(Received for publication, January 30, 1995; and in revised form, June 2, 1995)
From the
During studies on the regulation of rat ovarian steroidogenic
enzymes by interleukin-1 (IL-1
), we observed substantial
metabolism of 25-hydroxycholesterol to two unusual polar products. This
unexpected effect was observed both in isolated granulosa cells and in
whole ovarian dispersates and was also induced by tumor necrosis factor
, but not by insulin-like growth factor I or follicle-stimulating
hormone. The effect was dependent on time and the dose of IL-1
and
was blocked by an IL-1 receptor antagonist. The formation of the polar
metabolites was inhibited by ketoconazole and trilostane, but not by
aminoglutethimide. Subsequent purification of these novel metabolites
and analysis by gas chromatography/mass spectrometry, NMR, and high
performance liquid chromatography revealed them to be related
7
-hydroxylated hydroxycholesterols
(cholest-4-ene-7
,25-diol-3-one and
cholest-5-ene-3
,7
,25-triol). IL-1
-stimulated ovarian
7
-hydroxylase activity (3-10 pmol/min/mg of cellular
protein) was nearly 4-fold that of control levels using
25-hydroxycholesterol as substrate. Activities at or below control
levels were observed when IL-1
-treated cell sonicates were boiled
or assayed in the presence of NADH (rather than NADPH), indicating that
involvement of a nonenzymatic process was unlikely.
IL-1
-stimulated 7
-hydroxylase activity was inhibited to basal
levels by a 10-fold excess of unlabeled 25- or 27-hydroxycholesterol,
but not by cholesterol, pregnenolone, progesterone, testosterone, or
dehydroepiandrosterone, suggesting that ovarian 7
-hydroxylase is
specific for hydroxycholesterols. Furthermore, when IL-1
-treated
ovarian cultures were incubated with radiolabeled cholesterol or
testosterone, no 7
-hydroxylated products were observed. We were
also unable to detect any mRNA transcripts for liver cholesterol
7
-hydroxylase in IL-1
-stimulated ovarian cultures. This study
describes an ovarian hydroxycholesterol 7
-hydroxylase that differs
from liver cholesterol 7
-hydroxylase and from other nonhepatic
progestin/androgen 7
-hydroxylases. The novel finding of the
regulation of a 7
-hydroxylase by IL-1
(and tumor necrosis
factor
) suggests a unique role for cytokines in the regulation of
cholesterol metabolism in the ovary and possibly other tissues.
The 7-hydroxylation of steroids has potentially widespread
biological significance. At least three categories of steroid
7
-hydroxylases may be defined. By far the best understood is
cholesterol 7
-hydroxylase (cholesterol 7
-monooxygenase, EC
1.14.13.17), a putative liver-specific (1) microsomal enzyme (2) and member of the cytochrome P450 gene family that
catalyzes the rate-limiting step of bile acid biosynthesis(3) .
As such, cholesterol 7
-hydroxylase plays a critical dual role as
the primary enzyme promoting both cholesterol catabolism and bile acid
formation. A second category of 7
-hydroxylases includes several
recently described nonhepatic enzymes that act on side chain-cleaved
steroids. A dehydroepiandrosterone/pregnenolone 7
-hydroxylase has
been reported in brain and other tissues (4, 5) and in
adipose stromal cells(6, 7) , and a testosterone
7
-hydroxylase in testis, lung, and kidney(8, 9) .
The functional significance of these 7
-hydroxylated steroids in
nonhepatic tissues has not yet been established. The metabolites may
regulate the availability of their biologically active precursors or
could have biological activity themselves. For example, Morfin and
Courchay (5) have suggested that 7
-hydroxylated
metabolites of pregnenolone and dehydroepiandrosterone may regulate the
immune response in mice.
