Concerted regulation of free arachidonic acid and hormone-induced steroid synthesis by acyl-CoA thioesterases and acyl-CoA synthetases in adrenal cells Paula Maloberti, Rocı´o Castilla L
Trang 1Concerted regulation of free arachidonic acid and hormone-induced steroid synthesis by acyl-CoA thioesterases and acyl-CoA
synthetases in adrenal cells
Paula Maloberti, Rocı´o Castilla Lozano, Pablo G Mele, Florencia Cano, Cecilia Colonna,
Carlos F Mendez, Cristina Paz and Ernesto J Podesta´
Department of Biochemistry, School of Medicine, University of Buenos Aires
Although the role of arachidonic acid (AA) in the regulation
of steroidogenesis is well documented, the mechanism for
AA release is not clear Therefore, the aim of this study was to
characterize the role of an acyl-CoA thioesterase (ARTISt)
and an acyl-CoA synthetase as members of an alternative
pathway in the regulation of the intracellular levels of AA in
steroidogenesis Purified recombinant ARTISt releases AA
from arachidonoyl-CoA (AA-CoA) with a Km of 2 lM
Antibodies raised against recombinant acyl-CoA
thioest-erase recognize the endogenous protein in both adrenal tissue
and Y1 adrenal tumor cells by immunohistochemistry and
immunocytochemistry and Western blot Stimulation of
Y1 cells with ACTH significantly stimulated endogenous
mitochondrial thioesterases activity (1.8-fold)
Nordihydro-guaiaretic acid (NDGA), an inhibitor of AA release known
to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochond-rial acyl-CoA thioesterases ACTH-stimulated steroid syn-thesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase The apparent IC50for NDGA was reduced from
50 lMto 25, 7.5 and 4.5 lMin the presence of 0.1, 0.5 and
2 lM triacsin C, respectively Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells
Keywords: arachidonic acid; steroidogenesis; acyl-CoA thio-esterases; acyl-CoA synthetase; arachidonoyl-CoA
The rate-limiting step, which initiates the synthesis of all
steroids, depends on the availability of cholesterol to
cytochrome P450scc [1,2] The involvement of a
cAMP-dependent protein kinase (PKA) phosphorylation event is
accepted as an intermediate step in the cAMP-mediated
stimulation of cholesterol availability [3–9] and particularly
in the transport of cholesterol from the outer to the inner
mitochondrial membrane [1,2,10,11] The latter event is, in
turn, controlled by the steroidogenic acute regulatory
protein (StAR protein) [12–16]
Several reports have shown that arachidonic acid (AA)
and its metabolites play an essential role in the regulation of
steroidogenesis and both the expression and function of
StAR [17–26] Adrenal cholesterol metabolism is inhibited
by nordihydroguaiaretic acid (NDGA) [19–25], a lipoxy-genase inhibitor that also acts as phospholipase A2inhibitor [27] NDGA blocks the acute response of bovine cells to ACTH particularly when cAMP release is low [24], and it also decreases the expression of StAR mRNA in rodent steroidogenic cells [23,25,26] Those effects can be reversed
by AA hydroperoxides, a fact that suggests the involvement
of lipoxygenases [23,25]
Although the role of AA in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented [17–26], the mechanism responsible for the release of AA remains unknown Previous studies have reported that phospholipase A2(PLA2) inhibitors abrogate the effect of LH- and ACTH-stimulated steroid production thereby suggesting the involvement of PLA2 in the mech-anism of action of trophic hormones [18,19,23] However,
no evidence has been reported demonstrating the activation
of PLA2by steroidogenic hormones
We have previously identified a hormone-dependent phosphoprotein involved in steroid synthesis through the release of AA The protein was identified by its capacity to increase mitochondrial steroidogenesis in a cell-free assay [28,29] The activity of the protein was dependent on cAMP and PKA and blocked by the use of 4-bromophenacyl-bromide (BPB) and NDGA, both inhibitors of AA release Importantly, this inhibition could be overcome by the addition of AA [29,30], an indication that this protein regulates steroid synthesis through the (direct or indirect) activation of AA release The protein was later purified to homogeneity and identified as a 43-kDa phosphoprotein (p43) [31] Further cloning and sequencing of a cDNA
Correspondence to E J Podesta´, Depto de Bioquı´mica,
Facultad de Medicina, Paraguay 2155, 5 piso, C1121ABG
Buenos Aires, Argentina Fax: 5411 45083672 ext 31
E-mail: biohrdc@fmed.uba.ar
Abbreviations: PKA, cAMP-dependent protein kinase; AA,
arachi-donic acid; AA-CoA, arachidonoyl-CoA; NDGA,
nordihydro-guaiaretic acid; StAR protein, steroidogenic acute regulatory
protein; PLA 2 , phospholipase A 2 ; ATK, arachidonoyl trifluoromethyl
ketone; BPB, 4-bromophenbromide; MTE-I, mitochondrial
acyl-CoA thioesterase; CTE-I, cytosolic acyl-acyl-CoA thioesterase; ACS4,
arachidonic acid preferred acyl-CoA synthetase; Y1, murine
adreno-cortical tumor cell line; RIA, radioimmunoassay; IPTG, isopropyl
thio-b- D -galactoside; DBI, diazepam binding inhibitor; PBR,
periph-eral benzodiazepine receptor; HDL, high density lipoprotein; ACBP,
acyl-CoA binding protein.
