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Original Studies |
Departments of Pediatrics (T.C.L., W.L.M., R.J.A.) and Internal Medicine (R.J.A.), and the Metabolic Research Unit (W.L.M.), University of California, San Francisco, California 94143-0978
Address all correspondence and requests for reprints to: Dr. Richard J. Auchus, Department of Pediatrics, Building MR-IV, Room 209, University of California, San Francisco, California 94143-0978. E-mail: richa{at}itsa.ucsf.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/17,20-lyase (P450c17), and type II
3ß-hydroxysteroid dehydrogenase/isomerase (3ßHSDII). We found no
effect of either drug on P450scc in intact human choriocarcinoma JEG-3
cells. Using microsomes from yeast expressing human P450c17 or
microsomes from human adrenals, we found that dexamethasone inhibited
P450c17 with a Ki of 87 µmol/L, which is about 1000 times
higher than typical therapeutic concentrations, but that MPA has no
detectable action on P450c17. Using microsomes from yeast expressing
human 3ßHSDII, we found that this enzyme has indistinguishable
apparent Km values of 5.2–5.5 µmol/L and similar maximum
velocities of 0.34–0.56 pmol steroid/min·µg microsomal protein for
the three principal endogenous substrates, pregnenolone,
17-hydroxypregnenolone, and dehydroepiandrosterone. In this system, MPA
inhibited 3ßHSDII with a Ki of 3.0 µmol/L, which is
near concentrations achieved by high therapeutic doses of 5–20 mg
MPA/kg·day. These data establish the mechanism of action of MPA as an
inhibitor of human steroidogenesis, and are in contrast with the
results of earlier studies indicating that MPA inhibited both P450c17
and 3ßHSD in rat Leydig cells. These studies establish the
"humanized yeast" system as a model for studying the actions of
drugs on human steroidogenic enzymes and suggest that 3ßHSDII may be
an appropriate target for pharmacological interventions in human
disorders characterized by androgen excess or sex steroid dependency. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). High doses of MPA reduce circulating
androgen and estrogen concentrations in gonadotropin-independent
sexual precocity due to testotoxicosis (1) or to functional ovarian
cysts (2, 3) as found in the McCune-Albright syndrome. High doses
are also used to treat breast cancer, even in postmenopausal women (4);
responses improve when plasma MPA concentrations exceed 0.1 µmol/L
(5) and when adrenal androstenedione secretion is suppressed (6, 7).
Dex may suppress steroidogenesis by the human fetal adrenal before the
hypothalamic-pituitary-adrenal axis is established (8), which may
explain how prenatally administered Dex limits the virilization of
female fetuses with 21-hydroxylase deficiency. Preliminary experiments
indicated that Dex can suppress transcription of the human gene for
P450c17, which catalyzes both the 17-hydroxylase and 17,20-lyase
reactions (9), but the mechanism(s) by which Dex and MPA directly
inhibit human steroidogenesis is unclear.
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-hydroxylase, 3ß-hydroxysteroid
dehydrogenase/
5-4-isomerase (3ßHSD),
and 17ß-hydroxysteroid dehydrogenase (17ßHSD) (10). However, these
studies may not apply to human enzymes because there are substantial
differences between corresponding rodent and human steroidogenic
enzymes. Rat and human P450c17 have different substrate specificities;
rat P450c17 efficiently catalyzes 17,20-lyase activity with
4-17-hydroxyprogesterone, whereas human P450c17 does not
(11). In addition, there are multiple different human (and rodent)
isoforms of 17ßHSD with diverse functions and substrate specificities
(12); furthermore, rodents have at least four to six functional 3ßHSD
genes and isoenzymes (13), whereas human beings have only two (14, 15).