The existence of a third class of steroid
7-hydroxylases that preferentially metabolize hydroxycholesterols
is controversial. It is well established that the liver is able to use
hydroxycholesterols as well as cholesterol as substrates for bile acid
biosynthesis(3, 10, 11) . Recently, it has
been suggested that this activity involves a hepatic hydroxycholesterol
7
-hydroxylase that is different from cholesterol
7
-hydroxylase(12, 13, 14) . Using
porcine liver microsomes, Toll et al.(14) were able
to separate 25- and 27-hydroxycholesterol 7
-hydroxylase from
cholesterol 7
-hydroxylase activity. Oxysterols such as 25- and
27-hydroxycholesterols (as distinct from cholesterol itself) act via a
putative receptor (cf.(15) ) as potent regulators of
cholesterol homeostasis, inhibiting 3-hydroxy-3-methylglutaryl-CoA
reductase activity and LDL (
)receptor levels in
vitro(16, 17, 18) . Oxysterols also
affect cell growth and viability presumably via their inhibitory
effects on cholesterol availability(19, 20) . Given
the regulatory importance of hydroxysterols, their further metabolism
to 7
-hydroxylated products is of considerable interest. Apart from
serving as intermediates in bile acid synthesis, 7
-hydroxylated
hydroxysterols may themselves regulate cholesterol homeostasis. Dueland et al.(21) have made the intriguing proposal that
7
-hydroxylase may indirectly induce the LDL receptor gene through
inactivation of oxysterol inhibition.
Both 25- and
27-hydroxycholesterols circulate in the
blood(22, 23) , and sterol 27-hydroxylase ()mRNA is widely distributed(25) . Both activity and
mRNA for 27-hydroxylase have been detected in human and rat
ovary(26, 27) . Rat luteal cells can metabolize
25-hydroxycholesterol(28) . 27-Hydroxycholesterol has been
shown to inhibit human ovarian cell sterol synthesis(27) .
In this work, we describe a novel ovarian 7-hydroxylase with
apparent substrate specificity for 25- and 27-hydroxycholesterols, thus
differentiating it from liver cholesterol 7
-hydroxylase and from
nonhepatic 7
-hydroxylases, which catalyze C
and
C
steroids. We further describe the dramatic enhancement
of ovarian 7
-hydroxylase activity by interleukin-1
(IL-1
) and tumor necrosis factor
(TNF-
), cytokines that
have multiple biological effects in the
ovary(29, 30, 31, 32, 33, 34, 35, 36, 37) .
The physiologic significance of cytokine-induced 7
-hydroxylated
hydroxycholesterols is unknown at present. However, these studies
suggest a possible role for cytokines in the regulation of cholesterol
homeostasis in the ovary and perhaps other tissues.
2-Hydroxypropyl--cyclodextran (2-HPBCD) was obtained from
Pharmatec, Inc. (Alachua, FL) and as a gift from George Reed (American
Maize Products Co., Hammond, IN). Recombinant human IL-1
(2
10
units/mg) was generously provided by Drs. Errol
B. DeSouza and C. E. Newton (DuPont Merck Pharmaceutical Co.)
Recombinantly expressed, naturally occurring human IL-1 receptor
antagonist (38) was generously provided by Dr. Daniel E. Tracey
(The Upjohn Co.). Recombinant human TNF-
was generously provided
by Dr. H. M. Shepard (Genentech, South San Francisco, CA). Recombinant
human insulin-like growth factor I (IGF-I) was from Bachem California
(Torrance, CA).
Authentic cholest-5-ene-3,27-diol
(27-hydroxycholesterol) and cholest-4-ene-7
,25-diol-3-one
(7
,25-dihydroxycholestenone) were the generous gifts of Dr. Norman
Javitt (New York University, New York). Cholest-5-ene-3
,25-diol
(25-hydroxycholesterol) and other steroids were obtained from
Steraloids, Inc. (Wilton, NH).
25-[26,27-
H]Hydroxycholesterol (83 Ci/mmol),
[1,2-
H]cholesterol (51.7 Ci/mmol),
[1
,2
-
H]testosterone (42.5 Ci/mmol), and
25-[16,17-
H]hydroxycholesterol (custom
preparation) were obtained from DuPont NEN.
Radioactivity was detected on line by a Flo-One/Beta detector (Packard Instruments), and absorbance at 240 nm was simultaneously monitored by a flow-through spectrophotometer (Lambda-Max, Waters). The column was calibrated with more than 35 steroids(40) , including most of those expected to be found in the ovary. The column tends to separate steroids based on the number of their hydroxyl groups, with less polar steroids eluting first.
Figure 1:
Polar hydroxycholesterol metabolites in
granulosa cells: stimulation by IL-1. Granulosa cells (5
10
cells/ml) were initially cultured for 72 h in the
absence or presence of IL-1
(50 ng/ml), with and without FSH (2
milliunits/ml). Cells were then washed, and
25-[
H]hydroxycholesterol (0.2 µM)
plus the same treatment protocols were added for an additional 24 h.
