Assessment of porcine and human 16-ene-synthase, a third activity
of P450c17, in the formation of an androstenol precursor
Role of recombinant cytochrome
b
5
and P450 reductase
Penny Soucy, Lucille Lacoste and Van Luu-The
Molecular Endocrinology and Oncology Research Center, Laval University Medical Center (CHUL) and Laval University,
Que
´
bec, Canada
Recently, we have shown that the biosynthesis of androste-
nol, a potential endogenous ligand for the orphan receptors
constitutive androstane receptor and pregnane-X-receptor,
requires the presence of enzymes of the steroidogenic path-
way, such as 3b-hydroxysteroid dehydrogenase, 5a-reduc-
tase and 3a-hydroxysteroid dehydrogenase. In this report,
we examine at the molecular level whether the enzyme 17a-
hydroxylase/17,20-lyase (P450c17), which possesses dual
17a-hydroxylase and 17,20-lyase activities and catalyzes the
production of precursors for glucocorticoids and sex ster-
oids, is also able to catalyze the formation of a third class of
active steroids, 16-ene steroids (including androstenol). The
role of components of the P450 complex is also assessed. We
transfected human embryonic kidney (HEK-293) cells with
various amounts of vectors expressing P450c17, NADPH-
cytochrome P450 reductase, and cytochrome b
5
. Our results
showed that P450c17 possesses a 16-ene-synthase activity
able to transform pregnenolone into 5,16-androstadien-

5
), an allosteric effector
[3–6].
As for 16-androstenes, the precise mechanism by which
they are biosynthesized has been until now subject for
debate. Early studies reported testosterone to be a precursor
for these steroids [7–12]. Later, however, testosterone and
a large number of other compounds, including epitestos-
terone, DHEA, 16a-hydroxypregnenolone and 16a-
hydroxyprogesterone, were excluded as precursors for
16-androstenes, whereas preg and progesterone were found
to be putative precursors. Multiple pathways have been
suggested for the transformation of C21-steroids into
16-unsaturated C19-steroids in porcine testicular homogen-
ates. These included 20b-reduction (preg fi pregnenediol fi
5,16-androstadien-3b-ol) [12], 21-hydroxylation (preg fi
21-OHpreg fi 5,16-androstadien-3b-ol) [10,13] and 16–
17-dehydrogenation (preg fi 17a-OHpreg fi 16-dehydro-
preg fi 5,16-androstadien-3b-ol) [7,14]. A concerted
process (preg fi 5,16-androstadien-3b-ol) has also been
suggested. Finally, results published by Weusten et al.
provided evidence that androstadienol was synthesized from
preg in a single step by a 16-ene-synthase enzyme system in
human testicular homogenates [15]. However, the molecular
mechanism responsible for this biosynthesis remains to be
elucidated.
It is well recognized that 16-androstenes are produced by
Leydig cells of porcine testis [8] and that these steroids have
Correspondence to V. Luu-The, Oncology and Molecular Endocrino-
logy Research Center, Laval University Medical Center (CHUL),

show that androstenol (5a-16-androsten-3a-ol) could
modulate the activity of two orphan receptors, the recently
renamed CAR (constitutive androstane receptor) [18],
previously known as constitutively active receptor [19],
and PXR (pregnane-X-receptor) [20]. It has been suggested
that androstenol is an endogenous ligand for these receptors
[20], which share a common hetero-dimerization partner,
RXR (retinoid-X-receptor), and are subject to cross talk
interactions with other nuclear receptors and with a broad
range of other intracellular signaling pathways [21,22].
The purpose of this research is to examine, at the
molecular level, whether the human P450c17 overexpressed
in HEK-293 cells possesses 16-ene-synthase activity and
how it differs from the 17a-hydroxylase and 17,20-lyase
activities. We also compare the human P450c17 with its
porcine counterpart.
Experimental procedures
Construction of P450red, cyt
b
5
and P450c17
expression vectors
The cDNA fragments containing the entire coding regions
of human NADPH-cytochrome P450reductase (P450red,
EC 1.6.2.4) [23,24] and cyt b
5
were isolated as previously
described [25]. The cDNAs were then subcloned into a
pCMV expression vector. Porcine P450c17 cDNA was
amplified by PCR using Taq DNA polymerase (Perkin-

