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 metabolites, and thereby the role of CYP7B1 activity, in estrogenic and androgenic 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 separate expression patterns. ER is expressed in breast, liver, bone, brain, urogenital tract and in the cardiovascular system while ER is expressed in
breast, bone, urogenital tract, gastrointestinal tract, lung, cardiovascular system 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 estrogen 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 musculoskeletal 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 receptor 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 carcinogenesis.
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 conversion of androgenic precursors to estrogens. The gene expression of aro16
matase is regulated by different promoters in a tissue-specific manner [24,
45]. The aromatase expression is higher in breast cancer tissue than in normal 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 processes, 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 strategy to decrease androgen production, a CYP17A1 inhibitor has undergone a
phase III clinical trial as a treatment against castration-resistant prostate cancer [52]. Furthermore, ER is expressed in the prostate and has been proposed 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 vitamin 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 vitamin D can be obtained from the diet or be de novo synthesized from 7dehydrocholesterol. Further, the active form of vitamin D, 1 ,25dihydroxyvitamin 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 being a vitamin solely involved in bone metabolism and calcium homeostasis,
to being a multifunctional hormone known to affect a broad range of physiological 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 recep17
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 ,25dihydroxyvitamin D3 may affect the expression of up to 200 genes in humans [61].
Production, bioactivation and metabolism
There are two forms of vitamin D, vitamin D2 (ergocalciferol) which is synthesized in plants, yeast and fungi and vitamin D3 (cholecalciferol) which is
synthesized in animals. Vitamin D3 is synthesized in the skin from 7dehydrocholesterol 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 25hydroxyvitamin 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 circulation. 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 ,25dihydroxyvitamin 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 50125 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 differentiation and gene expression in a way resembling the one of 1 ,2518
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 , 25dihydroxyvitamin 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 vitamin D receptor (VDR) which forms a heterodimer with the retinoid X receptor (RXR). Extensive research has been conducted to understand the
mechanism for 1 ,25-dihydroxyvitamin D3-mediated induction of gene expression. The mechanism is now well known and based on a direct interaction between the ligand-activated VDR-RXR complex and a vitamin D responsive 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 transcriptional rate by recruiting coactivators [57].
The mechanism for vitamin D-mediated downregulation of gene expression, 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 deacetylation and DNA methylation [73-77]. The negative vitamin D responsive elements (nVDRE) that have been described only vaguely resembles the
pVDRE described [77]. It has been suggested that the mechanism for vitamin D-mediated downregulation of gene expression does not include a direct
19
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 between vitamin D and the adrenal steroidogenesis [79, 80]. De Toni et al. [79]
reported in 1959 several clinical cases describing children with rickets having 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 disturbance and the sensitivity or resistance of organisms to antirachitic vitamins. 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 osteomalacia had elevated serum levels of aldosterone and low levels of 25hydroxyvitamin D [81]. The condition was normalized after 24 months of
treatment with vitamin D. Chatterjee and collaborators [82, 83] have reported that ligand-activated VDR upregulates the expression of sulfotransferase 2A1 (SULT2A1), an enzyme that catalyzes the conversion of dehydroepiandrosterone (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 ,25dihydroxyvitamin 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 ,25dihydroxyvitamin 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 expression, while the same treatment increased the aromatase gene expression in
osteosarcoma cell lines. 1 ,25-Dihydroxyvitamin D3 has therefore been
20
proposed to be a tissue-selective aromatase modulator [92]. Furthermore,
1 ,25-dihydroxyvitamin D3 and analogs have been reported to alter sex hormone signaling in breast cancer cells by suppressing the expression of estrogen 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 potential 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 cancer. 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 importance for the discussion of vitamin D as an anti-cancer agent against breast
cancer.
21
Aims of the present investigation
The overall aim of the present investigation was to study the enzymatic regulation 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 steroid hormone production and expression and activity of key adrenocortical 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
22
Experimental procedures
Materials
1 ,25-Dihydroxyvitamin D3 was obtained from Solvay, Duphar, The Netherlands. 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 Shigeaki 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, Howard 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 receptor 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 university. 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% antibiotic/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
23
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. HEK293 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 dissolved 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, testosterone and dihydrotestosterone was measured. The ELISA kits were purchased 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 recommendations.
