Reduction in the expres-sion of the mitochondrial acyl-CoA thioesterase MTE-I by antisense or small interfering RNA siRNA and of the arachidonic acid-preferring acyl-CoA synthetase ACS4
Trang 1thioesterase I and acyl-CoA synthetase 4 inhibits
hormone-induced steroidogenesis
Paula Maloberti*, Rocı´o Castilla*, Fernanda Castillo, Fabiana Cornejo Maciel, Carlos F Mendez, Cristina Paz and Ernesto J Podesta´
Department of Biochemistry, School of Medicine, University of Buenos Aires, Argentina
Steroid hormones are synthesized in specialized
steroido-genic cells in the adrenal gland, ovary, testis, placenta
and brain and are essential for maintaining normal
body homeostasis and reproductive capacity The
bio-synthesis of all steroid hormones begins at the
mito-chondria with the conversion of cholesterol into
pregnenolone by the cholesterol side-chain cleavage
cytochrome P-450 enzyme (P450scc) [1,2] The
trans-port of cholesterol to the inner mitochondrial
mem-brane and the availability of cholesterol for P450scc
constitutes the rate-limiting step of steroidogenesis, a
step controlled by the steroidogenic acute regulatory protein (StAR) [3,4] and the peripheral benzodiazepine receptor (PBR) [5,6]
Arachidonic acid (AA) can act as a signaling messenger itself or through its metabolites exerting numerous effects on different cellular processes [7–9] Several reports have shown that AA and lipoxyge-nase metabolites play an essential role in the regula-tion of steroidogenesis In steroidogenic cells, AA participates in hormone-stimulated induction of StAR expression [10,11] However, the manner in which
Keywords
ACS4; acyl-CoA; arachidonic acid;
mitochondrial acyl-CoA thioesterase I
(MTE-I); steroidogenesis
Correspondence
E J Podesta´, Depto de Bioquı´mica,
Facultad de Medicina, Paraguay 2155,
piso 5, C1121ABG Buenos Aires, Argentina
Tel ⁄ Fax: +54 11 4508 3672, ext 31
E-mail: biohrdc@fmed.uba.ar
*Note
These authors contributed equally to this
work
(Received 27 October 2004, revised 9
February 2005, accepted 16 February 2005)
doi:10.1111/j.1742-4658.2005.04616.x
Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis Reduction in the expres-sion of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells This effect was blunted
by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane The inhibition of steroidogenesis was overcome
by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis Knocking down the expression of MTE-I leads to a signifi-cant reduction in the expression of steroidogenic acute regulatory protein This protein is induced by arachidonic acid and controls the rate-limiting step Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospho-lipase A2cascade
Abbreviations
AA, arachidonic acid; ACS4, arachidonic acid-preferring acyl-CoA synthetase 4; ACTH, adrenocorticotrophin; 8Br-cAMP, 8-bromo-3¢,5¢-cAMP; 22(R)-OH-cholesterol, 22a-hydroxycholesterol; DAPI, 4¢,6-diamidino-2-phenylindole; DBI, diazepam binding inhibitor; EGFP, enhanced green fluorescent protein; MTE-I, mitochondrial acyl-CoA thioesterase I; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; P450scc, cholesterol side-chain cleavage cytochrome P-450 enzyme; PBR, peripheral benzodiazepine receptor; siRNA, small interfering RNA; StAR protein, steroidogenic acute regulatory protein.