Media steroids were extracted and analyzed by HPLC as described under
``Materials and Methods.'' Standards are indicated by arrows as follows: Po, progesterone; Pe,
pregnenolone; M, 25-hydroxycholestenone; 20
-DHP,
20
-dihydroprogesterone. X1 and X2 are polar metabolites eluting at
15.7 and 20.4 min, respectively. Chromatograms from the same
representative experiment are shown. The inset in the secondpanel shows the IL-1
dose-dependent
formation of X1 and X2 (mean ± S.E., n = four
values from two separate experiments).
The IL-1-induced metabolism of 25-hydroxycholesterol to polar
metabolites was also apparent in comparable experiments using whole
ovarian dispersates (Fig.2). Significant quantities of X1
accumulated in the absence of exogenous IL-1
(Fig.2, toppanel). The formation of X1 and X2 by control
cells was reduced (Fig.2, secondpanel) by
the concomitant application of a naturally occurring IL-1 receptor
antagonist(38) , suggesting that the formation of the polar
metabolites in control cells could be related to endogenous production
of IL-1
. We have observed similar evidence of receptor-mediated,
endogenous IL-1
activity for other end points of IL-1
activity in whole ovarian dispersates (e.g. nitrite and
prostaglandin production; cf.(33) ). The accumulation
of both X1 and X2 was markedly increased above control levels (2.9
± 0.5-fold, p < 0.05, n = 4) by the
addition of IL-1
(Fig.2, thirdpanel).
IL-1
-induced formation of the X2 metabolite was consistently more
prominent in whole ovarian dispersates compared with granulosa cells.
The accumulation of X1 and X2 was time-dependent in both whole ovarian
dispersates (Fig.2, secondpanel, inset) and granulosa cells (not shown). The addition of
receptor antagonist to cells that were exogenously stimulated by
IL-1
dramatically inhibited the formation of the polar products (Fig.2, bottompanel), suggesting that the
hydroxycholesterol metabolizing action of IL-1
is mediated via its
receptor. Data from replicate experiments are summarized in Fig.2(bottompanel, inset),
demonstrating that the addition of receptor antagonist promotes a 51% (p < 0.05) and a 66% (p < 0.005) reduction in
the formation of X1 and X2 by control and IL-1
-stimulated cells,
respectively.
Figure 2:
IL-1-stimulated polar
hydroxycholesterol metabolites in whole ovarian dispersates: inhibition
by receptor antagonist. Whole ovarian dispersates (2.5
10
cells/ml) were initially cultured for 72 h in the absence or
presence of IL-1
(50 ng/ml), IL-1 receptor antagonist (RA; 5 µg/ml), or trilostane (3
10
M). Cells were then washed, and
25-[
H]hydroxycholesterol (10 µM)
plus the same treatment protocols were added for an additional 24 h.
Media steroids were extracted and analyzed by HPLC. Steroid elution
times are indicated by arrows as described for Fig.1.
Chromatograms from the same representative experiment are shown. The inset in the secondpanel shows the
time-dependent formation of X1 and X2 in control (brokenlines) and IL-1
-treated (solidlines; mean ± difference, n = two
values from one experiment) cells. The inset in the bottompanel shows the accumulation of X1 plus X2 in control,
IL-1
-treated, and IL-1
+ trilostane (IL+T)-treated cultures in the absence (solidbars) and presence (hatchedbars) of
receptor antagonist. Results are the mean ± S.E. or difference
of two to five separate experiments. Significance by ANOVA analysis,
relative to IL-1
in the absence of receptor antagonist, is
indicated (*).
The formation of X1 and X2 by IL-1 was also
inhibited (47%, p < 0.05) when ovarian cells were cultured
in the presence of trilostane (Fig.2, bottompanel, inset), an agent that reportedly inhibits
3
-hydroxysteroid dehydrogenase/isomerase activity(47) .