Reverse transcriptase-polymerase chain reaction (RTPCR)
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 receptor RNA activator (SRA) mRNA in different cell lines were measured by RTPCR. RNA was isolated using RNeasy Mini kit (Qiagen) and reversed tran24
scribed to cDNA by Reverse Transcription System (Promega). The PCR reaction 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 RTPCR 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 NCIH295R 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). 11deoxycorticosterone was then added and metabolized to 11-deoxycortisol.
The substrate and metabolite was separated in a reversed phase high performance 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
25
estradiol while preventing the 5 -reductase-catalyzed conversion of testosterone 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 standardize 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 response 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 measured 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 containing 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 transfection efficiency. Cells were transfected using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s recommendations. In some experiments, 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 re26
porter activity is expressed as relative light units (RLU) divided by galactosidase activity. Reporter activity is presented as fold change compared to the control group standard deviation.
Gene silencing by small interfering RNA
Transfection of siRNA was used to silence the expression of VDIR in NCIH295R cells. Transfection was carried out with DharmaFECT transfection
reagent 1 (Thermo Scientific) in accordance with the manufacturer’s recommendations. As siRNA, the siGENOME SMARTpool M-009384-000005 (Thermo Scientific) was used. Following gene silencing, the effect of
1 ,25-dihydroxyvitamin D3 on the mRNA level of CYP21A2 was determined, 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 promotoers 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. QuickChange 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 statistically significant.
27
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 steroids. 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 stimulate estrogen receptor (ER ) and estrogen receptor (ER ). Previously,
conflicting data have been published regarding the effect of CYP7B1mediated 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 potent 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 kidney HEK-293 cells. Our results showed a significant stimulation of both
estrogen receptor and estrogen receptor by the CYP7B1 substrates Aenediol and 3 -Adiol while DHEA did not stimulate any of the estrogen receptors. To study the effects of CYP7B1-mediated steroid metabolism on a steroid´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 signaling 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 metabolite exerted significantly less effect on HEM45 gene expression, lending
further support to the conclusion that 7 -hydroxylation abolishes the estrogenic effect of Aene-diol. CYP7B1-mediated metabolism of 3 -Adiol has
been proposed to influence ER -mediated growth suppression [33]. Our
28
results indicate that Aene-diol, a steroid reported to be present in higher intraprostatic 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 signaling 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. Furthermore, information regarding the regulation of estrogenic and androgenic
signaling can be used in the development of new treatment strategies against
prostate cancer.
29
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 signaling. 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 mechanisms that regulate the levels of androgens in the prostate. CYP7B1 is a
widely expressed steroid hydroxylase involved in the metabolism of a number 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
30
androgen-dependent luciferase reporter system. These experiments have
been conducted in different cell lines derived from human embryonic kidney, human adrenal cortex and human prostate. The results indicate significantly 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 converting AR ligands into less active metabolites. Furthermore, we found cell linespecific 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 cellline specific manner. In human embryonic kidney HEK-293 cells, dihydrotestosterone stimulated the AR-dependent reporter system but neither treatment 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, dihydrotestosterone 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 importance 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 androgenic 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
31
comodulators have been overexpressed, demonstrate that the expression
level of comodulators could be of importance for AR´s ability to trigger androgenic 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 response. 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 understand the role of comodulator expression in cell line-specific regulation of
androgenic signaling.
Another possible mechanism for the observed cell line differences in androgenic 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 testosterone, 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. However, we found that all four studied cell lines did express 3 -HSD. Furthermore, 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 signaling 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 androgen receptor activation.