or cyt b
5
as previously described [25]. Briefly, [
3
H]preg,
[
3
H]17a-OHpreg or [
3
H]DHEA was added to freshly
changed culture medium in six-well culture plates. For
Fig. 1. Central role of cytochrome P450c17 in the biosynthetic pathways leading to the formation of 16-ene steroids, sex steroids and glucocorticoids.
1350 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
enzymatic assays performed with intact cells in culture, we
have previously established 16 h to be an appropriate
incubation time period, as the activity vs. time graph still
shows linearity. After 16 h of incubation, the steroids were
extracted twice with 2 mL of ether. The organic phases
were pooled and evaporated to dryness. The steroids were
Fig. 2. Identification by HPLC of pregnenolone metabolites from HEK-293 cells transfected with P450c17 and cyt b
5
. (A) [
3
H]standard preg (left
panel), 17a-OHpreg (middle panel) and DHEA (right panel), (B) nonlabeled preg (left panel) and androstadienol (right panel). Products extracted
from cells transfected with 1 lg of pCMV-cyt b
5
and 0.1 lg of (C) human or (D) porcine pCMV-P450c17. Separation and identification of
metabolites were performed as described in Experimental procedures.
Ó FEBS 2003 Biosynthesis of 5,16-androstadien-3b-ol by human P450c17 (Eur. J. Biochem. 270) 1351

transfected with P450c17 and cyt b
5
.
3
H-Labeled preg,
17a-OHpreg, and DHEA, used as standards, showed
elution peaks at 4.70, 2.30 and 2.50 min, respectively
(Fig. 2). In both porcine and human assays using preg as
a substrate, an additional peak of elution appeared at
15 min (panels C and D, respectively). This additional peak
coincides with the elution time of nonlabeled commercial
androstadienol monitored using UV at 216 nm. This data
shows that one of the metabolites obtained in assays using
human and porcine P450c17 is androstadienol. In addition
to comigratory behavior on both HPLC and TLC analyses,
the identity of the radiolabeled androstadienol product was
confirmed by cocrystallization with commercial steroid
(data not shown).
Assessment of the 16-ene-synthase, 17a-hydroxylase
and 17,20-lyase activities of human
and porcine P450c17
In order to produce DHEA from preg, P450c17 first
transforms preg into 17a-OHpreg through its 17a-hydroxy-
lase activity and then transforms this intermediate into
DHEA through its 17,20-lyase activity. In order to deter-
mine whether the transformation of preg into androstadie-
nol requires prior synthesis of 17a-OHpreg or DHEA, we
performed enzymatic assays using human and porcine
P450c17 in the presence of various substrates – preg, 17a-
OHpreg and DHEA – and analyzed androstadienol

human P450c17 were incubated with 5 n
M
of [
3
H]preg in the absence/
presence of 1 l
M
ketoconazole. Metabolites were analyzed after
overnight incubation (16 h). (D) Nonlabeled standards revealed with
molybdate/sulfuric acid (10 : 10, v/v).
1352 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
androstadienol from preg in the pig and the human. As
illustrated in Fig. 4, both human and porcine enzymes have
the ability to produce androstadienol in presence of cyt b
5
.
When exogenous cyt b
5
is omitted from the transfection
assays, both human and porcine P450c17 poorly catalyze
the formation of androstadienol from preg (less than 2% of
preg transformation). Porcine P450c17 shows a slightly
stronger stimulation by cyt b
5
, its activity increasing to 15%
of preg transformation while the activity of human P450c17
increases to 12%. These results show that human and
porcine P450c17 have similar catalytic activities and that, in
both species, P450c17 is involved in the biosynthesis of
androstadienol.

5
changes dramatically as the cyt b
5
/P450c17 ratio varies.
Effect of P450red on DHEA and androstadienol synthesis
To further investigate the modulation of human P450c17
16-ene-synthase activity, we proceeded to analyze the
relative effect of P450red on 17a-hydroxylase/17,20-lyase
and 16-ene-synthase activities. To do so, we cotransfected
P450c17 and cyt b
5
in amounts determined to be optimal for
androstadienol formation along with increasing amounts of
P450red. It can clearly be seen in Fig. 6 that the addition of
P450red, even in small amounts, has a profound effect on
17a-hydroxylase/17,20-lyase activities. In presence of only
endogenous P450red levels, DHEA formation from preg is
below 10%. Increasing P450red up to 0.25 lgcausesa
drastic increase of DHEA formation, reaching levels up to
50% of preg transformation. On the other hand, increasing
amounts of P450red do not significantly stimulate 16-ene-
synthase activity. For this activity, endogenous levels of
P450red seem to be sufficient for optimal cyt b
5
stimulation
as increasing amounts of P450red do not further stimulate
androstadienol production. These results clearly show a
differential modulation of 17a-hydroxylase/17,20-lyase and
16-ene-synthase activities by P450red and cyt b
5