Furthermore, a UV
peak (characteristic of
-3-oxosteroids) was always coincident with formation
of the radiolabeled X1 metabolite in both granulosa cells and whole
ovarian dispersates. Taken together, these data suggest that the
formation of X1 involves, in part, oxidation of the C-3 moiety of
25-hydroxycholesterol by the abundant 3
-hydroxysteroid
dehydrogenase/isomerase activity present in these ovarian cells. A
3-oxo moiety on X1 was later confirmed as described below (cf.Fig.5and Fig. 7). However, inhibition of X2
formation by trilostane is not explained by trilostane inhibition of
3
-hydroxysteroid dehydrogenase. Trilostane inhibition of both X1
and X2 is suggestive of a more generalized inhibition, perhaps of the
7
-hydroxylase activity we report herein.
Figure 5:
Identification of X1 by GC/MS. X1 was
purified from ovarian cultures and analyzed by GC/MS. Representative
mass spectra for the methyloxime-trimethylsilyl derivatives of isolated
X1 (toppanel) and authentic
cholest-4-ene-7,25-diol-3-one (7
,25-dihydroxycholestenone) (bottompanel) are shown. The molecular ion (M
) is indicated at m/z 589, and
removal of methyloxime and trimethylsilyl groups is noted by 31- and
90-mass unit losses, respectively. The m/z 131 ion is composed
of the terminal three side chain carbons with the derivatized C
hydroxyl group.
Figure 7:
Coelution of X1 and authentic
7,25-dihydroxycholestenone on HPLC. Authentic
7
,25-dihydroxycholestenone (10 nmol) (toppanel)
and the 25-[
H]hydroxycholesterol metabolites (bottompanel) from IL-1
-stimulated whole
ovarian dispersates (prepared as described for Fig.2) were
analyzed by HPLC. Elution was simultaneously monitored by absorbance at
240 nm (solidlines) and on-line scintillation
counting (brokenline).
Figure 3:
Polar hydroxycholesterol metabolites in
whole ovarian dispersates: peptide specificity. Whole ovarian
dispersates were cultured and analyzed as described for Fig.2,
except in the absence or presence of TNF- (10 ng/ml) or IGF-I (50
ng/ml). The doses used were maximally effective in modulating ovarian
steroidogenesis (not shown). Results were replicated in an additional
experiment. In three other similar experiments (not shown) using
granulosa cells rather than whole ovarian dispersates, TNF-
stimulated accumulation of X1 and X2. M,
25-hydroxycholestenone.
To determine whether the ovarian
metabolism of 25-hydroxycholesterol is sensitive to P450 enzyme
inhibitors, granulosa cells were cultured in the absence or presence of
IL-1 with and without ketoconazole (5-15 µM), a
well-known inhibitor of cholesterol hydroxylases and other P450
enzymes(17, 49, 50, 51) , or with
aminoglutethimide (15-30 µM), a potent inhibitor of
the P450
enzyme(52, 53) . As shown in Fig.4, IL-1
-dependent formation of X1 and X2 was inhibited
by ketoconazole in a dose-dependent manner, with nearly complete
inhibition at the 15 µM dose. Simultaneous assessment of
cell viability by tetrazolium dye reduction (54) indicated that
ketoconazole was not toxic to these cells at the doses used (not
shown). In contrast to the inhibitory action of ketoconazole,
aminoglutethimide had no effect on the formation of X1 and X2. In a
parallel experiment (not shown), FSH-dependent P450
activity was inhibited to control levels by aminoglutethimide (15
µM), as expected. These data demonstrate that the ovarian
enzyme that catalyzes the formation of X1 and X2 is
ketoconazole-sensitive and aminoglutethimide-insensitive.
Figure 4:
Formation of polar hydroxycholesterol
metabolites: effect of P450 inhibitors. Granulosa cells (5
10
cells/ml) were initially cultured for 72 h in the
presence of IL-1
(50 ng/ml), with and without ketoconazole
(5-15 µM) or aminoglutethimide (15 and 30
µM). Cells were then washed, and
25-[
H]hydroxycholesterol (0.2 µM)
plus the same treatment protocols were added for an additional 24 h.
Accumulation of X1 (solidlines) and X2 (brokenlines) was determined by HPLC analysis. Data are the mean
± S.E. or difference (n = two to five separate
experiments).
Figure 6:
Identification of X2 by GC/MS: comparison
with reduced X1. X2 was purified from ovarian cultures and analyzed by
GC/MS. The comparison spectrum for the trimethylsilyl derivatives of X2 (topsection) and NaBH-reduced X1 (bottomsection, inverted) is shown. The molecular
ion (M
) is at m/z634, and
the base peak is at m/z 544. The representative spectrum of X2 (topsection) is identical to that of authentic
cholest-5-ene-3
,7
,25triol (7
,25-dihydroxycholesterol; cf.(14) ). Removal of each trimethylsilyl group is
noted by a 90-mass unit loss.