32
30
Reporter activity
- fold change vs EtOH
25
*
20
*
Aene-diol
Aene-triol
15
10
*
5
*
0
HEK-293
NCI-H295R
LNCaP
DU-145
Figure 6. Effects of CYP7B1 substrate Aene-diol and CYP7B1 metabolite Aenetriol 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 cultured 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
33
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 steroidogenesis. + 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 ,25dihydroxyvitamin 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 observed 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 production of corticosterone, androstenedione, dehydroepiandrosterone (DHEA)
and DHEA-sulfate (DHEA-S), while the production of aldosterone and cortisol 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
34
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 regulated via the expression of the CYP17A1 gene, while the 17,20-lyase activity
is regulated by posttranscriptional mechanisms [1, 4-7]. Three posttranscriptional mechanism for regulation of 17,20-lyase activity have been described;
the abundance of P450 oxidoreductase (POR) [8, 9], allosteric action of cytochrome 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 ,25dihydroxyvitamin 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 20hydroxyvitamin D3 and the conversion of 1 -hydroxyvitamin D3 to 1 ,20dihydroxyvitamin D3. 1 -hydroxyvitamin D3 is an exogenous substance
used to treat hypovitaminosis D caused by insufficient 1 -hydroxylase capacity. 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 ,25dihydroxyvitamin 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.
35
Interestingly, we found that 1 ,25-dihydroxyvitamin D3 exerted cell linespecific effects on estrogen and androgen metabolism. In breast cancer
MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In adrenocortical 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 aromatase 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. Furthermore, this paper reports important knowledge about 1 ,25-dihydroxyvitamin
D3 as an interesting substance for further research in the field of breast cancer prevention and treatment.
Table 1. Effects of 1 ,25-dihydroxyvitamin D3 on estrogen and androgen metabolism, summary of results reported in paper IV. represents increased; represents
decreased; - represents unaltered.
Cell line
Aromatase mRNA level
Effect of treatment with
1 ,25-dihydroxyvitamin D3
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
LNCaP
NCI-H295R
36
(p I.3, p I.4 and p II)
No statistically significant
alterations
(p I.3 and p I.4), (p II)
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. Epidemiological studies have revealed relationships between solar UV-B exposure, 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 estrogens 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 ,25dihydroxyvitamin D3 alters the gene expression and enzyme activity of aromatase 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 tissueselective manner. This lends further support to the hypothesis that the bioactivated form of vitamin D acts as a selective aromatase modulator [92]. Furthermore, the suppressing effect of 1 ,25-dihydroxyvitamin D3 on the aromatase 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 associated with risk of adverse effects due to effects on peripheral estrogen metabolism (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 ,25dihydroxyvitamin D3 treatment in a different way than breast cancer MCF-7
cells and prostate cancer LNCaP cells. The observed differences between
37
NCI-H295R cells and cells derived from important endocrine target tissues,
and the molecular basis for these differences, need to be addressed and clarified if NCI-H295R cells should be used as a model for estrogen and androgen 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 proposed 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 promoter 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 tissueselective 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 D3mediated effects on CYP21A2. In this paper, the effect of 1 ,25dihydroxyvitamin D3 on the promoter activity of CYP21A2 has been studied
in a luciferase reporter assay system in cell lines derived from human adrenal 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 ,25dihydroxyvitamin 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
38
key comodulators may shift a suppressing effect of vitamin D to a stimulatory 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 influenced 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 decreased 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 expression 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 expression 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 suppressing 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 effect of 1 ,25-dihydroxyvitamin D3 to a stimulatory effect.
Epigenetic mechanisms
It has been proposed that epigenetic mechanisms, such as DNA demethylation and histone deacetylation, may affect 1 ,25-dihydroxyvitamin D3mediated suppression of transcription [75]. Furthermore, epigenetic components 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,
39
NCI-H295R cells transfected with the CYP21A2 promoter-luciferase construct and expression vectors for VDR and RXR, were treated with 1 ,25dihydroxyvitamin 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 ,25dihydroxyvitamin 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 modifications are involved in the effects of vitamin D on CYP21A2. This indicates
that the complex formed by nuclear receptors and comodulators that mediates the effect of vitamin D on CYP21A2 also may include factors that function 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 treatment. The other deletion constructs were not affected to the same extent.