Trang 2trophic hormones, such as adrenocorticotrophin
(ACTH) and luteinizing hormone, regulate AA release
is not entirely clear
We have proposed the involvement of acyl-CoA
synthetase 4 (ACS4) and mitochondrial acyl-CoA
thio-esterase I (MTE-I) as important regulators of AA release
in the mechanism of action of trophic
hormone-stimu-lated cholesterol metabolism [12] ACS4 has been
des-cribed as an AA-preferring acyl-CoA synthetase that is
preferentially expressed in steroidogenic tissues such
as adrenal cortex, luteal and stromal cells of the ovary
and Leydig cells of the testis [13] Moreover, it has
been demonstrated that ACS4 expression in the murine
adrenocortical tumor cell line Y1 is induced by ACTH
and suppressed by glucocorticoids [14]
The acyl-CoA thioesterase was first identified as a
43-kDa phosphoprotein by its capacity to increase
mito-chondrial steroidogenesis in a cell-free assay [15] The
protein was then purified to homogeneity [15] Further
cloning and sequencing of its cDNA revealed that it is
member of a thioesterase family with long-chain
acyl-CoA thioesterase activity [16] which includes four
iso-forms with different subcellular localization and a high
degree of homology [17,18] In particular, MTE-I was
shown by immunoelectron microscopy to associate with
the matrix face of mitochondrial cristae [17] In
accord-ance with the postulated role of MTE-I in
steroido-genesis, we detected the protein and its mRNA in the
adrenal gland, ovary, testis, placenta and brain [16]
Although it is known that acyl-CoA thioesterases
are a group of enzymes that catalyze the hydrolysis of
acyl-CoA to the nonesterified fatty acid and CoA [19]
and that they can release AA from the
arachidonoyl-CoA, so far, the phospholipase A2pathway is the most
commonly accepted mechanism operating to produce
lipoxygenated products from plasma membrane
signa-ling [7]
Using inhibitors of the acyl-CoA synthetase and the
acyl-CoA thioesterase, we have previously postulated
the existence of a new pathway for AA release that
operates in the regulation of steroid synthesis in
adre-nal cells [12] Here we address the question of whether
MTE-I and ACS4 are indeed essential for AA release
and cholesterol metabolism in steroidogenic cells We
show for the first time that silencing the expression of
MTE-I by antisense cDNA or small interfering RNA
(siRNA) or of ACS4 by siRNA results in the
inhibi-tion of steroid synthesis, an effect that is overcome by
the addition of exogenous AA In summary, our
results demonstrate a critical role for both ACS4 and
MTE-I in the hormonal regulation of steroidogenesis
as a new pathway of AA release different from the
classical phospholipase A2
Results
MTE-I knock down and overexpression
in MA-10 cells
To provide definitive proof about the role played by the mitochondrial acyl-CoA thioesterase in steroido-genesis, we performed experiments aimed at silencing the expression of MTE-I or at overexpressing the pro-tein in steroidogenic cells For this purpose, we transi-ently transfected MA-10 cells with pRc⁄ CMVi plasmid containing either an antisense or the full-sense MTE-I cDNA (accession No Y09333) The efficiency of trans-fection using the protocol described in Experimental Procedures was 50–70%
The effect of sense and antisense plasmid transfec-tion on MTE-I protein concentratransfec-tions was studied
by immunocytochemistry, using a specific antibody against the MTE-I and b-tubulin as control (Fig 1)
As expected, antisense-transfected cells showed a strong reduction in MTE-I protein concentrations, whereas cells transfected with full MTE-I sense cDNA showed a clear increase compared with cells trans-fected with vector alone The expression of b-tubulin remained unchanged in spite of the treatments used Neither MTE-I antisense or sense expression affected cell viability, as assessed by the trypan blue exclusion method and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (0.68 ± 0.02, 0.66 ± 0.01 and 0.65 ± 0.01 absorbance units for antisense-transfected, sense-transfected and mock-trans-fected cells, respectively) As the MTT assay is based
on MTT reduction by the mitochondrial diaphorase enzyme, an index of mitochondrial integrity [20], these results indicate that neither antisense nor sense transfection affect mitochondrial function Moreover, staining of the cells with the nuclear dye 4¢,6-diami-dino-2-phenylindole (DAPI) showed no changes in nuclear morphology, indicating that neither of the treatments used induced apoptosis in MA-10 cells (Fig 1)
Effect of MTE-I knock down on steroidogenesis Steroid hormone production in Leydig cells involves increases in intracellular cAMP concentrations Thus,
we studied the effect of MTE-I antisense on 8-bromo-3¢,5¢-cAMP (8Br-cAMP)-stimulated progesterone pro-duction (the major steroid produced by the MA-10 cell line) and correlated the steroid-producing capability with the level of protein expression as assessed by western blot The expression of MTE-I protein in MTE-I antisense-transfected MA-10 cells was reduced
Trang 3by 69 ± 6% as compared with mock-transfected or
enhanced green fluorescent protein (EGFP)-transfected
cells (Fig 2A,B) Accordingly, progesterone production
was reduced by 70 ± 1.3% in MTE-I
antisense-trans-fected MA-10 cells as compared with mock-transantisense-trans-fected
or EGFP-transfected cells (Fig 2C) As mentioned in
Experimental Procedures, the inhibition of MTE-I
expression by antisense treatment improves after
elec-troporation 2 or 3 times Progesterone inhibition
followed the same pattern To investigate if the
reduc-tion in MTE-I expression produced by the
anti-sense-impaired cholesterol-transport mechanism, we
determined progesterone production in cells incubated
with the water-soluble derivative of cholesterol,
22(R)-OH-cholesterol, which travels freely across the
membranes to reach the inner mitochondrial
choles-terol side-chain cleavage cytochrome P-450 enzyme
(P450scc) Steroidogenesis stimulated by
22(R)-OH-cholesterol was assayed under conditions of time and
substrate concentration in the linear range No
signi-ficant difference in 22(R)-OH-cholesterol-sustained
progesterone production was detected among the
dif-ferent treatments (Fig 2C, inset) This result provides
evidence that the reduction in MTE-I expression
impaired cholesterol transport without affecting mito-chondrial integrity
Effect of MTE-I knock down on StAR protein concentrations
The observation that 22(R)-OH-cholesterol-sustained steroid synthesis is not affected by a reduction in MTE-I expression confirms a previously expected role
of MTE-I in the regulation of the rate-limiting step in steroidogenesis through the release of AA [12] Consid-ering the crucial role of StAR in that step and given the fact that AA regulates the expression of this protein in steroidogenic cells [10], we hypothesized a decrease in StAR expression after MTE-I silencing Thus, we examined the expression of StAR protein in MTE-I-deficient cells StAR protein was detected as a 30-kDa protein after 6 h of 8Br-cAMP treatment in mock-transfected cells (Fig 3A) The effect of 8Br-cAMP on StAR protein concentrations was strongly reduced in cells transfected with the plasmid contain-ing MTE-I antisense The reduction in StAR protein concentrations in antisense-transfected cells reached
56 ± 14% as compared with mock-transfected cells
Fig 1 MTE-I expression in MA-10 Leydig cells transiently transfected with sense or antisense MTE-I cDNA Mock-transfected, MTE-I antisense-transfected (MTEas) and MTE-I sense-transfected (MTEs) MA-10 cells were grown on coverslips, stained with antibody against MTE-I (green), antibody against b-tubulin (red) and DAPI (light blue) and subjected to immunofluorescence microscopy Arrows indicate the effect of the transfections in single cells.