The spectrum for the methyloxime-trimethylsilyl derivative of X1 (Fig.5, toppanel) was characterized by a molecular ion at m/z 589 and ions formed by loss of the oxime (m/z 558 (M - 31)) and trimethylsilyl (m/z 468 (M - 31 - 90); m/z 378 (M - 31 - 90 - 90)) groups, among others. These data indicate a cholestenediolone structure with an underivatized molecular mass of 416 Da.
The spectrum for the
trimethylsilyl derivative of X2 (Fig.6, topsection of comparison spectrum) demonstrated it to be a
cholestenetriol with a derivatized molecular mass of 634 Da to include
three trimethylsilyl groups. The underivatized molecular mass would be
418 Da. The most prominent ion in the spectrum was at m/z 544
(M - 90), although M - 90 - 90 and M - 90
- 90 - 90 ions were also present. The high abundance of the
M - 90 ion immediately gave a strong indication of the presence
of a 7-hydroxy moiety since virtually all steroid classes
(androstanes, pregnanes, cholestanes) give base peaks at M - 90
for trimethylsilyl derivatives of 7
-hydroxy compounds.
Furthermore, comparison with standards available at the time of
analysis indicated that the additional hydroxyl group on X2 was not at
C-23, C-24, or C-26.
Sodium borohydride reduction of X1 gave two
compounds following GC with similar spectra to X2, but slightly shorter
and longer retention times, respectively. These almost certainly
represented reduction of the carbonyl group to - and
-hydrogens. The comparison spectra for reduced X1 (Fig.6, bottomsection, inverted) and X2 (topsection) show that the only difference is the presence of
an ion at m/z 196 in the spectrum for reduced X1. This ion is
very distinctive for trimethylsilyl derivatives of steroids with
3,7
-dihydroxy-4-ene structures(56) . The presence of a
4-ene group in reduced X1 strongly indicated that the moiety prior to
reduction was a 3-carbonyl. The lack of the m/z 196 ion in X2
with an otherwise identical spectrum to reduced X1 indicated that X2
had a 5-ene group.
Subsequent analysis by NMR (see below)
definitively identified X1 as cholest-4-ene-7,25-diol-3-one
(7
,25-dihydroxycholestenone). As shown, isolated X1 has an
identical spectrum to that of authentic 7
,25-dihydroxycholestenone (Fig.5, bottompanel). Furthermore, the X1
metabolite of 25-[
H]hydroxycholesterol from
IL-1
-stimulated whole ovarian dispersates (Fig.7, bottompanel, brokenline) coeluted
with authentic 7
,25-dihydroxycholestenone (toppanel) in our HPLC system. Note that only X1 and
7
,25-dihydroxycholestenone show the UV
absorbance (solidlines) that is typical of
-3-oxosteroids. NMR analysis (see below) identified
both X1 and X2 as specifically 7
(rather than 7
or 7-keto)
metabolites. The X2 metabolite was identified by NMR as
cholest-5-ene-3
,7
,25-triol (7
,25-dihydroxycholesterol).
Isolated X2 has a spectrum (Fig.6, topsection) identical to that reported for authentic
7
,25-dihydroxycholesterol (cf.(14) ). Taken
together, these data suggest that X2 is a 7
-hydroxy metabolite of
25-hydroxycholesterol that is additionally oxidized at C-3 to form X1.
The X2 metabolite was identified by NMR as
cholest-5-ene-3,7
,25-triol. At the time of analysis, a
suitable reference compound (i.e. one containing the
5-ene-3
,7
-dihydroxy structure) was not available. Assignment
was further complicated by the presence of an impurity (
60%), at a
similar concentration to the steroid, which was probably some type of
carbohydrate. The characteristic signals for this compound are listed
in Table2. As with X1, it was possible to assign chemical shifts
to all of the ring protons.
The identification of X1 and X2 as
specifically 7-hydroxy is confirmed by the characteristic
multiplet pattern and chemical shift (cf.(44) ) of
the 7
-proton in the NMR spectrum. A 7
-proton in a
7
-hydroxy compound has a different pattern and shift. A 7-keto
compound would show no 7-proton signal. That the relevant signal in
each spectrum is a 7-proton is confirmed by the cross-peaks present in
the two-dimensional COSY spectrum.