(Received 1 July 2002, revised 29 August 2002,
accepted 18 September 2002)
Trang 2encoding p43 revealed its primary structure [32] The protein
resulted 100% homologous to a
mitochondrial-peroxisome-proliferator-induced acyl-CoA thioesterase (MTE-I) and
92.5% homologous to a cytosolic thioesterase (CTE-I)
[33,34] CTE-I and MTE-I are members of an acyl-CoA
thioesterase family with very long chain and long chain
acyl-CoA thioesterase activity [33,34] that includes four isoforms
with different subcellular locations and a high degree of
homology [35] The family includes a cytosolic (CTE-I), a
mitochondrial (MTE-I) and peroxisomal forms (PTE-Ia
and Ib) of the enzyme [35] Recently, this gene family was
cloned and characterized in mouse showing that all isoforms
are encoded by three exons spaced by two introns [35,36]
Like StAR, p43 is targeted to the inner mitochondrial
membrane [15,32,33] In accordance with the postulated
obligatory role of the protein in steroidogenesis, we detected
the protein and its mRNA in all steroidogenic tissues
including placenta and brain [32] Inhibition of ACTH
release and steroid synthesis by dexamethasone produced a
dose-dependent decrease in the abundance of the adrenal
transcript The transcript was induced by in vivo stimulation
of the adrenal with ACTH The effect had a rapid onset
(5 min), reached maximal stimulation (62%) at 15 min and
returned to basal levels at 30 min The effect of ACTH on
the transcript was inhibited by actinomycin D and
enhanced by cycloheximide [32] Given the obligatory role
of the protein in the activation of steroidogenesis through
the release of AA, we proposed the name arachidonic
acid-related thioesterase involved in steroidogenesis (ARTISt)
for p43 [32]
Recently, the expression and the activity of different
isoforms of acyl-CoA synthetases was described in the rat
liver [37,38] In addition, a report by Kang [39] implicates
the participation of an AA-preferring acyl-CoA synthetase
named ACS4, in steroidogenic tissues The expression of
ACS4 was observed in adrenal cortex cells, luteal and
stromal cells of the ovary and Leydig cells of the testis [39]
Moreover, it was demonstrated that ACS4 expression in the
murine adrenocortical tumor cell line Y1 (Y1) is induced by
ACTH and suppressed by glucocorticoids [40]
Here, we address the question of whether AA-CoA,
acyl-CoA synthetase and thioesterase are, indeed, essential for
AA release and adrenal cholesterol metabolism We
dem-onstrate that recombinant MTE-I and CTE-I release AA
from arachidonoyl-CoA in vitro and that ACTH increases
the activity of the endogenous enzyme and promotes AA
release from AA-CoA ACTH-stimulated steroid synthesis
in Y1 cells was significantly inhibited by a synergistic effect
of NDGA and triacsin C, an inhibitor of the AA-CoA
synthetase Our results are consistent with the involvement
of an acyl-CoA synthetase and an acyl-CoA thioesterase
as important regulators of AA release in the
mecha-nism of action of trophic hormone-stimulated cholesterol
metabolism
E X P E R I M E N T A L P R O C E D U R E S
Materials
The Bulk GST Purification Module and pGEX-4T3 vector
were purchased from Amersham Pharmacia Biotech, Little
Chalfont, UK Restriction enzymes were obtained from
Promega Corp Madison, WI, USA and [1-14
C]arachido-noyl-CoA from NEN Life Science Products, Inc (Boston,
MA, USA) Arachidonoyl trifluoromethyl ketone (ATK) was purchased from Cayman Chemicals (Ann Arbor, MI, USA) NDGA and 22R-OH-cholesterol were purchased from Sigma Chemicals Co., St Louis, MO, USA Triacsin C was from BIOMOL Research Laboratories Inc (Plymouth Meeting, PA, USA) and Ham-F10 cell culture medium was from Life Technologies Inc (Gaithersburg, MD, USA) All other reagents were of highest grade available
Tissue culture Murine Y1 adrenocortical tumor cells, generously provided