Trang 4when quantified by densitometry related to the
b-tubu-lin signal
As was the case for StAR protein concentrations,
knock down of MTE-I resulted in a significant reduction
in progesterone production in cells that had been
stimu-lated for 6 h with 8Br-cAMP (data not shown) This
result demonstrates the participation of MTE-I in the
regulation of the rate-limiting step in steroidogenesis
To provide additional insights into the sequential
steps involved in down-regulation of StAR protein
concentrations, we investigated whether treatment with MTE-I antisense had any effect on StAR mRNA con-centrations Down-regulation of MTE-I produced a marked inhibition of StAR mRNA concentrations, which was already detectable after 1 h of 8Br-cAMP stimulation (Fig 3B) These results are in agreement with the inhibition of steroidogenesis produced by actinomycin D in this cell type, as this drug inhibits only 30% of steroid synthesis induced by 8Br-cAMP
or AA for 1 h (data not shown) However, the effect
of cycloheximide on steroidogenesis stimulated with 8Br-cAMP or AA produced inhibition before 1 h of stimulation These results suggest that AA regulates StAR protein at transcriptional and protein levels
Effect of MTE-I overexpression on steroidogenesis
To further explore the role of MTE-I in the regulation
of steroidogenesis, we examined the effect of MTE-I
A
B
C
Fig 2 Effect of MTE-I knock down on steroidogenesis in MA-10
cells Mock-transfected (h), EGFP-transfected ( ) or MTE-I
anti-sense-transfected (MTEas, ) MA-10 cells were incubated for 1 h
in serum-free medium in the presence or absence of 8Br-cAMP
(0.5 m M ) (A) MTE-I expression was assayed by immunoblotting.
The membrane was incubated sequentially with anti-MTE-I and
anti-(b-tubulin) sera (B) Western blot quantification by
densitome-try Bars denote relative levels of MTE-I expression (C)
Determin-ation of progesterone production by RIA Inset: MA-10-transfected
cells were incubated for 1 h in serum-free medium in the presence
of 22(R)-OH-cholesterol (5 l M ) Results are expressed as the mean
± SD from one representative (n ¼ 3) experiment performed in
triplicate ***P < 0.001 vs mock-transfected or EGFP-transfected
8Br-cAMP-treated cells.
A
B
Fig 3 Effect of MTE-I knock down on StAR expression in steroido-genic cells Mock-transfected or MTE-I antisense-transfected (MTEas) MA-10 cells were incubated in the presence or absence of 8Br-cAMP (0.5 m M ) (A) Representative western blot of MA-10 transfected cells stimulated for 6 h with 8Br-cAMP The membrane was blotted sequentially with anti-MTE-I, anti-StAR and antib-tubu-lin sera (B) Representative northern blot analysis of total RNA from MA-10 transfected cells stimulated for 1 and 6 h with 8Br-cAMP The membrane was developed using probes against StAR and 28S.