Thus, it is impossible to determine the mode of action of MPA on human
steroidogenesis from studies in rodents. Furthermore, a recent study
concluded that Dex was a substrate for human P450c17 (16) based on the
conversion of Dex to
9-fluoro-androsta-1,4-diene-11ß-hydroxy-16-methyl-3,17-dione by
human kidney microsomes, but not by liver microsomes (16). Although
conversion of Dex to the 9-fluroandrostane derivative is a
17,20-lyase reaction, the expression of P450c17 in kidney has not been
described. To ascertain the modes by which MPA and Dex might affect human steroidogenesis, we quantitatively evaluated MPA and Dex as substrates and inhibitors of the three early steps that are common to both adrenal and gonadal steroidogenesis: P450scc, P450c17, and 3ßHSD type II (3ßHSDII).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construct pcDNA-3ßHSD (17), was used as a template for PCR amplification of the human 3ßHSDII complementary DNA (cDNA) using pfu polymerase (Stratagene, La Jolla, CA) and the primers 5'-CGGGATCCATGGGCTGGAGCTGCCTTGTGACAGG-3' (sense) and 5'-CCGAATTCAATCACTGAGTCTTGGACTTCAGGG-3' (antisense). The yeast expression vector V10 (18) was digested with BglII, rendered blunt ended with pfu polymerase, and digested with EcoRI. The 3'-end of the 3ßHSD PCR product was digested with EcoRI, and the 5'-end was left blunt for cloning into the correspondingly digested V10 vector, yielding vector V10–3ß. Saccharomyces cerevisiae strain W303B (18) was transformed with the V10–3ß construct using the lithium acetate protocol (19). Subsequent yeast culturing and microsome preparation were also performed according to our previously described procedures (11).
Enzyme assays
The 17-hydroxylase and 17,20-lyase activities of P450c17 were
measured using microsomes from transfected yeast expressing both human
P450c17 and P450 oxidoreductase or microsomes from human adrenal glands
(11). Microsomes were incubated with [3H]- or
[14C]pregnenolone,
[3H]17-hydroxypregnenolone, or
[3H]dehydroepiandrosterone
([3H]DHEA) as previously described (20). In
assaying P450c17 or 3ßHSD activity, reactions were initiated by
adding NADPH or NAD+, respectively, to a final
concentration of 1 mmol/L. Results were analyzed by thin layer
chromatography (TLC) (21) and quantitated by liquid scintillation
counting for 3H-labeled steroids or by phosphorimaging
(Storm 860, Molecular Dynamics, Inc., Sunnyvale, CA) for
14C-labeled steroids. Dex and MPA (Sigma Chemical Co., St. Louis, MO) were dissolved in ethanol and added to the
incubations at the indicated concentrations; the same volume of ethanol
was added to control incubations. Ki values were calculated
from the equation K'm =
(Km/Ki)[I] + Km, where
K'm is the measured Km in the presence of
inhibitor at concentration [I] (22). Incubations with Dex alone were
analyzed by TLC using 3:1, 2:1, 1:1, and 1:2 dilutions of
chloroform-ethyl acetate and short wave UV light to identify Dex and
any metabolites.
Cell culture
Human choriocarcinoma JEG-3 cells were grown in monolayer in T-125 flasks with 15 mL DMEM-Ham’s 21 (DMEM-H21) containing 5% FCS, 5% horse serum, and 50 mg/mL gentamicin. For RIA of pregnenolone, JEG-3 cells were grown to confluence in six-well plates (9.4-cm2 wells) and incubated with 10 µmol/L Dex, 10 and 100 µmol/L MPA, or ethanol vehicle alone in 2.5 mL DMEM-H21 medium. A 1-mL aliquot was removed after 8 h and frozen at -20 C until assayed. A 200-µL aliquot was extracted with 400 µL ethyl acetate-isooctane (1:1) and analyzed by TLC using 1:0, 4:1, 2:1, 1:1, and 1:2 dilutions of chloroform-ethyl acetate; short wave UV light was used to identify Dex, MPA, and their metabolites.