by B Schimmer (University of Toronto, Toronto, Canada), were maintained in Ham-F10 medium, supplemented with 12.5% heat-inactivated horse serum and 2.5% heat-inac-tivated fetal bovine serum, 1.2 gÆL)1NaHCO3, 200 IUÆmL)1 penicillin and 200 lgÆmL)1 streptomycin sulfate [41] ACTH stimulation was performed in culture medium containing 0.1% bovine serum albumin Steroids produced were measured by radioimmunoassay (RIA) Determin-ation of progesterone production by RIA and of steroids by fluorometry showed comparable results Therefore, data are shown as progesterone production (ngÆmL)1) in the incu-bation medium
Expression and purification of CTE-I inE coli The full-length CTE-I cDNA, generously provided by
S Alexson (Karolinska Institute, Stockholm, Sweden), was amplified and modified by PCR to include restriction sites for BamHI and EcoRI enzymes The subsequent amplified cDNA was cloned into the BamHI and EcoRI sites of vector pGEX-4T3 and used for transformation of XL1 cells Cloning into the BamHI and EcoRI sites creates an open reading frame for the expression of the mouse CTE-I
as a GST-tagged fusion protein Plasmids were isolated, partially sequenced, and subsequently used for the trans-formation of BL21 cells For the expression of the fusion protein, bacteria were cultured at 30C in YTA medium until an D600of 0.6–2.0 was reached Induction of protein expression was carried out for 2 h by adding 0.1 mM
isopropyl thio-b-D-galactoside (IPTG) After recovery by centrifugation, cells were resuspended and lysed by sonica-tion (4· 15 s) in NaCl/Pi, containing 0.1% 2-mercaptoeth-anol, 1 mgÆmL)1lysozyme, 10 lMleupeptin, 1 lMpepstatin
A and 1 mMEGTA The sonicate was incubated for 30 min with 1% Triton X-100, and subsequently clarified by centrifugation at 12 000 g for 15 min at 4C The purifi-cation of recombinant CTE-I was performed by the GST Purification Module according to manufacturer’s instruc-tions (Amersham Pharmacia), by thrombin cleavage and DEAE ionic exchange chromatography Protein content was determined according to Bradford [42]
Expression and Purification of MTE-I inE coli The recombinant MTE-I was first expressed in E coli using the GST tag system The yield of protein expression obtained by this procedure was extremely low Therefore,
we made use of the HIS tag system for the expression and purification of the recombinant protein pET22b (+) bearing MTE-I sequence was transformed into BL21 cells
Trang 3according to standard procedures Transformants were
grown overnight at 37C in 3 mL of LB medium
contain-ing 50 lgÆmL)1ampicillin These precultures were then used
to inoculate 100 mL of fresh LB/ampicillin medium
Expression of the MTE-I was induced by the addition of
1 mM IPTG After a 3-h incubation period, cells were
harvested and used for protein extraction following
Nova-gen’s recommendations The His-tag domain adjacent to
the cloning site borne by the pET22b(+) vector, allowed
purification of the expressed fusion proteins using a Ni2+
chelation resin Purification of the recombinant His-tagged
MTE-I enzyme was performed according to the
manufac-turer’s instructions Even though the yield of purified
protein was again low, the amount of protein obtained was
sufficient to perform the experiments
Production of polyclonal antibodies against
recombinant CTE-I
Rabbits were injected once with 500 lg of recombinant
CTE-I as antigen dissolved in 0.5 mL of distilled water and
mixed with equal volumes of Freund’s complete adjuvant,
and three times with 500 lg of antigen mixed with equal
volumes of Freund’s incomplete adjuvant Antibody titre
against recombinant CTE-I was determined by ELISA
Western blot
Proteins were separated on 12% SDS/PAGE and
electro-phoretically transferred to nitrocellulose membranes in a
buffer containing 25 mMTris, 192 mMglycine, pH 8.3 and
20% methanol at a constant voltage of 15 mA for 30 min
Membranes were then incubated with 5% fat-free powdered
milk in NaCl/Tris/Tween (500 mMNaCl, 20 mMTris/HCl
pH 7.5; 0.5% Tween-20) for 30 min at room temperature
with gentle shaking The membranes were then rinsed twice