Trang 5overexpression in MA-10 cells As predicted,
transfec-tion of the cells with the full MTE-I cDNA construct
resulted in a marked increase in protein abundance as
compared with mock-transfected cells, as shown by
western blot in Fig 4A Protein concentrations
increased by 78 ± 20% as compared with
mock-trans-fected cells (Fig 4B)
Also, we studied the effect of submaximal doses of 8Br-cAMP on steroidogenesis in MA-10 cells over-expressing MTE-I protein Along with the increased protein abundance, MTE-I overexpression resulted in
a significant increase in the levels of steroid synthesis (Fig 4C) reaching concentrations of progesterone equivalent to maximal steroid synthesis, although it was used at submaximal 8Br-cAMP concentration Once again, incubation of the cells in the presence of 22(R)-OH-cholesterol produced no significant differ-ences in steroid production among the different treat-ments (Fig 4C, inset)
Effect of AA on steroid production inhibited
by MTE-I knock down
We have previously demonstrated that recombinant MTE-I releases AA from arachidonoyl-CoA [12] Thus, we analyzed here if addition of exogenous AA restores the steroid-synthesizing capacity of cells trans-fected with MTE-I antisense cDNA As shown in Fig 5, MTE-I antisense produced a significant inhibi-tion of 8Br-cAMP-induced progesterone producinhibi-tion,
an effect that was overcome by the addition of AA The effect of AA was specific, as indicated by the lack
of effect of other fatty acids such as oleic and arachi-dic acid Moreover, oleic acid and arachiarachi-dic acid did not affect the stimulated steroidogenesis in control-transfected or noncontrol-transfected MA-10 cells (data not shown)
Knock down of ACS4 and MTE-I by siRNA
on MA-10 cells
To confirm the effect of MTE-I knock down by anti-sense experiments, we also designed siRNA duplexes directed against this acyl-CoA thioesterase Transfec-tion with siRNA against MTE-I produced a marked reduction (39 ± 8%) in acyl-CoA thioesterase activity
as assayed by western blot (Fig 6A,B) Also, pro-gesterone production by MA-10 cells transfected with siRNA against MTE-I was inhibited when compared with control cells (Fig 6C) As shown in Experimental procedures, the amount of cells used for siRNA is less than the amount used in the antisense experiment Therefore, progesterone production in the siRNA-transfected cells is lower than the corresponding in Fig 2
In addition, it is observed that down-regulation of MTE-I by siRNA is accompanied by a small but signi-ficant increase in ACS4 expression
We have previously demonstrated a concerted action
of ACS4 and MTE-I on hormone-induced steroid
A
B
C
Fig 4 Effect of MTE overexpression on steroidogenesis in MA-10
cells EGFP-transfected (h), MTE-I antisense-transfected (MTEas,
) and MTE-I sense-transfected (MTEs, ) MA-10 cells were
incu-bated for 1 h in serum-free medium in the presence or absence of
8Br-cAMP (0.5 m M ) (A) MTE-I expression was analyzed by western
blot The membrane was incubated sequentially with anti-MTE-I
and anti-(b-tubulin) sera A representative experiment is shown
(n ¼ 3) (B) Western blot quantification by densitometry Bars
denote relative levels of MTE-I expression Results are
expres-sed as the mean ± SD from three independent experiments
(P < 0.0001; analysis of variance) ***P < 0.001 MTE-I antisense
and MTE-I sense-transfected vs EGFP-transfected
8Br-cAMP-trea-ted cells, respectively (C) Determination of progesterone
produc-tion by RIA Inset: MA-10 transfected cells were incubated for 1 h
in serum-free medium in the presence of 22(R)-OH-cholesterol
(5 l M ) Results are expressed as the mean ± SD from three
independent experiments (P < 0.0001; analysis of variance).
***P < 0.001 MTE-I antisense-transfected vs EGFP-transfected
8Br-cAMP-treated cells; **P < 0.01 MTE-I sense-transfected vs.
EGFP-transfected 8Br-cAMP-treated cells.