This result indicates that the promoter sequence most important for 1 ,25dihydroxyvitamin 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 ,25dihydroxyvitamin D3 was abolished. We conclude from this, that the sequence CACCTG located at -8205 to -8210 in the CYP21A2 promoter (figure 8) is important for the effects of 1 ,25-dihydroxyvitamin D3 on the promoter activity and that it might function as a VDRE.
-8214
ggaCACCTGggc-8202
Figure 8. The functional VDRE identified in the CYP21A2 promoter.
40
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 metabolism of ER and ER ligands to less active metabolites
Androgenic signaling may be altered by CYP7B1-mediated metabolism 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 ,25dihydroxyvitamin D3 in a cell line-specific manner
The transcriptional rate of CYP21A2 is repressed by 1 ,25dihydroxyvitamin D3. The effect is mediated by nuclear receptors
VDR and RXR and comodulators VDIR and WSTF via a functional
VDRE in the CYP21A2 promoter
41
Populärvetenskaplig sammanfattning
Enzymer är ämnen som påskyndar kemiska reaktioner. De kemiska reaktionerna i en viss vävnad, till exempel tillverkningen av hormoner, kan styras
genom att reglera mängden av ett eller flera enzymer som finns i den vävnaden. 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 ytterligare kunskap om vad som orsakar bröstcancer och prostatacancer samt hur
nya behandlingsstrategier för dessa sjukdomar kan utvecklas. I kroppen tillverkas 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 5androsten-3 ,17 -diol kan fungera både som östrogen och som androgen, i
vart fall i vissa vävnader. Ytterligare forskning behövs för att klarlägga varför 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.
42
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 dehydroepiandrosterone, 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 könshormoner. Mina resultat visar att vitamin D påverkar tillverkningen av östrogener 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 användas för att i framtiden utveckla vitamin D-liknande läkemedel mot bröstcancer.
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.
43
Acknowledgements
The work presented in this thesis was carried out at the Division of Biochemistry, Department of Pharmaceutical Biosciences, Faculty of Pharmacy,
Uppsala University, Sweden. The Swedish Academy of Pharmaceutical Sciences, 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 acknowledged 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 openminded 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.
44
Professor Per Artursson and professor Ingvar Brandt, both at Uppsala University, 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 constructs, 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.
45
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
46
W. Miller, Steroidogenic Enzymes, Endocr Dev. 13 (2008) 1-18.
W. Miller, Androgen synthesis in adrenarche, Reviews in Endocrine
& Metabolic Disorders 10 (2009) 3-17.
W.E. Rainey, Y. Nakamura, Regulation of the adrenal androgen
biosynthesis, The Journal of Steroid Biochemistry and Molecular
Biology 108 (2008) 281-286.
A. Nguyen, A. Conley, Adrenal androgens in humans and nonhuman
primates: production, zonation and regulation, Endocr Dev. 13
(2008) 33-54.
W.L. Miller, N. Huang, V. Agrawal, K.M. Giacomini, Genetic variation in human P450 oxidoreductase, Molecular and Cellular Endocrinology 300 (2009) 180-184.
W.L. Miller, R.J. Auchus, D.H. Geller, The regulation of 17,20 lyase
activity, Steroids 62 (1997) 133-142.
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.
D. Lin, S.M. Black, Y. Nagahama, W.L. Miller, Steroid 17 alphahydroxylase and 17,20-lyase activities of P450c17: contributions of
serine106 and P450 reductase, Endocrinology 132 (1993) 24982506.
K. Yanagibashi, P.F. Hall, Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes, Journal of Biological Chemistry 261 (1986) 8429-8433.
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.
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.
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.
L.H. Zhang, H. Rodriguez, S. Ohno, W.L. Miller, Serine phosphorylation 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.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
Y.-H. Wang, M.K. Tee, W.L. Miller, Human Cytochrome P450c17:
Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A, Endocrinology 151 (2010) 1677-1684.
M. Zachmann, J. Vollmin, W. Hamilton, A. Prader, Steroid 17, 20desmolase deficiency: a new cause fo male pseudohermaphroditism,
Clinical Endocrinology 1 (1972) 369-385.
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.