in NaCl/Tris/Tween and incubated overnight with the
appropriate dilutions of primary antibody at 4C Bound
antibodies were detected by chemiluminescence using the
ECL kit (Amersham Pharmacia Biotech)
Immunohistochemistry and immunocytochemistry
Rat adrenals were dissected out and fixed by immersion in
4% paraformaldehyde in 0.01Mphosphate buffered saline
(NaCl/Pi) for 2 h at room temperature and left overnight at
4C The tissue was stored in NaCl/Pi containing 12%
sucrose at 4C, sectioned at 16 lm in a cryostat and
thaw-mounted onto gelatinized glass slides
Y1 cells grown on poly-L-lysine glass cover slips were
washed once with NaCl/Piand then fixed overnight at 4C
with 4% (w/v) paraformaldehyde in NaCl/Pi Both cover
slips and glass slides were processed for indirect
immuno-cytochemistry and immunohistochemistry, respectively
Briefly, sections were rinsed in NaCl/Piand incubated with
blocking solution (1.5% goat serum in 0.3% Triton-X100
NaCl/Pi) for 1 h at room temperature and incubated with
rabbit polyclonal antibodies against recombinant acyl-CoA
thioesterase (1/100), or vehicle (control) in a humidified
chamber for 24 h at 4C Detection of the primary antibody
was done by means of a cy3-conjugated goat anti-rabbit
IgG (Molecular Probes, Eugene, OR) After rinsing with
NaCl/P, the sections were mounted in FluorSave reagent
(Calbiochem, CA, USA) and examined in an Olympus BX
50 epifluorescence Microscope Kodak T-400 C41 film was used for photography
Preparation of postmitochondrial and mitochondrial fractions
Isolation of mitochondria was carried out as described [28,43] Briefly, Y1 cell cultures were washed with NaCl/Pi, scraped in 10 mM Tris/HCl, pH 7.4, 250 mM sucrose, 0.1 mM EDTA, 10 lM leupeptin, 1 lM pepstatin A and
1 mMEGTA (buffer A), homogenized with a Pellet pestle motor homogeniser (Kontes), and centrifuged at 600 g during 15 min A second centrifugation at 10 000 g during
15 min rendered a mitochondrial pellet and a supernatant (postmitochondrial fraction) The mitochondrial pellet was washed once with buffer A, and resuspended in 10 mMTris/ HCl pH 7.4, 10 lMleupeptin, 1 lMpepstatin A and 1 mM
EGTA
Thioesterase activity determination Acyl-CoA thioesterase activity was determined using [1-14C]arachidonoyl-CoA (specific activity: 51.6 mCiÆ mmol)1, concentration: 0.02 mCiÆmL)1) as substrate The reaction was carried out at 37C and under vigorous shaking, using 0.1 lg of the recombinant protein in a buffer containing 10 mMHepes, 50 mMKCl, pH 7.4 and 15 lMof the substrate Arachidonic acid released during the reaction was extracted from the aqueous phase with n-hexane and quantified by scintillation counting
Statistical analysis The results of the studies of different inhibitors on enzyme activity are expressed as progesterone produced (ngÆmL)1) Comparison of mean values was performed using either the analysis of variance (ANOVA) or two-way analysis of variance (two-wayANOVA) followed by the Student-New-man-Kuels test
R E S U L T S
Detection of recombinant CTE-I and MTE-I
by antibodies The recombinant cytosolic and mitochondrial acyl-CoA thioesterases were produced in E coli and purified to homogeneity as described in Experimental procedures In order to identify the recombinant proteins we used antibodies raised against a synthetic peptide corresponding
to the catalytic domain of ARTISt (G11K antibody) [31] The antibody recognized the protein from adrenal zona fasciculata (Fig 1A, lane 3) as it was previously described [31] and also CTE-I and MTE-I recombinant proteins (Fig 1A, lanes 1 and 2)
In another set of experiments we obtained an antibody using the purified recombinant CTE-I protein as described
in Experimental procedures The immune rabbit serum recognized both recombinant proteins (Fig 1A, lanes 4 and 5) and the 43-kDa protein from mitochondrial and postmitochondrial fractions of Y1 tumor cells (Fig 1A, lanes 6 and 7) Preadsorbed antibody with the purified
Trang 4recombinant CTE-I, did not recognize the 43-kDa protein
