Báo cáo khoa học: Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor doc

Assessment of porcine and human 16-ene-synthase, a third activityof P450c17, in the formation of an androstenol precursor Penny Soucy, Lucille Lacoste and Van Luu-The Molecular Endocrino

Assessment of porcine and human 16-ene-synthase, a third activity

of P450c17, in the formation of an androstenol precursor

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 NADPH-cytochrome b5 Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3b-ol, without the formation of the precursor 17-hydroxy-pregnenolone Cytochrome b5has a much stronger effect on the 16-ene-synthase activity than on the 17a-hydroxylase/ 17,20-lyase activities On the other hand, P450reductase has

a drastic effect on the latter, but a negligible one on 5,16-androstadien-3b-ol synthesis Our results therefore demon-strate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol

Keywords: 16-ene-synthase; 17a-hydroxylase/17,20-lyase; cytochrome b5; 5,16-androstadien-3b-ol; pregnenolone

It has been established that human cytochrome P450c17

(product of the CYP17 gene) has two distinct activities

responsible for the synthesis of glucocorticoid and sex

steroid precursors from pregnenolone (preg) A

17a-hydroxylase activity, which converts preg into 17a-OHpreg,

is necessary for cortisol synthesis, and a 17,20-lyase activity

further transforms 17a-OHpreg into

dehydroepiandroster-one (DHEA), the precursor of sex steroids (Fig 1) Several

different studies have revealed that these two activities are

differentially modulated by many factors, two of the most

important being the abundance of the redox partner cytochrome P450reductase (P450red) [1,2] and the inter-action with cytochrome b5 (cyt b5), 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 (pregfi pregnenediol fi 5,16-androstadien-3b-ol) [12], 21-hydroxylation (pregfi 21-OHpreg fi 5,16-androstadien-3b-ol) [10,13] and 16– 17-dehydrogenation (pregfi 17a-OHpreg fi 16-dehydro-pregfi 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),

2705 Laurier Boulevard, Que´bec (QC) G1V 4G2, Canada.

Fax: + 1 418 654 2761, Tel.: + 1 418 654 2296,

E-mail: van.luu-the@crchul.ulaval.ca

Abbreviations: P450c17, 17a-hydroxylase/17,20-lyase; P450red,

NADPH cytochrome P450 reductase; cyt b 5 , cytochrome b 5 ; 3b-HSD,

3b-hydroxysteroid dehydrogenase/D5fiD4 isomerase; 3a-HSD,

3a-hydroxysteroid dehydrogenase; preg, pregnenolone; DHEA,

dehydroepiandrosterone; 17a-OHpreg, 17a-hydroxypregnenolone;

androstadienol, 5,16-androstadien-3b-ol; androstenol,

5a-16-androsten-3a-ol; CAR, constitutive androstane receptor; PXR,

pregnane-X-receptor; RXR, retinoid-X-receptor; HEK-293,

trans-formed human embryonic kidney 293 cells.

Enzyme: NADPH-cytochrome P450reductase (P450red, EC 1.6.2.4).

(Received 4 November 2002, revised 23 January 2003,

accepted 10 February 2003)

pheromonal activity in pigs In humans, the physiological

role of 16-androstene steroids is still ill-defined It has been

proposed that these compounds may have significant effects

on behavior, namely reducing nervousness, tension and

other negative emotional states in women [16] Another

study demonstrated a positive relationship between

menstrual synchrony and the ability to smell certain

16-androstene steroids [17] Recent reports in the literature

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, cytb5and 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 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-Elmer Cetus, Emerville, CA, USA) [26] and an oligo-primer pair (5¢-GGGGTCGACATGTGGGTGCTCTTGGTTT TCTTCTTG-3¢ and 5¢-GGGGTCGACTCAGGAGGT ACTCCCCTCAGTGTGGGC-3¢) and poly(A)+ RNA isolated from pig testis The cDNA was then subcloned into a pCMV expression vector The cDNA coding for human P450c17 (EC 1.14.99.9) was kindly provided by Y Tremblay (CHUL Research Center, Quebec, Canada)