/0.1 lgof
P450c17). Transfections and enzymatic assays were performed as
described in Experimental procedures. The results are the mean ±
SEM of three independent experiments.
Fig. 4. Role of cyt b
5
in the formation of androstadienol from preg-
nenolone by human and porcine P450c17. HEK-293 cells were trans-
fected with 0.1 lg human or porcine pCMV-P450c17 in the presence
or absence of 1 lgpCMV-cytb
5
. Their ability to catalyze the trans-
formation of 5 n
M
of [
3
H]preg into androstadienol after overnight
incubation (16 h) was determined. Transfections and enzymatic assays
were performed as described in Experimental procedures. The results
are the mean ± SEM of three independent experiments.
Ó FEBS 2003 Biosynthesis of 5,16-androstadien-3b-ol by human P450c17 (Eur. J. Biochem. 270) 1353
androstenol (Fig. 1). This reaction differs from the produc-
tion of DHEA through the 17a-hydroxylase/17,20-lyase
activity, in that it does not require the 17a-OH-preg
formation step and it is strongly stimulated by optimal
amounts of cyt b
5
. Indeed, results obtained from assays
using 17a-hydroxypregnenolone as a substrate demonstrate
that the synthesis of androstadienol diverges from the

Although it is well known that androstenol is a phero-
mone in the pig, its role in the human is still ill-defined.
Recent findings show that androstenol is able to modulate
the expression of certain cytochrome P450s and alcohol
dehydrogenases through interactions with the orphan
receptors CAR and PXR [18,20]. These receptors belong
to the P450-regulatory nuclear receptors, in the subfamily
NR1 (nuclear binding site 1) [21]. Other members of this
NR1 orphan nuclear receptor gene subfamily are PPAR
(peroxisome proliferator-activated receptor), LXR (liver X
receptor) and FXR (farnesol-X-receptor). They share a
common hetero-dimerization partner, the RXR, and are
subject to cross-talk interactions with other nuclear recep-
tors and with a broad range of other intracellular signaling
pathways, including those activated by certain cytokines
and growth factors [21,22]. It has been shown that the
steroids androstenol [16(5a)-androsten-3a-ol] and 5b-preg-
nanedione (5b-pregnane-3,20-dione) modulate the action of
these receptors and thus are putative endogenous ligands for
these receptors [20]. Upon binding to the ligands, PXR and
CAR bind DNA as a heterodimer with the RXR and
modulate the expression of cytochromes P450, especially
CYP2B and CYP3A families. Because P450s play an
essential role in the detoxification of drugs and of a large
series of exogenous compounds from the environment,
androstenol and 5b-pregnanedione that modulate cyto-
chrome P450 levels could have a profound effect on the
detoxification process.
Because of the low affinity of these orphan nuclear
receptors (in the range of 1–10 · 10

are produced locally or accumulated in the tissue (probably
through active transport or hydrophobicity) is the concen-
tration high enough to modulate the receptor activity.
As suggested above, local biosynthesis in various tissues
such as the liver, could constitute a way to selectively
regulate the activity of nuclear orphan receptors such as
CAR and PXR. Although there is no evidence of P450c17
expression in the human liver, many other enzymes such as
3b-HSD, 5a-reductase and 3a-HSD, whose activities lead to
androstenol synthesis from androstadienol, are present in
Fig. 6. Influence of increasing amounts of P450red on the relative for-
mation of DHEA and androstadienol by P450c17. HEK-293 cells were
transfected with 0.1 lg pCMV-P450c17, 1 lg pCMV-cyt b
5
and the
indicated amounts of pCMV-P450red. The transfected cells were
analyzed for their ability to catalyze the transformation of 5 n
M
of
[
3
H]preg into DHEA (j) and androstadienol (n). Transfections
and enzymatic assays were performed as described in Experimental
procedures. The results are the mean ± SEM of three independent
experiments.
1354 P. Soucy et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the liver and many other peripheral tissues. Furthermore,
androstadienol is found in circulation suggesting that it is
synthesized at the sites of expression of P450c17 and
eventually converted to androstenol by different enzymes in

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