Figure 8:
IL-1-stimulated 7
-hydroxylase
activity in whole ovarian dispersates. Whole ovarian dispersates (5
10
cells/ml) were cultured for 72 h in the absence (solidbar) or presence (hatchedbars) of IL-1
(50 ng/ml). Cells were then sonicated
and assayed for 7
-hydroxylase activity using
25-[
H]hydroxycholesterol (0.2 µM) as
substrate. Heat-treated sonicates (100 °C, 10 min) or NADH (0.5
mM)-substituted reactions (rather than NADPH) monitored basal
assay activity as indicated (mean ± S.E., n =
three to five separate experiments). Significance by ANOVA analysis,
relative to maximal activity (IL-1
, standard conditions), is
indicated (*). The inset is a representative experiment
showing a double reciprocal plot of activity (V;
picomoles/hour/10
cells) for IL-1
-treated cells in the
presence of different concentrations of 25-hydroxycholesterol (S; micromolar).
Figure 9:
IL-1-stimulated 7
-hydroxylase
activity in whole ovarian dispersates: substrate specificity. Whole
ovarian dispersates were cultured in the presence of IL-1
and
assayed for 7
-hydroxylase activity as described for Fig.8.
Activity was measured in the absence (solidbar) or
presence (hatchedbars) of a 10-fold excess of
unlabeled steroids (2 µM), which were potential
competitors of the activity determined using
25-[
H]hydroxycholesterol as substrate. Data are
normalized relative to activity in the absence of excess steroid (solidbar, 100%), and significance by ANOVA analysis
relative to this value is indicated (*). 25-OH,
25-hydroxycholesterol; 27-OH, 27-hydroxycholesterol; Chol, cholesterol; Pe, pregnenolone; Po,
progesterone; Testo, testosterone; DHEA,
dehydroepiandrosterone. Data are the mean ± S.E. or difference
from two to three separate experiments, each in
duplicate.
To more directly
examine the specificity of ovarian 7-hydroxylase, whole ovarian
dispersates were initially cultured for 72 h in the absence or presence
of IL-1
, after which cells were labeled with
[
H]cholesterol or
[
H]testosterone (Fig.10). No polar
metabolites of cholesterol (indicative of 7
-hydroxylase activity)
were observed in either the absence (brokenlines) or
presence (solidlines) of IL-1
, the
chromatograms being qualitatively similar. An increased accumulation of
an unidentified, less polar metabolite was consistently observed in
IL-1
-treated cells. Importantly, in parallel experiments using
FSH-pretreated cultures, we observed substantial metabolism of
[
H]cholesterol to P450
products
(not shown), demonstrating that solubility/cellular availability of the
added cholesterol was not a problem.
Figure 10:
IL-1 does not promote ovarian
7
-hydroxylation of cholesterol or testosterone. Whole ovarian
dispersates (2.5
10
cells/ml) were initially
cultured for 72 h in the absence (brokenlines) or
presence (solid lines) of IL-1
(50 ng/ml), after which
cells were washed, and either [
H]cholesterol (3
µM; upperpanel) or
[
H]testosterone (3 µM; lowerpanel) was added for an additional 24 h. Media steroids
were extracted and analyzed by HPLC. Elution times of standards are
indicated by arrows as follows: A1, androstanedione; A2, androstenedione; A3, androsterone and
dihydrotestosterone; DHEA, epiandrosterone; Diols,
androstenediol, 3
-androstanediol, and 3
-androstanediol; 7
-Testo, 7
-hydroxytestosterone. Representative
chromatograms are shown.
As shown in Fig.10(bottompanel), a variety of
[H]testosterone metabolites were observed in both
control (brokenline) and IL-1
-treated (solidline) cells. However, no metabolism to
7
-hydroxytestosterone was apparent. These data directly confirm
the conclusion of the enzyme competition assays (Fig.9), that
ovarian 7
-hydroxylase is specific for hydroxycholesterols.