Transient expression in transformed human embryonic kidney (HEK-293) cells

Vectors expressing P450c17 (pCMV-P450c17), P450red (pCMV-P450red) and cyt b5 (pCMV-cyt b5) were trans-fected into HEK-293 cells using the Ex-gene kit according to the manufacturer’s instructions (MBIFermentas, Amherst,

NY, USA) Cells were initially plated at 5· 105cells per well in six-well falcon flasks and grown in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA) at 37C under a 95% air, 5% CO2

humidified atmosphere

Assay of enzymatic activity Determination of the activities was performed in intact cells transiently transfected with P450c17 and/or P450red and/

or cyt b5 as previously described [25] Briefly, [3H]preg, [3H]17a-OHpreg or [3H]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.

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) [3H]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.

· 150 mm, 4 lm).

The mobile phase was MeOH/H2O (80 : 20, v/v) with 2 mM

ammonium acetate and 0.1% acetic acid, with a flow rate of

1 mLÆmin)1 Radioactivity was monitored in the eluent

using Beckman 171 HPLC Radioactivity Monitoring

System Nonlabeled androstadienol and preg standards

were monitored using UV at 216 nm

Results

Identification of metabolites by HPLC analysis

and corecrystallization

To verify the nature of the metabolites obtained from the

transformation of preg by human and porcine P450c17, we

identified by HPLC analysis, extracts of HEK-293 cells

transfected with P450c17 and cyt b5 3H-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

formation from each substrate As observed in Fig 3, the

biosynthesis of androstadienol in humans (A) and pigs (B)

does not require prior formation of 17a-OH-preg and

DHEA The lack of androstadienol synthesis in the presence

of 1 l of ketoconazole (C), an inhibitor of cytochrome

P450, further demonstrates the specific implication of P450c17 in the formation of this metabolite

Formation of androstadienol by human and porcine P450c17, with and without cytb5

Using porcine and human P450c17 expressed in

HEK-293 cells in culture, we compared the formation of

Fig 3 Thin layer chromatography showing the transformation of pregnenolone,17a-OHpregnenolone and DHEA by human and porcine P450c17 HEK-293 cells transfected with 1 lg pCMV-cyt b 5 and 0.1 lg (A) human, or (B) porcine pCMV-P450c17 were treated with

5 n M of the indicated3H-labeled substrates and analyzed for their ability to produce androstadienol after overnight incubation (16 h) (C) HEK-293 cells transfected with 1 lg pCMV-cyt b 5 and 0.1 lg human P450c17 were incubated with 5 n M of [3H]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).

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 b5

When exogenous cyt b5 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 b5, 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

Effect of cytb5on DHEA and androstadienol

biosynthesis

In order to determine the effect of cyt b5 in P450c17

16-ene-synthase and 17a-hydroxylase/17,20-lyase activities,

we performed transfection assays with increasing amounts

of DNA fragments encoding cyt b5 and monitored the

formation of androstadienol and DHEA As shown in

Fig 5 the stimulation of DHEA and androstadienol

production from preg increases with increasing amounts

of cyt b5 in presence of endogenous levels of P450red In

presence of these low levels of P450red, cyt b5 shows a

slight stimulatory effect on DHEA formation However,

we observe a more profound effect on the synthesis of

androstadienol More precisely, an increase of

androsta-dienol formation was observed at a cyt b5/P450c17 ratio

of 5 : 1 (Fig 5) The activity reached a maximum at a

ratio of 12 : 1 Thus, the influence of human cyt b5 changes dramatically as the cyt b5/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 b5in 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 lg causes a 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 b5stimulation