in both fractions (Fig 1A, lanes 8 and 9)
The expression of the thioesterase protein in Y1 cells was
also studied by immunocytochemistry Cells were uniformly
stained with the exception of nuclei as observed in Fig 1B,
panel A No signal was detected when the cells were
incubated in the presence of secondary antibody alone
(Fig 1B, panel C)
Taken together, these results clearly indicate that the
endogenous protein is indeed similar to the recombinant
proteins
The expression of the thioesterase was also studied by
immunohistochemistry in rat adrenals Intense fluorescence
was detected throughout the adrenal cortex, while the
medulla was devoid of signal (Fig 2, panel A) The label was most intense in the zona fasciculata as compared with that shown by both zona glomerulosa and reticularis (Fig 2, panels C and D) The specificity of the label was corroborated when the tissue sections were incubated with preadsorbed primary antibody (Fig 2, panel B)
CTE-I and MTE-I thioesterase activity
To further characterize the recombinant CTE-I and MTE-I,
we studied the kinetic parameters of the enzymes by measuring thioesterase activity using [1-14C]AA-CoA as described in Experimental procedures Expectedly, incuba-tion of [1-14C]AA-CoA in the presence of the purified enzyme resulted in the release of AA Kmand Vmax values obtained for the reactions were 4.1 and 2 lMand 948 and
193 nmolÆmin)1Æmg)1for CTE-I and a MTE-I, respectively Next, we measured the effect of ACTH on mitochondrial thioesterase activity in Y1 adrenal cells For that purpose, mitochondria were isolated from cultures of confluent Y1 cells incubated in the presence or absence of 5 mIUÆmL)1 ACTH and enzyme activity was determined as AA released from AA-CoA ACTH significantly stimulated enzyme activity in the mitochondrial fraction from 1.59 ± 0.22
to 3.0 ± 0.25 and from 1.67 ± 0.28 to 4.21 ± 0.29 pmol AAÆmin)1 for control and ACTH at 5 and 30 min, respectively A similar effect in enzyme activity was observed when the postmitochondrial fraction was used as
a source of enzyme (data not shown)
Effect of NDGA on acyl-CoA thioesterase activity Although NDGA is commonly used as an inhibitor of the lipoxygenase pathway, it is also known to inhibit PLA2 activity [27] Since NDGA strongly inhibited both steroid production and StAR protein expression [23,25,26], we tested here whether NDGA inhibits acyl-CoA thioesterase
Fig 2 Immunohistochemical analysis of acyl-CoA thioesterases in the adrenal gland Tissue sections from rat adrenal gland were incubated with anti-(CTE-I) (A, C and D) or antibody preadsorbed with purified CTE-I (B) Specific binding was detected using a cy3-conjugated goat anti-rabbit IgG as secondary antibody and observed under standard fluorescence microscopy Original magnifications are: A and B, · 100;
C and D · 200 Acyl-CoA thioesterase immunoreactivity was found in the zona fasciculata (f), zona glomerulosa (g) and zona reticularis (r).
No signal was detected in the medulla (m).
Fig 1 Immunodetection of CTE-I and MTE-I (A) Samples of purified
recombinant CTE-I (CTE-I) and MTE-I (MTE-I), homogenates from
adrenal zona fasciculata (Fasciculata), mitochondrial (MF) or
post-mitochondrial fractions (PMF) obtained from Y1 cells, as indicated,
were resolved on SDS/PAGE and immunoblotted as described in
Experimental procedures Antibodies used were: anti-G11K (lanes
1–3), anti-(CTE-I) (lanes 4–7) or antibody preadsorbed with purified
recombinant CTE-I (control) (lanes 8 and 9) (B)
Immunocytochemi-cal detection of acyl-CoA thioesterase in Y1 cells Cells were fixed and
stained using anti-(CTE-I) serum as described in Experimental
pro-cedures Specific binding was detected by means of a cy3-conjugated
secondary antibody and fluorescence observed under a standard
epifluorescence microscope (A) (C) Cells stained only with secondary
antibody (B) and (D), phase contrast micrographs of cells observed in
(A) and (C), respectively Original magnification · 400.