Trang 6synthesis [12] On the basis of these results, silencing
the expression of ACS4 should also result in inhibition
of hormone-induced steroid synthesis siRNA duplexes
directed against ACS4 were also produced for
transfec-tion of MA-10 cells siRNA transfectransfec-tion resulted in a
marked reduction (56 ± 6%) in ACS4 protein
concen-trations and steroidogenesis as detected by western
blot and RIA (Fig 6A,B,C, respectively)
As demonstrated for MTE-I antisense cDNA effect,
addition of exogenous AA blunted the inhibition of
progesterone production produced by both siRNAs
(Fig 6B) As shown in Fig 2, the reduction in protein
expression correlates with the inhibition of
progester-one production As was the case in previous
experi-ments, the 22(R)-OH-cholesterol effect on steroid
synthesis was not modified by ACS4 or MTE-I siRNA
transfection (Fig 6B, inset) Dose–response curves of
progesterone production and cell viability for both
siRNAs are shown in Fig 6D,E The maximal
inhi-bition of progesterone production was observed at
500 nm siRNA Importantly, cell viability measured by
the MTT assay remained unchanged after siRNA
transfection independently of the concentrations of
siRNA used (Fig 6E) Transfection with control
siRNA remained unchanged for progesterone
produc-tion and cell viability (data not shown)
Discussion
We have previously reported that recombinant MTE-I releases AA from arachidonoyl-CoA in vitro and that ACTH increases the activity of the endogenous enzyme
in adrenal cells [12] Moreover, inhibitors of MTE-I and ACS4 inhibited ACTH-induced steroidogenesis in adrenal cells [12] These results are consistent with the involvement of ACS4 and MTE-I as important regula-tors in the mechanism of action of trophic hormone-stimulated cholesterol metabolism
To provide definitive proof about the role of MTE-I and ACS4 in the regulation of hormone-stimulated steroid synthesis, we performed experiments aimed at studying the effect of suppressing the expression of both enzymes Immunocytochemistry and western blot experiments demonstrate that the expression of MTE-I was strongly reduced when MA-10 cells were
transfect-ed with a vector containing an MTE-I antisense cDNA Importantly, the transfection procedure devel-oped and used in this study did not affect cell viability and⁄ or morphology of sense-transfected or antisense-transfected cells as compared with mock-antisense-transfected cells Moreover, a possible negative effect of the treat-ments on mitochondrial activity was ruled out by using the MTT assay Finally, a possible apoptotic effect produced by the manipulation of MTE-I protein con-centrations was also discarded after staining of the cells with DAPI showed no changes in nuclear mor-phology
Knocking down MTE-I expression levels leads to a strong reduction in 8Br-cAMP-stimulated steroidogen-esis The reduction in protein expression does not affect steroid biosynthesis when it is induced by a per-meable analog of cholesterol, 22(R)-OH-cholesterol, which bypasses the rate-limiting step of steroidogene-sis Given the role of StAR in cholesterol transport and the fact that AA induces StAR expression, we studied here whether the reduction in MTE-I expres-sion could affect steroid hormone synthesis by affect-ing StAR protein concentrations We demonstrate that
a reduction in MTE-I expression in MA-10 cells leads
to a marked reduction in 8Br-cAMP-mediated StAR induction at the RNA and protein level Together, these results provide evidence that the reduction in MTE-I expression impairs cholesterol transport with-out affecting mitochondrial integrity and are in line with previous reports that AA regulates the rate-limit-ing step in steroid biosynthesis [10]
Importantly, the reduction in steroid biosynthesis was restored by addition of exogenous AA, clearly demons-trating that the effect produced by MTE-I protein reduction is based on decreased AA concentrations The
Fig 5 Effect of AA on steroid production inhibited by MTE-I knock
down EGFP-transfected (h) or MTE-I antisense-transfected
(MTEas, ) MA-10 cells were incubated for 1 h in the presence or
absence of 8Br-cAMP (0.5 m M ) and with or without 300 l M
differ-ent fatty acids (arachidonic, arachidic and oleic acid) as indicated.
Progesterone concentrations were determined by RIA Results are
expressed as the mean ± SD from three independent experiments
(P < 0.0019; analysis of variance) **P < 0.01 MTE-I
antisense-transfected vs EGFP-antisense-transfected 8Br-cAMP-treated cells; *P < 0.05
MTE-I antisense-transfected 8Br-cAMP-treated cells vs MTE-I
antisense-transfected cells treated with 8Br-cAMP and arachidonic
acid.
Trang 7correction of the signal abnormality in MTE-I-deficient
cells by the addition of exogenous AA, although subject
to caveats concerning specific effects, clearly supports a
physiological role for this enzyme in AA release and
steroid biosynthesis Moreover, the effect of AA in
restoring the steroidogenic capability of cells in which