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 b5

Discussion

It is already known that human cytochrome P450c17 possesses two distinct activities, a 17a-hydroxylase and a 17,20-lyase activity, responsible for the biosynthesis of glucocorticoid and sex steroid precursors In this report we show that human and porcine P450c17 also possess a 16-ene-synthase activity that catalyzes the transformation of preg into androstadienol, a precursor in the biosynthesis of

Fig 5 Influence of increasing concentrations of cyt b 5 on the relative formation of androstadienol and DHEA by human P450c17 HEK-293 cells were transfected with 0.1 lg pCMV-P450c17 and the indicated amounts of pCMV-cyt b 5 The transfected cells were analyzed for their ability to catalyze the transformation of 5 n M of [3H]preg to andro-stadienol An increase in 16-ene-synthase activity is observed at a cytb 5 / P450c17 ratio of 5 : 1 (0.25 lg cytb 5 /0.1 lg P450c17) while the optimal stimulation is observed at a ratio of 12 : 1 (1 lg of cytb 5 /0.1 lg of 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 lg pCMV-cyt b 5 Their ability to catalyze the

trans-formation of 5 n M of [3H]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.

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 b5 Indeed, results obtained from assays

using 17a-hydroxypregnenolone as a substrate demonstrate

that the synthesis of androstadienol diverges from the

biosynthetic pathway of sex steroids at the level of preg

transformation and that it does not involve the

17a-hydroxy-lase activity Furthermore, time course experiments (data not

shown) using preg as a substrate do not show the production

of any intermediates in the formation of androstadienol

which suggests that it is synthesized from pregnenolone in a

single step Therefore, although the synthesis of

glucocorti-coid/sex steroid precursors and 16-ene-steroids result

from the same enzyme, the activities responsible for their

formation and the regulation of these activities are distinct

Gower et al had previously demonstrated, using porcine

testis microsomes, the formation of 16-androstenes from

17a-hydroxypregnenolone [8] However, this situation is not

observed in the intact transfected cell system using porcine or

human P450c17 In combination, these results suggest the

presence, in pig testis, of another system that can use

17a-hydroxypregnenolone to form a different product

P450c17 is therefore a crucial enzyme, not only in the

formation of sex steroid precursors, but also in the

production of androstadienol which is considered to be an

intermediate in the 16-androstene pathway leading to the

biosynthesis of androstenol and of 5a(16)androsten-3-one, a

pheromonally active steroid in the pig [8,14] A recent report

by our group [27] shows that further transformation of

androstadienol into androstenol involves the classic

enzymes of the steroidogenic pathway, namely 3b-HSD,

5a-reductase and 3a-HSD Interestingly, because of the lack

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)6M) and their relatively broad spectrum of ligand specificity, many researchers that are familiar with classic steroid receptors, namely androgen, estrogen, progesterone, glucocorticoid and mineralocorti-coid receptors, that bind to their corresponding specific ligand with a very high affinity (10)10)10)9M), are skeptical about the idea of androstenol and 5b-pregnanedione being ligands for these orphan receptors However, it is notewor-thy that, as classic active steroids are diluted in the blood, their concentration is very low and thus they require high affinity receptors to pick them up On the other hand, the orphan receptor ligands are most probably produced locally

in the various tissues Because of the small volume of the cell, the production of a little amount of ligand will give a relatively high concentration (up to 1–10· 10)6M) We hypothesize that this low affinity combined with the local biosynthesis of ligands represents a mechanism allowing the selective regulation of the action of the receptor: ligands that enter the cell or tissue by chance will not have a high enough concentration to turn on the receptor Only for ligands that 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

[3H]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.

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

peripheral tissues such as the liver and adipose tissue Our

study aimed at elucidating the nature and the mechanism of

the reactions involved in the local formation of androstenol

is thus of major importance

Acknowledgements

This work has been supported by a grant from the Canadian Institutes

of Health Research The authors would like to thank Guy Reimnitz,

Nathalie Paquet and Mei Wang for their technical assistance and Sylvie

Me´thot for careful reading of the manuscript.

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