Trang 5activity in vitro Figure 3 shows that a 5-min preincubation
of recombinant protein with NDGA inhibited thioesterase
activity Interestingly, NDGA produced only a 20%
inhibition of the mitochondrial enzyme activity when
measured in mitochondria isolated from ACTH-stimulated
Y1 cells (data not shown) This later result is in agreement
with the previous observation that NDGA inhibitory effect
is manifested only when the inhibitor is added prior to
ACTH [21]
Role of arachidonoyl-CoA and arachidonoyl-CoA
synthetase on ACTH-stimulated cholesterol metabolism
Next, we tested the effect of NDGA on ACTH-induced
steroid production in Y1 cells According with the effect
observed in isolated rat zona fasciculata cells [44], NDGA
significantly (P < 0.001) inhibited ACTH-induced steroid
production in a dose-dependent manner (Fig 4), with an
apparent IC50of 50 lM
The expression of an acyl-CoA synthetase specific for
AA, acyl-CoA synthetase 4 (ACS4), in steroidogenic tissues
has been reported [39] Triacsin C has been described as an
inhibitor of acyl-CoA synthetases with a preferential effect
for AA-CoA synthetases in intact cells [45,46] Thus, we
investigated a possible concerted regulatory role of
acyl-CoA synthetases and thioesterases in the regulation of
steroidogenesis by using triacsin C and NDGA on
ACTH-stimulated steroid synthesis in Y1 cells
For this purpose, ACTH-stimulated Y1 cells were treated
with ineffective concentrations of NDGA (5–25 lM, Fig 4)
alone or in combination with increasing concentrations of
triacsin C (0.1–2 lM) and steroids measured as indicated in
Experimental procedures Expectedly, NDGA alone had no
effect on ACTH-induced steroid production, while
triac-sin C alone produced a slight but significant (P¼ 0.0445)
inhibition of steroid output (Fig 5B) However, NDGA
significantly inhibited steroid biosynthesis when combined
with 0.1, 0.5 and 2 lM triacsin C (Fig 5A) A two-way
analysis of variance rendered a highly significant
(P¼ 0.0026) value for the combination of NDGA and
triacsin C, thereby indicating a synergistic effect on steroid production Moreover, the synergistic effect is evidenced when the IC50 for the inhibitors are analysed Thus, the apparent IC50for NDGA is reduced from 50 lMto 25, 7.5 and 4.5 lMin the presence of 0.1, 0.5 and 2 lMtriacsin C, respectively The apparent IC50for triacsin C is also reduced from 5.5 lMto 1.75, 0.275 and 0.1 lMin the presence of 5,
10 and 25 lMof NDGA, respectively (Fig 5B)
The inhibitory effect of both triacsin C and NDGA was clearly not due to an inhibition of P450sccactivity since no significant inhibition of steroid production was observed when 22R-OH-cholesterol was added to the Y1 cell culture (data not shown)
These results confirm the participation of ACS4 and of AA-CoA in the regulation of steroidogenesis in Y1 cells
D I S C U S S I O N
AA is present in the plasma membrane of most mammalian cells esterified to phospholipids Free cytosolic AA can be produced by the action of phospholipase A2or cholesterol esterase which release the fatty acid by cleavage from membrane phospholipids or cholesterol esters, respectively [47] Previous studies have demonstrated that inhibitors
of PLA2 activity affect hormone-induced steroidogenesis [17–19,25] Those studies have raised the possibility that PLA2 could be involved in the mechanism of action of hormones that control steroid production However, there
is no direct evidence showing that AA is released by PLA2in steroidogenic tissues
The present study is the first one to provide evidence for
an alternative pathway of AA generation Our results are consistent with the hypothesis that, in steroidogenic cells,
AA is released by the action of an acyl-CoA thioesterase activity We show here that the mitochondrial acyl-CoA thioesterase activity hydrolyses AA-CoA to release free AA
Fig 3 Effect of NDGA on recombinant thioesterase activity Activity
of recombinant CTE-I (open bars) and MTE-I (filled bars) was
measured in the presence of increasing doses of NDGA Acyl-CoA
thioesterase activity was determined using [1-14C]AA-CoA as
sub-strate Results are expressed as the mean ± SD of one representative
experiment performed in triplicate.
Fig 4 Effect of NDGA on steroid hormone synthesis Y1 cells were preincubated with variable concentrations (5–100 l M ) of NDGA for
15 min at 37 C and further incubated in the absence or the presence of
2 mIUÆmL)1of ACTH for 60 min Determination of progesterone production by RIA and of steroids by fluorometry showed comparable results Therefore, data are shown as progesterone production (ngÆmL)1) in the incubation medium Basal steroid production is denoted by the dotted line The effect of NDGA was highly significant (P < 0.001) by ANOVA , and indicated values (*) significantly different (P < 0.05) by Student-Newman-Keuls post test.