MTE-I expression was knocked down was specific, as
other fatty acids were unable to reproduce its effect The
role of MTE-I in the regulation of steroid synthesis was
further confirmed by the observation that
overexpres-sion of this acyl-CoA thioesterase leads to a significant
increase in steroidogenesis stimulated by 8Br-cAMP
whereas it did not affect basal steroidogenesis
Steroido-genic cells express MTE-I but also a cytosolic isoform
(CTE-I) which is 92.5% homologous to the mitochond-rial enzyme From the expression profile of the enzyme,
a possible role for CTE-I in steroidogenesis has been suggested [19] Although we cannot rule out the partici-pation of CTE-I in our antisense approach, the results obtained with overexpression of MTE-I strongly sup-port the participation of this enzyme in AA release and hormone-induced steroid synthesis
Our results are in line with reports showing the hor-monal regulation of MTE-I activity and the function
of ACS4 in supplying arachidonoyl-CoA to that enzyme [12] We also demonstrate here the role of ACS4 and MTE-I using siRNA technology Transfec-tion with either siRNA duplex directed against ACS4
A
B
D
C
E
Fig 6 Effect of knock down of ACS4 and MTE-I by siRNA on MA-10 cells (A) Western blot of MA-10 cells transfected with scramble, ACS4 or MTE-I siRNA The membrane was incubated sequentially with anti-MTE-I, anti-ACS4 and anti-(b-tubulin) sera A representative experiment is shown (n ¼ 3) (B) Western blot quantification by densitometry Bars denote relative concentrations of ACS4 and MTE-I (C) MA-10 cells transfected with scramble (h), ACS4 ( ) or MTE-I ( ) siRNA were incubated for 1 h in serum-free medium in the presence
or absence of 8Br-cAMP (0.5 m M ) and with or without AA (300 l M ) Progesterone concentrations were determined by RIA Inset: Transfected MA-10 cells were incubated for 1 h in serum-free medium in the presence of 22(R)-OH-cholesterol (5 l M ) Results are expressed as the mean ± SD from three independent experiments (P < 0.0033; analysis of variance) a and b, P < 0.01 vs scramble siRNA-transfected treated cells; c, P < 0.05 vs ACS4 siRNA-transfected 8Br-treated cells; d, P < 0.01 vs MTE-I siRNA-transfected 8Br-cAMP-treated cells (D) MA-10 cells transfected with increasing concentrations of ACS4 ( ) or MTE-I ( ) or without (j) siRNA were incubated for
1 h in serum-free medium in the presence of 8Br-cAMP (0.5 m M ) Progesterone concentrations were determined by RIA (E) Cell viability of MA-10 cells transfected with increasing concentrations of ACS4 ( ) or MTE-I ( ) or without siRNA (j) was measured by MTT assay Results of (D) and (E) are expressed as the mean ± SD from one representative (n ¼ 3) experiment performed in triplicate.
Trang 8or MTE-I produced a significant inhibition in
8-Br-cAMP-induced steroid synthesis These results together
with our finding that AA or 22(R)-OH-cholesterol can
bypass the effect of siRNA strongly indicate that
ACS4 and MTE-I act in the same signaling pathway
at a step before the rate-limiting passage of cholesterol
from the outer to inner mitochondrial Therefore, a
concerted action of ACS4 and MTE-I in regulating
the concentrations of AA during steroidogenesis seems
plausible
Down-regulation of MTE-I by siRNA is
accompan-ied by an increase in ACS4 expression We do not
know the mechanisms involved in this regulation It
can be speculated that either the reduction in steroid
synthesis or the reduction in arachidonic acid release
may regulate the expression of ACS4
The human gene encoding ACS4 is located in the
chromosome Xq 22–23 region close to the a5 chain of
the type 4 collagen gene which is related to X-linked
Alport syndrome Genetic analysis of the gene revealed
that it was deleted in a family with Alport syndrome,
ellipoptocytosis and mental retardation [21]
ACS4-deficient mice have been generated [22]
Female mice heterozygous for ACS4 deficiency became
pregnant less frequently and produced small litters
with extremely low transmission of the disrupted
alle-les with a high frequency of uterus embryonic death
ACS4+females showed marked accumulation of
pros-taglandin in the uteruses of the heterozygous females,
suggesting that ACS4 modulates female fertility and
uterus prostaglandin production ACS4 -⁄ Y
hemizy-gous males presented an apparently normal phenotype
The authors suggest that ACS3, an isoform of the
enzyme that also prefers arachidonate [23], may
com-pensate for the ACS4 deficiency
Under the experimental conditions used in this study,
the knock down of ACS4 expression was performed in
isolated cells Under these conditions, cells respond to
the acute hormonal stimulus in a time frame in which no
compensatory mechanisms may occur Thus, our system
seems more appropriate to exploration of the actual
physiological role of ACS4 in AA release, leukotriene
formation and steroid synthesis
It is known that AA has to be metabolized through
the lipoxygenase pathway in order to stimulate StAR
expression and steroidogenesis [11,24] This raises the