RIA
Pregnenolone synthesis was measured with a [3H]pregnenolone RIA kit (ICN Biomedicals, Inc., Costa Mesa, CA), using half the amount of all reagents specified in the manufacturer’s protocol and omitting the chromatography step required for serum samples. Each sample and standard was assayed in duplicate. Inter- and intraassay coefficients of variation were 10–13% and 6–7%, respectively, over the range of concentrations encountered. Assays of fresh medium confirmed negligible cross-reactivity with Dex and MPA at the concentrations used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the actions of Dex and MPA on human
P450c17. This enzyme is the qualitative regulator of steroidogenesis:
in its absence, the adrenal zona glomerulosa produces 17-deoxysteroids
such as aldosterone; when its 17-hydroxylase activity is expressed
in the zona fasciculata, the 17-hydroxysteroid cortisol is produced;
when both the 17-hydroxylase and 17,20-lyase activities are present,
such as in the zona reticularis (11, 23, 24), DHEA, the
C19 precursor of sex steroids, is produced. The 17,20-lyase
activity of P450c17 can be augmented by phosphorylation (20) and
allosteric interaction with cytochrome b5 (11).
Thus, it was necessary to study in detail how Dex and MPA modulate both
the 17-hydroxylase and 17,20-lyase activities of P450c17.
Yeast microsomes containing human P450c17 and its obligate electron
donor P450 oxidoreductase (OR) (11) were incubated with
[14C]pregnenolone in the presence of various
concentrations of Dex or MPA. Concentrations up to 10 µmol/L Dex or
100 µmol/L MPA failed to inhibit 17-hydroxylase activity, and only
incubation with 100 µmol/L Dex reduced activity (Fig. 2). We then compared the effects of 100
µmol/L Dex, 100 µmol/L MPA, and 10 µmol/L progesterone on the
17-hydroxylase and 17,20-lyase activities of human P450c17 and
examined their effects on human P450c17 in both our yeast microsome
system (Fig. 3A) and native human P450c17
in microsomes prepared from human adrenals (Fig. 3B).
Progesterone, which is a substrate for P450c17 (Fig. 1
) and hence binds
competitively with pregnenolone to P450c17, inhibited the
17-hydroxylation of pregnenolone to about 60% of the control value
in both yeast and human microsomes, validating the assay system.
Consistent with the data in Fig. 2, 100 µmol/L MPA had little if any
effect on either the hydroxylase or lyase activity of P450c17, whereas
100 µmol/L Dex inhibited both reactions. Dex inhibited
17-hydroxylase activity to 73.0 ± 1.1% or 62.2 ± 2.8%
of the control value and inhibited 17,20-lyase activity to 64.9 ±
3.7% or 56.0 ± 3.0% in yeast and human microsomes,
respectively, indicating that the action of Dex is on human P450c17 and
not on some other component of yeast or human microsomes. Thus,
although previous studies showed that low micromolar concentrations of
MPA inhibit rat P450c17, MPA does not exert the same effect on the
human enzyme. Furthermore, Dex is only a weak inhibitor of P450c17.
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-hydroxylase reaction using Lineweaver-Burk
plots (Fig. 4A). The plots of 1/V
vs. 1/[S] with and without 100 µmol/L Dex intersect near
1/Vmax (Vmax = maximum velocity), showing that
the mode of inhibition by Dex is competitive. The calculated
Ki of 87 µmol/L quantitatively demonstrates that Dex is
not a significant inhibitor of P450c17 at doses used clinically,
because even when 4 mg Dex is given orally, plasma concentrations
rarely exceed 0.1 µmol/L (25). In addition, because a previous study
concluded that Dex is metabolized by human P450c17 (16), we incubated
yeast and human adrenal microsomes with 100 µmol/L Dex but observed
no turnover of Dex regardless of the amount of P450 used or the source
of the microsomes (Fig. 4B). Thus, in contrast to earlier studies, we
found no evidence that Dex is metabolized by human P450c17.
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Because MPA inhibited rat testicular 3ßHSD activity (10), we
examined the effects of Dex and MPA on human 3ßHSDII, which is
the only form of 3ßHSD expressed in human adrenals and gonads (17).