To
begin to delineate the substrate specificity of ovarian
7-hydroxylase at the molecular level, total RNAs from immature
female rat liver and from cultured whole ovarian dispersates were
probed with rat liver cholesterol 7
-hydroxylase
[
P]UTP-labeled riboprobe using a highly
sensitive and specific liquid hybridization/RNase protection assay (Fig.11). A strong signal (protected fragment) of the
appropriate size (346 nucleotides) was apparent for the rat liver
positive control. In contrast, no cholesterol 7
-hydroxylase mRNA
transcripts were apparent for either control or IL-1
-treated
ovarian cells in spite of an obvious protected fragment for the
normalizing probe (RPL19, 153 nucleotides) in both these lanes. (The
band seen in the control ovary lane is undigested RPL19.) Given the
sensitivity inherent in the liquid hybridization/RNase protection assay
and since we were able to detect mRNA transcripts for ovarian
5
-reductase (not shown; an enzyme of comparable abundance in this
system), it seems likely that immature rat ovaries lack liver
cholesterol 7
-hydroxylase transcripts.
Figure 11:
Analysis of cholesterol
7-hydroxylase transcripts in rat liver and ovary. Whole ovarian
dispersates (1.5
10
cells/3 ml) were cultured for
72 h in the absence or presence of IL-1
(50 ng/ml). Thereafter,
total RNA was prepared from cultured cells (RNA from 1.5
10
cells, each lane) and from immature female rat liver (20
ng/lane) and subjected to liquid hybridization/RNase protection assay.
Both 7
-hydroxylase and RPL19 (to monitor equality of loading)
transcripts were simultaneously probed. Also shown are nucleotide (nt) size markers (Ambion Inc., Austin, TX) and the
undigested, full-length probes for 7
-hydroxylase and RPL19.
Results were replicated in an additional
experiment.
Herein, we describe a novel, cytokine-regulated, nonhepatic
hydroxycholesterol 7-hydroxylase. Activity was initially observed
in cultures of rat granulosa cells or whole ovarian dispersates as the
IL-1
-dependent, receptor-mediated accumulation of two unknown
polar metabolites (X1 and X2) of
25-[
H]hydroxycholesterol. X1 and X2 were
subsequently identified by GC/MS, NMR, and HPLC as
cholest-4-ene-7
,25-diol-3-one (7
,25-dihydroxycholestenone)
and cholest-5-ene-3
,7
,25-triol
(7
,25-dihydroxycholesterol). Although autoxidation of cholesterol
at C-7 (primarily to 7-oxo or 7
-hydroxyl moieties; cf.(58) ) is well known, it is unlikely that the
hydroxycholesterol 7
-hydroxylation described herein is a
nonenzymatic event. IL-1
-stimulated ovarian 7
-hydroxylase
activity demonstrated all the hallmarks of a typical enzyme reaction.
It displayed coenzyme specificity (NADH is not effective),
Michaelis-Menten kinetics, and heat instability. Product accumulation
was time- and IL-1
dose-dependent and did not occur in the absence
of living cells. Furthermore, ovarian 7
-hydroxylase activity was
subject to dose-dependent inhibition by ketoconazole, a well-known
inhibitor of P450 enzymes(17, 50, 51) , at a
dose range similar to that reported for inhibition of rat hepatocyte
cholesterol 7
-hydroxylase(49) . In contrast, ovarian
7
-hydroxylase activity was not inhibited by aminoglutethimide, a
potent inhibitor of the P450
enzyme(52, 53) .
The ovarian
hydroxycholesterol 7-hydroxylase enzyme differs from hepatic
cholesterol 7
-hydroxylase (1, 2, 3) since cholesterol does not compete
in assays using 25-hydroxycholesterol as substrate and is not
metabolized to any polar products and since no mRNA for hepatic
cholesterol 7
-hydroxylase can be detected in stimulated or
unstimulated ovaries. The ovarian hydroxycholesterol 7
-hydroxylase
enzyme also differs from nonhepatic enzymes that act on side
chain-cleaved
steroids(4, 5, 6, 7, 8, 9) ,
based on indirect and direct substrate specificity studies. However,
the identity of the physiologically pertinent substrate for ovarian
7
-hydroxylase is unknown at this time. 27- and
25-hydroxycholesterols are good candidates since activity and mRNA for
27-hydroxylase have been detected in human and rat
ovary(25, 27) , and rat luteal cells can metabolize
25-hydroxycholesterol to reproductive steroids(28) .