question of whether the existence of a different
AA-releasing pathway may provide AA metabolites in
a special compartment of the cell (e.g mitochondria)
The reason why the action of the two opposite
enzymes, namely the arachidonoyl-CoA synthetase and
the arachidonoyl-CoA thioesterase, is needed may be
the need to sequester AA from the free pool in the
form of arachidonoyl-CoA to bring this substrate to a special compartment of the cells The compartmental-ization of long-chain acyl-CoA esters is an important unsolved problem The high degree of sequestration
of CoA into long-chain acyl-CoA suggests that AA is limiting for diverse roles in specific compartments of the cells
The role of long-chain acyl-CoA esters in signal-transduction pathways is an important issue [25] It is known that an acyl-CoA-binding protein known also
as DBI (diazepam-binding inhibitor) is expressed at high concentrations in steroidogenic cells [26,27] and interacts with the PBR in the mitochondria [5] Both DBI and PBR have been reported to be essential for normal steroid biosynthesis [28,29] Moreover, the site
of action of these two proteins is the rate-limiting step
in steroidogenesis [6] Fatty acyl-CoAs bind to the acyl-CoA-binding protein, which can bind and may thereby activate PBR This interaction favors the accumulation of fatty acids near StAR, perhaps by promoting fatty acyl-CoA transfer into the inner mitochondria
Although it is clear from our results that the acyl-CoA thioesterase is critical in steroidogenesis and that the AA produced promotes, at least, the stimulation of StAR protein expression, a possible action of the fatty acid at a different level of the steroidogenic pathway cannot be ruled out In accordance with this sugges-tion, a positive action on cholesterol metabolism of nonesterified fatty acids in the mitochondrial mem-brane has been suggested [30] It was demonstrated that cholesterol binding to P450scc in lipid vesicles is greatly potentiated when the local membrane is ren-dered more fluid by the addition of nonesterified fatty acids [31] P450scc interactions with cholesterol, but not with hydroxycholesterol, are strongly affected by the lipid environment of the inner mitochondrial mem-brane Cholesterol interacts strongly with the fatty acid chains of many phospholipids and is thereby con-strained from interacting with P450scc The increase in membrane fluidity in the presence of these fatty acids possibly favors the interaction of cholesterol with StAR or P450scc [30,31] All these observations may explain why the free cytosolic AA released from cho-lesterol esters or phospholipids should be re-esterified
by the AA-preferring acyl-CoA synthetase in order to
be released in mitochondria by the specific acyl-CoA thioesterase
In summary, the present work shows for the first time that knocking down MTE-I and ACS4 mRNA
by antisense or siRNA in steroidogenic cells results in
a reduction in steroid biosynthesis This provides evi-dence for the pivotal role played by ACS4 and MTE-I
Trang 9in AA release, StAR protein expression, and
steroido-genesis
Experimental procedures
Materials
8Br-cAMP, 22(R)-OH-cholesterol, fatty acid-free BSA,
arachidonic, arachidic and oleic acids were purchased from
Sigma Chemical Co (St Louis, MO, USA) Ham-F10 and
Waymouth MB752⁄ 1 cell culture media were from Life
Technologies Inc (Gaithersburg, MD, USA) All other
rea-gents were of the highest grade available
Cell culture
The MA-10 cell line is a clonal strain of mouse Leydig
tumor cells that produce progesterone rather than
testoster-one as the major steroid [32] MA-10 cells were generously
provided by Mario Ascoli, University of Iowa, College of
Medicine (Iowa City, IA, USA) and were handled as
ori-ginally described [30] The growth medium consisted of
Waymouth MB752⁄ 1 containing 1.1 gÆL)1NaHCO3, 20 mm
Hepes, 50 lgÆmL)1 gentamicin, and 15% horse serum
Flasks and multiwell plates were maintained at 36C in a
humidified atmosphere containing 5% CO2
Plasmid transfection
To develop optimized conditions for plasmid delivery to
MA-10 cells, several electroporation variables were tested
Electroporation experiments were performed using a Gene
Pulser II Electroporation System (Gibco, Grand Island,
NY, USA) MA-10 cells were routinely subcultured, and
cells were harvested and resuspended in NaCl⁄ Piat a
den-sity of 1.6· 107
cellsÆmL)1 pRc⁄ CMVi plasmid [33] con-taining the enhanced form of green fluorescent protein
(EGFP) (10 lg) was added to a 0.4-cm gap cuvette (Gibco)
containing 600 lL cell suspension This suspension was
electroporated with one pulse of 0.25 V and 600 lF Cells
were then cultured in complete fresh medium for 24 h and
electroporated under the same conditions twice more The
transfection efficiency of electroporation was estimated to
reach 50–70% by counting EGFP-transfected fluorescent
cells Approximately 24 h after transfection, cells were
stimulated with 8Br-cAMP (a permeable analog of cAMP)
in culture medium containing 0.1% fatty acid-free BSA