Trang 6and that the activity of the mitochondrial acyl-CoA
thioesterase increases significantly after ACTH stimulation
in Y1 cells We also demonstrate that inhibitors of AA
release and metabolism such as NDGA, are effective
inhibitors of recombinant CTE-I and MTE-I The
experi-ment using inhibitors of acyl-CoA synthetases in
combina-tion with NDGA demonstrates that the two enzymes, the
synthetase and the acyl-CoA thioesterase act in concert and
are essential for steroidogenesis
A possible explanation for the effect of classical PLA2
inhibitors on thioesterase activity could be the presence of
a serine-histidine-aspartic acid catalytic triad containing ab
hydrolases as determined by site-directed mutagenesis [48]
This possibility is supported by our previous results
showing that antibodies raised against a synthetic peptide
matching a sequence that contains the serine included in
the catalytic triad inhibit steroid synthesis in a recombinant
cell-free assay [32] These observations are also in
agree-ment with previous results showing that BPB, another
PLA2inhibitor, also blocks the activity of CTE-I [44] In
addition, another specific inhibitor of PLA2(100 lMATK)
was also effective to inhibit the activity of the enzyme (data
not shown)
NDGA is known to inhibit the activity of lipoxygenase
by binding to the reduced form (Fe2+) of the enzyme
thereby keeping it inactive [49] In our experiments NDGA
was effective in inhibiting the activity of purified
recombin-ant MTE-I in an in vitro assay performed in a Hepes-based
buffer containing only KCl Information about the tertiary
structure and of ion requirements of acyl-CoA thioesterases
is still lacking and the mechanism of inhibition of acyl-CoA
thioesterases by NDGA is unknown Nevertheless, we
cannot exclude an inhibitory mechanism for the thioesterase
involving electron transfer Noteworthy, the thioesterase
displays a decreased sensitivity for NDGA after ACTH
stimulation This latter effect could result from a
confor-mational change of the enzyme induced by protein
phos-phorylation
Blocking lipoxygenase-mediated AA metabolism by NDGA, greatly decreased Bt2cAMP-induced StAR protein expression and reduced cholesterol metabolism [25] How-ever, in view of our present results, the use of NDGA only
as lipoxygenase inhibitor has to be reviewed since it can also act as inhibitor of acyl-CoA thioesterases
Recombinant CTE-I and MTE-I expressed in E coli exhibited the expected molecular mass, showed high acyl-CoA thioesterase activity using AA-acyl-CoA as substrate and were detected by antipeptide antibodies that recognize the catalytic domain of the purified enzyme from adrenal In addition, antibodies raised against recombinant CTE-I recognize both recombinant proteins and the protein from cytosol and mitochondria from adrenal cells Using this antibody, immunohistochemical experiments show that the protein is exclusively located in steroidogenic cells of adrenal gland
The expression of a cytosolic and a mitochondrial thioesterase activity with different subcellular localizations suggests that these enzymes have different functions in vivo, although the role of CTE-I in steroidogenesis is currently not known The high degree of sequence similarity between the CTE-I and MTE-I genes suggests that they diverged relatively recently by gene duplication, possibly by duplica-tion of an MTE-I gene with loss of the 5¢-end encoding the mitochondrial targeting signal, resulting in a cytosolic enzyme [35]
In the present study we demonstrate that AA-CoA is an important intermediate in steroidogenesis The observation that triacsin C, an inhibitor of AA-CoA synthetase [44,45], affects hormone-induced steroid synthesis in Y1 cells supports this hypothesis The participation of the AA-CoA-mediated pathway for AA release in steroidogen-esis was further demonstrated by the combined inhibitory effect of triacsin C and NDGA Our results clearly show that addition of triacsin C in combination with ineffective doses of NDGA produced a marked reduction of the
IC for NDGA Therefore, the regulatory role of AA in
Fig 5 Combined effect of triacsin C and NDGA on steroid hormone synthesis Y1 cells were preincubated in the presence or the absence of increasing concentrations of triacsin C (4 h, 37 C) and of NDGA (15 min, 37 C) Cells were then incubated for 60 min with 2 mIUÆmL)1of ACTH or its vehicle Progesterone produced was determined in the incubation medium by RIA Data are expressed as the mean ± SD (n ¼ 3)
of different concentrations of triacsin C, 0.1 (d), 0.5 (m), 2 (j) lM or control (r) with indicated concentrations of NDGA (panel A), or variable concentrations of NDGA, 5 (s), 10 (n), 25 (h) l M or control (e) in the presence of triacsin C (panel B) Basal steroid production by Y1 cells is denoted by the dotted line.