M.I. New, Inborn errors of adrenal steroidogenesis, Molecular and
Cellular Endocrinology 211 (2003) 75-84.
W. Miller, Disorders of androgen synthesis from cholesterol to
dehydroepiandrosterone, Med Princ Pract. 14 (2005) 58-68.
C. Flück, A. Pandey, Clinical and biochemical consequences of
p450 oxidoreductase deficiency, Endocr Dev. 20 (2011) 63-79.
A.A. Gilep, T.A. Sushko, S.A. Usanov, At the crossroads of steroid
hormone biosynthesis: The role, substrate specificity and evolutionary development of CYP17, Biochimica et Biophysica Acta (BBA) Proteins & Proteomics 1814 (2011) 200-209.
N. Krone, W. Arlt, Genetics of congenital adrenal hyperplasia, Best
Practice & Research Clinical Endocrinology & Metabolism 23
(2009) 181-192.
S.J. Ellem, G.P. Risbridger, Aromatase and regulating the estrogen:androgen ratio in the prostate gland, The Journal of Steroid Biochemistry and Molecular Biology 118 (2010) 246-251.
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.
E.R. Simpson, C. Clyne, G. Rubin, W.C. Boon, K. Robertson, K.
Britt, C. Speed, M. Jones, Aromatase - a brief overview, Annual Review of Physiology 64 (2002) 93-127.
H. Lardy, A. Marwah, P. Marwah, L. Gerald, C19-5-ene Steroids in
Nature, Vitamins & Hormones 71 (2005) 263-299.
S. Ali, R.C. Coombes, Endocrine-responsive breast cancer and
strategies for combating resistance, Nat Rev Cancer 2 (2002) 101112.
H.I. Scher, C.L. Sawyers, Biology of Progressive, CastrationResistant Prostate Cancer: Directed Therapies Targeting the Androgen-Receptor Signaling Axis, Journal of Clinical Oncology 23
(2005) 8253-8261.
S. Balk, K. Knudsen, AR, the cell cycle, and prostate cancer, Nucl
Recept Signal. 6 (2008) e001.
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.
47
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
48
D.L. Auci, C.L. Reading, J.M. Frincke, 7-Hydroxy androstene steroids and a novel synthetic analogue with reduced side effects as a
potential agent to treat autoimmune diseases, Autoimmunity Reviews 8 (2009) 369-372.
R. Lathe, Steroid and sterol 7-hydroxylation: ancient pathways,
Steroids 67 (2002) 967-977.
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) - Molecular and Cell Biology of Lipids 1791 (2009) 1206-15.
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.
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.
Q. Mo, S.-f. Lu, N.G. Simon, Dehydroepiandrosterone and its metabolites: Differential effects on androgen receptor trafficking and
transcriptional activity, The Journal of Steroid Biochemistry and
Molecular Biology 99 (2006) 50-58.
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 Dehydroepiandrosterone, Endocrinology 146 (2005) 4568-4576.
K. Evaul, R. Li, M. Papari-Zareei, R.J. Auchus, N. Sharifi, 3 Hydroxysteroid Dehydrogenase Is a Possible Pharmacological Target in the Treatment of Castration-Resistant Prostate Cancer, Endocrinology 151 (2010) 3514-3520.
H. Miyamoto, S. Yeh, H. Lardy, E. Messing, C. Chang, 5Androstenediol is a natural hormone with androgenic activity in human prostate cancer cells, Proc Natl Acad Sci U S A. 95 (1998)
11083-8.
P. Marwah, A. Marwah, H.A. Lardy, H. Miyamoto, C. Chang, C19Steroids as androgen receptor modulators: Design, discovery, and
structure-activity relationship of new steroidal androgen receptor antagonists, Bioorganic & Medicinal Chemistry 14 (2006) 5933-5947.
A. Mizokami, E. Koh, H. Fujita, Y. Maeda, M. Egawa, K. Koshida,
S. Honma, E.T. Keller, M. Namiki, The Adrenal Androgen Androstenediol Is Present in Prostate Cancer Tissue after Androgen Deprivation Therapy and Activates Mutated Androgen Receptor, Cancer
Research 64 (2004) 765-771.