Progesterone produced was measured by RIA as previously
described [12,15]
SiRNA transfection
Two siRNAs were designed to target MTE-I (accession No
AF180798) and the ACS4 (accession No NM_207625)
coding sequence siRNA sequences 5¢-AAGAGCGAGT TCTATGCTGAT (nucleotides 322–342 of MTE-I cDNA), 5¢-AAATGACAGGCCAGTGTGAAC (nucleotides 1124–1143
of ACS4 cDNA), and 5¢-CGAGAAGACGTAAAGC (scramble siRNA of MTE-I) were custom-designed by Dharmacon (Lafayette, CO, USA) One day before trans-fection, MA-10 cells (5· 105
cells per well) were grown up
to 80% confluence on 24-well plates Transfection was per-formed using siRNA (800 nm) in Opti-MEM medium and
2 lL Lipofectamine 2000 reagent (Invitrogen, Carlsbad,
CA, USA) according to the instructions of the manufac-turer Cells were placed in normal culture medium 6 h after transfection and further grown for 48 h MA-10 cells were stimulated with 8Br-cAMP in culture medium containing 0.1% fatty acid-free BSA Progesterone production was measured by RIA, and data are shown as progesterone pro-duction (ngÆmL)1) in the incubation medium
Cell viability and mitochondrial integrity Cell viability was analyzed using the trypan blue exclusion method At the end of the incubation, the cells were washed three times with NaCl⁄ Pi and incubated for 15 min with 0.1% trypan blue stain After being washed, stained (dead) cells were counted by light microscopy Cell viability and mitochondrial integrity were also evaluated by measuring the levels of cellular MTT reduction [20]
SDS/PAGE and immunoblot assay Proteins were separated by SDS⁄ PAGE (12% gel) and electrophoretically transferred to poly(vinylidene difluoride) membrane (Bio-Rad Laboratories Inc, Hercules, CA, USA)
in buffer (25 mm Tris⁄ HCl, 192 mm glycine, pH 8.3, 20% methanol) at a constant voltage of 2.4 mAÆcm)2for 90 min Membranes were then incubated with 5% fat-free powdered milk in NaCl⁄ Tris ⁄ Tween (500 mm NaCl, 20 mm Tris ⁄ HCl,
pH 7.5, 0.5% Tween-20) for 60 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, Buenos Aires, Argentina)
Northern blot Total RNA from MA-10 cells was prepared by homogeni-zation in TRIzol reagent according to the manufacturer’s instructions Samples of RNA (24 lg) were resolved on 1.2% agarose⁄ 2.2 m formaldehyde gels and transferred on
to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) A cDNA probe for StAR was prepared by RT-PCR from total RNA from MA-10 cells Primers were
Trang 10designed according to the published sequence of mouse
StAR The forward (5¢-AAAGGATTAAGGCACCAA
GCTGTGC-3¢) and reverse (5¢-CTCTGATGACACCA
CTCTGCTCCGG-3¢) primers were used to amplify a
588-bp fragment The PCR product was sequenced to
con-firm its identity After prehybridization for 8 h at 42C,
blots were hybridized overnight with the [32
P]dCTP[aP]-radiolabeled cDNA probe at 42C The hybridization
solution contained 6· SCC, 5· Denhardt’s solution, 0.5%
formamide, and 100 lgÆmL)1 denatured salmon sperm
DNA Blots were subsequently washed twice with 2· SSPE
(150 mm NaCl, 10 mm NaH2PO4, 1 mm EDTA)⁄ 0.5%
SDS at room temperature and twice with 1· SSPE ⁄ 0.1%
SDS at 65C StAR hybridization signals were revelead
using a Storm PhosphorImager (Molecular Dynamics, Inc,
Sunnyvale, CA, USA) The membranes were then stripped
and rehybridized with 28S rRNA probe as loading control
Immunofluorescence and microscopy
MA-10 cells grown on poly(l-lysine) glass coverslips were
washed once with NaCl⁄ Pi and then fixed for 10 min at
room temperature with 4% (w⁄ v) paraformaldehyde in
NaCl⁄ Pi Briefly, MA-10 fixed cells were rinsed in NaCl⁄ Pi
and incubated with blocking solution (1.5% goat serum in
0.3% Triton X-100⁄ NaCl ⁄ Pi) for 1 h at room temperature
and incubated with rabbit polyclonal antibody against
recombinant acyl-CoA thioesterase I [12] and mouse
mono-clonal antibody against b-tubulin in a humidified chamber
for 24 h at 4C Primary antibodies were detected by
cy2-conjugated goat anti-(rabbit IgG) Ig or cy3-cy2-conjugated goat
anti-(mouse IgG) Ig (Molecular Probes, Eugene, OR,
USA) DNA was stained with DAPI The glass coverslips
were mounted in FluorSave reagent (Calbiochem) and
examined in an Olympus BX 50 epifluorescence
micro-scope
Statistical analysis
Data from the progesterone assay were analyzed for
statis-tical significance using analysis of variance followed by the
Student–Newman–Kuels test
Acknowledgements
Thanks are due to Douglas Stocco for the StAR
anti-body (Department of Cell Biology and Biochemistry,
Texas Tech University, Lubbock, TX, USA) We also
thank Ingo Leibiger (Department of Molecular
Medi-cine, Karolinska Hospital L3, Karolinska Institutet,
Stockholm, Sweden) for the pRc⁄ CMVi plasmid This
work was supported by grants from Agencia Nacional
de Promocio´n Cientı´fica y Tecnolo´gica (PICT6738, to
E.J.P.), Universidad de Buenos Aires (M034, to E.J.P.),
Fundacio´n Antorchasto E.J.P and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PEI02535, to C.P.)
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