Furthermore, we have detected 27-hydroxylase mRNA in cultured granulosa
cells and whole ovarian dispersates from both control and
IL-1
-treated cells (not shown). However, no information exists
regarding the presence or regulation of such hydroxysteroids in the
ovary. It is unclear whether the ovarian 7
-hydroxylase
preferentially catalyzes a hydroxycholesterol or a hydroxycholestenone
as substrate. Certainly, oxidation of hydroxycholesterols at C-3 by
3
-hydroxysteroid dehydrogenase/isomerase is prominent in the
ovary, as has been shown in the adrenal gland (59) and
liver(60) . Thus, any of a variety of 7
-hydroxylated
hydroxycholesterols or hydroxycholestenones may be of biological
significance in the ovary. The challenge in identifying a physiologic
role for ovarian 7
-hydroxylase will be to determine what are its
substrates in vivo.
We have previously reported rat ovarian
IL-1 gene expression (61) and a wide range of
receptor-mediated (62) biological activities for IL-1
in
the
ovary(29, 30, 31, 32, 33) .
TNF-
modulates rat ovarian
steroidogenesis(34, 35, 36, 37) .
Both these cytokines can also induce hydroxysterol 7
-hydroxylase
activity in the ovary. This effect is peptide-specific, however, since
other ovarian modulatory factors such as FSH and IGF-I (48) are
ineffective in this regard. Although this report extends our
understanding of the ovarian actions of cytokines, it is perhaps of
more interest as a demonstration of the existence of a previously
unreported class of 7
-hydroxylases that are regulated by IL-1
and TNF-
. Cholesterol 7
-hydroxylase is primarily regulated at
the level of gene transcription via feedback inhibition by bile acids,
but is also subject to regulation by diurnal, hormonal, and dietary
factors(1, 3, 57, 63) . Regulatory
mechanisms for liver hydroxycholesterol 7
-hydroxylase or for other
nonhepatic 7
-hydroxylases are unknown. Therefore, the potential
relevance of cytokines to the regulation of steroid 7
-hydroxylases
is of interest and remains to be determined.
A physiologic function
for 7-hydroxycholesterols or 7
-hydroxycholestenones can only
be speculated upon at this time. Although hydroxycholesterols are
substrates for P450
( (22) and (28) ; cf.Fig.1), preliminary studies (not shown) indicate
that 7
-hydroxylated hydroxycholesterols are not side
chain-cleaved, suggesting that such steroids are not precursors for
reproductive steroids. It is possible that 7
-hydroxy metabolites
mediate some of the biological actions of cytokines in the ovary or
play a role in ovarian cholesterol homeostasis. With respect to the
latter, Dueland et al.(21) showed that Chinese
hamster ovary cells, expressing hepatic 7
-hydroxylase, are
resistant to down-regulation of LDL receptor genes by
25-hydroxycholesterol. These authors propose that 7
-hydroxylase
increases the expression of LDL receptor by metabolic inactivation of
hydroxycholesterol inhibitors of this gene. This hypothesis also
suggests a mechanism by which liver cells (that express
7
-hydroxylase) are resistant to down-regulation of LDL
receptor(20) . However, the nature of the link between
7
-hydroxylase and cholesterol homeostasis is
controversial(14, 64) .
Treatments that stimulate
macrophages also increase hepatic cholesterol synthesis and mRNA levels
for 3-hydroxy-3-methylglutaryl-CoA reductase(65) . Macrophage
products (i.e. cytokines) increase serum cholesterol
levels(66) . The Dueland hypothesis(20, 21) proposes that cholesterol biosynthesis is stimulated by
7-hydroxylase-mediated inactivation of oxysterol repressors.
Perhaps cytokines enhance cholesterol biosynthesis (cf.(66) ) by just such a mechanism. The data described herein
are consistent with these previous observations and, taken together,
predict that cytokines could enhance cholesterol accumulation in the
ovary via the inactivation of inhibitory hydroxycholesterols by
cytokine-induced 7
hydroxylation.
Although we may speculate that
cytokine-induced 7-hydroxylation blocks the inhibitory action of
oxysterols on cholesterol biosynthesis, thereby enhancing cholesterol
availability, the significance of the IL-1
-stimulated
7
-hydroxylation of hydroxysterols in the ovary (or other tissues)
remains to be established. In any case, the finding of a nonhepatic
C
7
-hydroxylase that is regulated by IL-1
(and
TNF-
) suggests a unique role for cytokines in the metabolism of
C
steroids.