Unlike P450c17, the 3ßHSD protein is found in all major subcellular
compartments, including cytosol, endoplasmic reticulum, and
mitochondria (26). Thus, human 3ßHSDII activity can be examined in
microsomes from transfected yeast, and we found abundant 3ßHSDII
activity in these microsomes as well as other subcellular fractions. In
this yeast system, 3ßHSDII catalyzed the 3ß-dehydrogenation and
5 to 4 isomerization of all three major
substrates; the apparent Km for pregnenolone was 5.5
µmol/L, that for 17-hydroxypregnenolone was 5.2 µmol/L, and that
for DHEA was 5.4 µmol/L. The Vmax for
pregnenolone was 0.56 pmol steroid per min/µg protein; for
17-hydroxypregnenolone it was 0.44 pmol/min·µg, and for
DHEA it was 0.34 pmol/min·µg (Fig. 5). These very reliable values are
slightly higher than the 0.7–3.5 µmol/L values estimated for the
Km of this enzyme in transfected HeLa cells (17). Thus,
human 3ßHSDII has very similar affinities and catalytic activities
with all three major substrates.
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). This concentration of
DHEA inhibited 3ßHSD activity by 58%, but incubation
with 100 µmol/L Dex inhibited enzyme activity only 20%, which is
less than the effect of 100 µmol/L Dex on P450c17. By contrast, 100
µmol/L MPA had a substantial effect on 3ßHSD activity, inhibiting
the conversion of pregnenolone to progesterone by 90%. Experiments
using lower concentrations of MPA showed substantial inhibition of
3ßHSD activity by 1.0–10 µmol/L MPA (Fig. 6B). To determine the
mode of this inhibition, coincubation experiments were analyzed using
Lineweaver-Burk plots (Fig. 6C). Similar to the experiment in Fig. 3A, the data show that MPA acts as a competitive inhibitor of 3ßHSDII.
The lines derived from incubations with and without MPA intersect
at the y-axis, corresponding to 1/Vmax, and
yield a Ki for MPA of 3.0 µmol/L, which is very close to
the enzyme’s Km for its three physiological substrates.
Thus, MPA is a competitive inhibitor of 3ßHSDII at concentrations
achieved in human therapy (5).
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Cholesterol is the substrate for mitochondrial P450scc, but
radiolabeled cholesterol cannot be used effectively for enzyme assays
due to the poor solubility of cholesterol in water. Because yeast do
not normally synthesize cholesterol, and because it has been difficult
to express an active P450scc system in yeast (27), we determined the
effect of Dex and MPA on P450scc activity by assaying pregnenolone
production by human placental JEG-3 cells. The P450scc activity of
JEG-3 cells has been characterized (28), and JEG-3 cells do not express
P450c17 (9) (and hence have a limited steroidogenic pathway) or the
StAR protein (29, 30), which eliminates potentially confounding
variables. Pregnenolone production was not inhibited by Dex or MPA
during short term incubations (Table 1).
TLC analysis of steroids extracted from the conditioned medium using
1:0 to 1:2 dilutions of chloroform-ethyl acetate revealed only
unchanged Dex and MPA (data not shown). Thus, high concentrations of
Dex and MPA do not inhibit the P450scc system in JEG-3 cells, and these
steroids are not appreciably metabolized in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pharmacological inhibition of sex steroid biosynthesis is useful in the
treatment of gonadotropin-independent sexual precocity (1), cancers of
the breast (35) and prostate (36), gynecomastia (37), and possibly
congenital adrenal hyperplasia (38). Although potent, selective
inhibitors of late steps in sex steroid biosynthesis, such as steroid
5-reductase (39) and aromatase (31), are available, MPA may be
preferred when a more complete blockade is sought. For example, MPA is
used to treat gonadotropin-independent disorders of sex steroid excess,
but large doses of 2–5 mg MPA/kg·day are required (1), and the
responses of breast cancer patients to MPA improve when plasma MPA
concentrations remain above about 0.2 µmol/L (5). By contrast, the
physiological dose that induces withdrawal bleeding is 0.1 mg
MPA/kg·day, and the pharmacological dose used to treat endometriosis
is about 0.3–1 mg MPA/kg·day (40).