Trang 7steroidogenesis needs the concerted action of the acyl-CoA
synthetase and thioesterase
Although we cannot rule out a possible nonspecific effect
of both triacsin C and NDGA, our observation of a
synergistic effect on steroid production suggests this is
unlikely This, along with the fact that 22R-OH-cholesterol
by-passes the effect of the inhibitors strongly indicates that
the thioesterase and the acyl CoA-synthetase act in the same
signalling pathway in a step prior to the rate-limiting
passage of cholesterol from the outer to the inner
mito-chondrial membrane
The thioesterase activity requires an acyl-CoA pool as a
source of AA The presence of an acyl-CoA synthetase
specific for arachidonate described in steroidogenic tissues
suggests that such a mechanism is operable in steroid
biosynthesis The concept that long chain fatty acyl-CoA
esters are regulatory ligands as well as intermediates in
cellular metabolism is now well appreciated from results of a
number of investigations in a variety of organism and
tissues [50,51]
The question then arises as to why free cytosolic AA has
to be re-esterified in order to stimulate steroidogenesis One
possible explanation is the need for AA in a special
compartment of the cell (e.g mitochondria) The
compart-mentalization of long-chain acyl-CoA esters is an important
unsolved problem, and the actual cytosolic concentration of
free long-chain acyl-CoA esters is not known for any tissue
[51] The high degree of sequestration of CoA into long
chain acyl-CoA suggests that AA is likely to become
limiting for diverse roles in specific compartments of the cell
[51] Here we show that ACTH stimulates mitochondrial
thioesterase activity in Y1 cells This points to a direct effect
of ACTH upon enzyme activity However, we cannot rule
out a possible activation of the enzyme by an
ACTH-mediated increased availability of its substrate
It is known that an acyl-CoA binding protein (ACBP)
known also as DBI (diazepam binding inhibitor) is
expressed in high concentrations in specialized cells such
as steroid producing cells of the adrenal cortex and testis
[52,53] Thus it can be proposed that AA-CoA binds to
DBI, which in turns binds to the peripheral benzodiazepine
receptor (PBR) located in the outer mitochondrial
mem-brane [53,54] This will possibly lead to facilitated transfer of
AA-CoA into the mitochondria
Another important issue is the origin of cytosolic free AA
to be esterified into AA-CoA As already mentioned, AA
could derive from plasma membrane phospholipids or from
cholesterol esters The major source of cholesterol in the rat
adrenal is the cholesterol esterified in high-density
lipopro-teins (HDL) [55,56] In adrenocortical cells, HDL enhances
steroid production and increases cellular cholesterol
con-tent Rat HDL contains a high amount of arachidonate in
its cholesterol esters fatty acids This is an agreement with
the suggestion that free AA, which will be esterified to
acyl-CoA may come from the hydrolysis of cholesterol ester [34]
Nevertheless, since it has been shown that dexamethasone
inhibits cholesterol metabolism and that this effect is
reverted by free AA, we can not rule out the possibility
that the free AA that is esterified into acyl-CoA may come
from membrane phospholipids by the action of PLA2
However, in none of those studies, there is a demonstration
that dexamethasone is in fact working through the
inhibi-tion of PLA
Our current data indicate the presence of a new pathway that regulates intracellular levels of AA, in which ACS4 could act by sequestering free AA by esterification into AA-CoA CoA-esterified AA may bind ACBP/DBI thus forming an intracellular pool that could then be delivered to
an acyl-CoA thioesterase, which will, in turn, release AA in
a specific compartment of the cell upon hormone treatment Taken together the results shown here demonstrate the critical role of acyl-CoA synthetases and thioesterases in the regulation of intracellular levels of AA and steroid produc-tion This study further suggests a new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by a mechanism different than the classical PLA2-mediated pathway
A C K N O W L E D G E M E N T S Thanks are due to Dr Stefan Alexson for the cytosolic acyl-CoA thioesterase cDNA This work was supported by grants from Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, Universidad de Buenos Aires and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas.
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