S. Nilsson, J.A. Gustafsson, Estrogen Receptors: Therapies Targeted
to Receptor Subtypes, Clin Pharmacol Ther 89 (2011) 44-55.
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
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.
S. Koochekpour, Androgen receptor signaling and mutations in
prostate cancer, Asian Journal of Andrology 12 (2010) 639-657.
S. Hironobu, M. Yasuhiro, N. Shuji, S. Takashi, In situ estrogen
production and its regulation in human breast carcinoma: From endocrinology to intracrinology, Pathology International 59 (2009)
777-789.
S.E. Bulun, Z. Lin, H. Zhao, M. Lu, S. Amin, S. Reierstad, D. Chen,
Regulation of Aromatase Expression in Breast Cancer Tissue, Annals of the New York Academy of Sciences 1155 (2009) 121-131.
S. Chen, Aromatase and breast cancer, Frontiers in Bioscience 3
(1998) d922-d933.
W.R. Miller, T.J. Anderson, W.J.L. Jack, Relationship between tumour aromatase activity, tumour characteristics and response to
therapy, The Journal of Steroid Biochemistry and Molecular Biology
37 (1990) 1055-1059.
G. Mazziotti, E. Canalis, A. Giustina, Drug-induced Osteoporosis:
Mechanisms and Clinical Implications, The American Journal of
Medicine 123 (2010) 877-884.
Q. Khan, B. Kimler, C. Fabian, The Relationship Between Vitamin
D and Breast Cancer Incidence and Natural History, Current Oncology Reports 12 (2010) 136-142.
N. Ahmad, R. Kumar, Steroid hormone receptors in cancer development: A target for cancer therapeutics, Cancer Letters 300 (2011)
1-9.
E.J. Folkerd, M. Dowsett, Influence of Sex Hormones on Cancer
Progression, Journal of Clinical Oncology 28 (2010) 4038-4044.
M. Salem, J. Garcia, Abiraterone Acetate, A Novel Adrenal Inhibitor in Metastatic Castration-Resistant Prostate Cancer, Current Oncology Reports 13 (2011) 92-96.
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 Carcinoma, The American Journal of Pathology 159 (2001) 79-92.
S. Signoretti, M. Loda, Estrogen Receptor in Prostate Cancer:
Brake Pedal or Accelerator? The American Journal of Pathology 159
(2001) 13-16.
L.A. Plum, H.F. DeLuca, Vitamin D, disease and therapeutic opportunities, Nat Rev Drug Discov 9 (2010) 941-955.
A.S. Dusso, A.J. Brown, E. Slatopolsky, Vitamin D, Am J Physiol
Renal Physiol 289 (2005) F8-F28.
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.
49
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
50
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.
G. Jones, S.A. Strugnell, H.F. DeLuca, Current Understanding of the
Molecular Actions of Vitamin D, Physiol. Rev. 78 (1998) 11931231.
H.F. DeLuca, Overview of general physiologic features and functions of vitamin D, Am J Clin Nutr 80 (2004) 1689S-1696S.
H.A. Ramagopalan SV, Berlanga AJ, Maugeri NJ, Lincoln MR,
Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton SM, Watson 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.
M.F. Holick, Vitamin D: A millenium perspective, Journal of Cellular Biochemistry 88 (2003) 296-307.
D.E. Prosser, G. Jones, Enzymes involved in the activation and inactivation of vitamin D, Trends in Biochemical Sciences 29 (2004)
664-673.
M. Karlgren, S.-i. Miura, M. Ingelman-Sundberg, Novel extrahepatic cytochrome P450s, Toxicology and Applied Pharmacology
207 (2005) 57-61.
K.K. Deeb, D.L. Trump, C.S. Johnson, Vitamin D signalling pathways in cancer: potential for anticancer therapeutics, Nat Rev Cancer 7 (2007) 684-700.
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) 22712280.
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 ,20dihydroxyvitamin D3, The Journal of Steroid Biochemistry and Molecular Biology 112 (2008) 213-219.