In a careful study, Barbieri et al. (10) found that MPA
inhibits both P450c17 and 3ßHSD in rat Leydig cells, and hence it has
been thought that MPA directly inhibits multiple steps in human sex
steroid biosynthesis. However, the applicability of these data to human
systems was uncertain, as there are substantial differences between the
rodent and human forms of these enzymes. We found that MPA had no
effect on human P450c17 or P450scc; instead, MPA competitively inhibits
3ßHSDII with a Ki of only 3.0 µmol/L, which is similar
to its apparent Km of 5.2–5.5 µmol/L for its
5 substrates. Therapeutic doses of 5–20 mg MPA/kg·day
can produce plasma concentrations in this range (5), whereas
pregnenolone, 17-hydroxypregnenolone, and DHEA circulate
at concentrations below 0.1 µmol/L. Thus, our biochemical data
corroborate the clinical experience that doses above 1 mg MPA/kg·day
are needed to inhibit sex steroid biosynthesis. Our studies also
show that 3ßHSDII is the principal, if not the only, target. It is
likely that 3ßHSDI can also be inhibited by MPA, as the two enzymes
share 93.5% amino acid identity (17, 41), and their kinetics are
similar (42), although the Km values for the type I enzyme
are generally lower (17). 3ßHSDI is primarily expressed in the
placenta (17), but is also expressed in the brain, where it appears to
participate in the biosynthesis of allopregnanolone (43). However, it
is not known whether MPA, even when administered in high doses, will
reach 3ßHSDI in the brain.
MPA is a 4-steroid that is structurally similar to
17-hydroxyprogesterone; thus, the action of MPA to inhibit 3ßHSD
resembles product inhibition, and 3ßHSD product inhibition with
4-steroid products has been observed (42) (our
unpublished data). Thus, the high affinity of MPA for the active site
of human 3ßHSDII is not surprising, as the human 3ßHSDs can also
accommodate a variety of D-ring substituents in their natural
substrates. By contrast, human P450c17 has a lower affinity for
17-hydroxylated 4-steroids such as
17-hydroxyprogesterone than for 5-steroids (11, 44, 45),
so that the failure of MPA to inhibit P450c17 is consistent with
previous biochemical studies.
The clinical efficacy of MPA in the treatment of testotoxicosis (gonadotropin-independent sexual precocity) demonstrates that its inhibition of 3ßHSDII is sufficient to lower circulating sex steroid concentrations. Our results suggest that 3ßHSDII inhibition contributes to the therapeutic benefits of high doses of MPA and perhaps of other synthetic progestins in the treatment of breast cancer. These results also suggest that 3ßHSDs may be appropriate therapeutic targets for the management of androgen-dependent disorders such as prostatic hyperplasia and carcinoma and for disorders of androgen excess, such as 21-hydroxylase deficiency and polycystic ovary syndrome.
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Acknowledgments |
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Footnotes |
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Received November 24, 1998.
Revised January 14, 1999.
Accepted January 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-4-isomerase in human
adrenals and gonads. Mol Endocrinol. 5:1147–1157.-hydroxylase and 17,20 lyase activities of P450c17:
contributions of serine106 and P450 reductase. Endocrinology. 132:2498–2506.-hydroxylase/17,20 lyase): similarity to the gene for P450c21. DNA. 6:439–448.[Medline]
-hydroxylase/17,20 lyase): cloning of human adrenal and testis
cDNAs indicates the same gene is expressed in both tissues. Proc Natl
Acad Sci USA. 84:407–411.-hydroxylase/17,20 lyase) and P450c21
(21-hydroxylase) in the human fetus. J Clin Endocrinol Metab. 63:1145–1150.
4-ene-isomerase: purification from mitochondria and kinetic
profiles, biophysical characterization of the purified mitochondrial
and microsomal enzymes. Steroid Biochem. 33:209–217.
-hydroxylase-17,20-lyase (CYP17) by cytochrome b5:
endocrinological and mechanistic implications. Biochem J. 308:901–908.
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