A. Slominski, I. Semak, J. Wortsman, J. Zjawiony, W. Li, B.
Zbytek, R.C. Tuckey, An alternative pathway of vitamin D2 metabolism, FEBS Journal 273 (2006) 2891-2901.
A. Slominski, I. Semak, J. Zjawiony, J. Wortsman, M.N. Gandy, J.
Li, B. Zbytek, W. Li, R.C. Tuckey, Enzymatic Metabolism of Ergosterol by Cytochrome P450scc to Biologically Active 17 ,24Dihydroxyergosterol, Chemistry & Biology 12 (2005) 931-939.
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.
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
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.
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.
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.
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.
M. Kim, T. Kondo, I. Takada, M.-Y. Youn, Y. Yamamoto, S. Takahashi, T. Matsumoto, S. Fujiyama, Y. Shirode, I. Yamaoka, H. Kitagawa, K.-I. Takeyama, H. Shibuya, F. Ohtake, S. Kato, DNA demethylation in hormone-induced transcriptional derepression, Nature 461 (2009) 1007-1012.
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.
M. Kim, R. Fujiki, A. Murayama, H. Kitagawa, K. Yamaoka, Y.
Yamamoto, M. Mihara, K.-i. Takeyama, S. Kato, 1 ,25(OH)2D3Induced Transrepression by Vitamin D Receptor through E-BoxType Elements in the Human Parathyroid Hormone Gene Promoter,
Mol Endocrinol 21 (2007) 334-342.
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.
E. de Toni, S. Nordio, The Relationship between CalciumPhosphorus Metabolism, the `Krebs Cycle' and Steroid Metabolism,
Archives of Disease in Childhood 34 (1959) 371-382.
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.
H. Taylor, E. Elbadawy, Renal tubular acidosis type 2 with Fanconi's syndrome, osteomalacia, osteoporosis, and secondary hyperaldosteronism in an adult consequent to vitamin D and calcium deficiency: effect of vitamin D and calcium citrate therapy, Endocrine
practice 12 (2006) 559-567.
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.
I. Echchgadda, C.S. Song, A.K. Roy, B. Chatterjee, Dehydroepiandrosterone Sulfotransferase Is a Target for Transcriptional Induction
by the Vitamin D Receptor, Mol Pharmacol 65 (2004) 720-729.
51
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
52
J.-H. Wang, P. Tuohimaa, Regulation of 17 -hydroxysteroid dehydrogenase 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.
S.V. Hughes, E. Robinson, R. Bland, H.M. Lewis, P.M. Stewart, M.
Hewison, 1,25-Dihydroxyvitamin D3 Regulates Estrogen Metabolism in Cultured Keratinocytes, Endocrinology 138 (1997) 37113718.
D. Barrera, E. Avila, G. Hernández, A. Halhali, B. Biruete, F. Larrea, 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.
T. Sun, Y. Zhao, D.J. Mangelsdorf, E.R. Simpson, Characterization
of a Region Upstream of Exon I.1 of the Human CYP19 (Aromatase) Gene That Mediates Regulation by Retinoids in Human Choriocarcinoma Cells, Endocrinology 139 (1998) 1684-1691.
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.
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 1,25dihydroxyvitamin D3 in human osteoblasts, Mech Ageing Dev 123
(2002) 1107-1114.
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 cytochrome P450 synergistically with dexamethasone depending on
the vitamin D receptor level in cultured human osteoblasts, Endocrinology 137 (1996) 1860-1869.
K. Kinuta, H. Tanaka, T. Moriwake, K. Aya, S. Kato, Y. Seino, Vitamin D Is an Important Factor in Estrogen Biosynthesis of Both
Female and Male Gonads, Endocrinology 141 (2000) 1317-1324.
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.
S. Swami, A.V. Krishnan, D. Feldman, 1 ,25-Dihydroxyvitamin D3
Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells, Clinical Cancer Research 6 (2000) 3371-3379.
M. Simboli-Campbell, C.J. Narvaez, K. VanWeelden, M. Tenniswood, J. Welsh, Comparative effects of 1,25(OH)2D3 and EB1089
[95]
[96]
[97]
[98]
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells,
Breast Cancer Research and Treatment 42 (1997) 31-41.
S.Y. James, A.G. Mackay, L. Binderup, K.W. Colston, Effects of a
new synthetic vitamin D analogue, EB1089, on the oestrogenresponsive growth of human breast cancer cells, J Endocrinol 141
(1994) 555-563.
A. Stoica, M. Saceda, A. Fakhro, H.B. Solomon, B.D. Fenster, M.B.
Martin, Regulation of estrogen receptor- gene expression by 1,25dihydroxyvitamin D in MCF-7 cells, Journal of Cellular Biochemistry 75 (1999) 640-651.
B.P. Halloran, H.F. Deluca, Effect of Vitamin D Deficiency on Fertility and Reproductive Capacity in the Female Rat, J. Nutr. 110
(1980) 1573-1580.
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.
G.L. Christensen, J.S. Jepsen, C.K. Fog, I.J. Christensen, A.E. Lykkesfeldt, 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 Treatment, Breast Cancer Research and Treatment 85 (2004) 53-63.
A.V. Krishnan, S. Swami, D. Feldman, Vitamin D and breast cancer:
Inhibition of estrogen synthesis and signaling, The Journal of Steroid Biochemistry and Molecular Biology 121 (2010) 343-348.
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.
J. Welsh, Targets of Vitamin D Receptor Signaling in the Mammary
Gland, Journal of Bone and Mineral Research 22 (2007) V86-V90.
B.A. Ingraham, B. Bragdon, A. Nohe, Molecular basis of the potential of vitamin D to prevent cancer, Current Medical Research and
Opinion 24 (2008) 139-149.
H. Pettersson, L. Holmberg, M. Axelson, M. Norlin, CYP7B1mediated metabolism of dehydroepiandrosterone and 5 -androstane3 ,17 -diol – potential role(s) for estrogen signaling, FEBS Journal
275 (2008) 1778-1789.
W. Tang, G. Eggertsen, J.Y.L. Chiang, M. Norlin, Estrogenmediated 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.
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.
53
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[117]
[118]
54
K.-D. Voigt, W. Bartsch, Intratissular androgens in benign prostatic
hyperplasia and prostatic cancer, Journal of Steroid Biochemistry 25
(1986) 749-757.
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.
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 Adrenocortical Carcinoma Cell Line That Expresses Multiple Pathways of
Steroid Biosynthesis, Cancer Res 50 (1990) 5488-5496.
A. Oskarsson, E. Ullerås, K.E. Plant, J.P. Hinson, P.S. Goldfarb,
Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro, J Appl Toxicol 26
(2006) 484-492.
W.E. Rainey, I.M. Bird, J.I. Mason, The NCI-H295 cell line: a
pluripotent model for human adrenocortical studies, Mol Cell Endocrinol 100 (1994) 45-50.
W.E. Rainey, I.M. Bird, C. Sawetawan, N.A. Hanley, J.L.
McCarthy, E.A. McGee, R. Wester, J.I. Mason, Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids,
J Clin Endocrinol Metab 77 (1993) 731-737.
W.E. Rainey, K. Saner, B.P. Schimmer, Adrenocortical cell lines,
Mol Cell Endocrinol 228 (2004) 23-38.
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 effects, Ecotoxicology and Environmental Safety 65 (2006) 293-305.
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.
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 production, Toxicology and Applied Pharmacology 217 (2006) 114-24.
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) 11371148.
A. Gomez, M. Ingelman-Sundberg, Epigenetic and microRNAdependent control of cytochrome P450 expression: a gap between
DNA and protein, Pharmacogenomics 10 (2009) 1067-1076.
[119]
C. Rodriguez-Antona, A. Gomez, M. Karlgren, S. Sim, M. Ingelman-Sundberg, Molecular genetics and epigenetics of the cytochrome P450 gene family and its relevance for cancer risk and
treatment, Human Genetics 127 (2010) 1-17.
55