Silencing the expression of mitochondrial acyl-CoAthioesterase I and acyl-CoA synthetase 4 inhibitshormone-induced steroidogenesisPaula Maloberti*, Rocıo 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 Bioquımica,

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 A2 cascade.

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.

1804 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS

trophic 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 A2 pathway 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 concentrations 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-tubulinremained 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

P. Maloberti et al. Acyl-CoA thioesterase and synthetase

FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 1805

by 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-transfected

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.

Acyl-CoA thioesterase and synthetase P. Maloberti et al.

1806 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS

when 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 mM). (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 lM). 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 mM). (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.

P. Maloberti et al. Acyl-CoA thioesterase and synthetase

FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 1807

overexpression 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 production,

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 arachidic acid did

not affect the stimulated steroidogenesis in control-

transfected or nontransfected 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 mM). (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 lM). 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.

Acyl-CoA thioesterase and synthetase P. Maloberti et al.

1808 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS

synthesis [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 transfection 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-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 mM) and with or without 300 lM 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-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.

P. Maloberti et al. Acyl-CoA thioesterase and synthetase

FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 1809

correction 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 mM) and with or without AA (300 lM). 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 lM). 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 8Br-

cAMP-treated cells; c, P < 0.05 vs. ACS4 siRNA-transfected 8Br-cAMP-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 mM). 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.

Acyl-CoA thioesterase and synthetase P. Maloberti et al.

1810 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS

or 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

P. Maloberti et al. Acyl-CoA thioesterase and synthetase

FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 1811

in 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)1 NaHCO3, 20 mm

Hepes, 50 lgÆmL)1 gentamicin, and 15% horse serum.

Flasks and multiwell plates were maintained at 36 �C 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 ⁄Pi at 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. Cellswere 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)2 for 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 4 �C. Boundantibodies 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

Acyl-CoA thioesterase and synthetase P. Maloberti et al.

1812 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS

designed 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 42 �C,blots were hybridized overnight with the [32P]dCTP[aP]-radiolabeled cDNA probe at 42 �C. 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 65 �C. 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 4 �C. Primary antibodies were detected by cy2-

conjugated goat anti-(rabbit IgG) Ig or cy3-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 Promocion Cientıfica y Tecnologica (PICT6738, to

E.J.P.), Universidad de Buenos Aires (M034, to E.J.P.),

Fundacion Antorchas to E.J.P. and Consejo Nacional de

Investigaciones Cientıficas y Tecnicas (PEI02535, to

C.P.).

References

1 Privalle CT, Crivello JF & Jefcoate CR (1983) Regula-

tion of intramitochondrial cholesterol transfer to side-

chain cleavage cytochrome P-450 in rat adrenal gland.

Proc Natl Acad Sci USA 80, 702–706.

2 Crivello JF & Jefcoate CR (1980) Intracellular move-

ment of cholesterol in rat adrenal cells. Kinetics and

effects of inhibitors. J Biol Chem 255, 8144–8151.

3 Stocco DM (2000) The role of the StAR protein in ste-

roidogenesis: challenges for the future. J Endocrinol 164,

247–253.

4 Stocco DM (2001) Tracking the role of a star in the

sky of the new millennium. Mol Endocrinol 15, 1245–

1254.

5 Papadopoulos V, Amri H, Li H, Yao Z, Brown RC,

Vidic B & Culty M (2001) Structure, function and regu-

lation of the mitochondrial peripheral-type benzodiaze-

pine receptor. Therapie 56, 549–556.

6 Lacapere JJ & Papadopoulos V (2003) Peripheral-type

benzodiazepine receptor: structure and function of a

cholesterol-binding protein in steroid and bile acid

biosynthesis. Steroids 68, 569–585.

7 Khan WA, Blobe GC & Hannun YA (1995) Arachido-

nic acid and free fatty acids as second messengers and

the role of protein kinase C. Cell Signal 7, 171–184.

8 Hartl WH & Wolfe RR (1990) The phospholipid ⁄arachidonic acid second messenger system: its possible

role in physiology and pathophysiology of metabolism.

JPEN J Parenter Enteral Nutr 14, 416–427.

9 Whatley RE, Zimmerman GA, McIntyre TM &

Prescott SM (1990) Lipid metabolism and signal trans-

duction in endothelial cells. Prog Lipid Res 29, 45–63.

10 Wang X & Stocco DM (1999) Cyclic AMP and arachi-

donic acid: a tale of two pathways. Mol Cell Endocrinol

158, 7–12.

11 Wang XJ, Dyson MT, Jo Y, Eubank DW & Stocco

DM (2003) Involvement of 5-lipoxygenase metabolites

of arachidonic acid in cyclic AMP-stimulated steroido-

genesis and steroidogenic acute regulatory protein gene

expression. J Steroid Biochem Mol Biol 85, 159–166.

12 Maloberti P, Lozano RC, Mele PG, Cano F, Colonna

C, Mendez CF, Paz C & Podesta EJ (2002) Concerted

regulation of free arachidonic acid and hormone-

induced steroid synthesis by acyl-CoA thioesterases and

acyl-CoA synthetases in adrenal cells. Eur J Biochem

269, 5599–5607.

13 Kang MJ, Fujino T, Sasano H, Minekura H, Yabuki

N, Nagura H, Iijima H & Yamamoto TT (1997) A

novel arachidonate-preferring acyl-CoA synthetase is

P. Maloberti et al. Acyl-CoA thioesterase and synthetase

FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 1813

present in steroidogenic cells of the rat adrenal, ovary,

and testis. Proc Natl Acad Sci USA 94, 2880–2884.

14 Cho YY, Kang MJ, Ogawa S, Yamashita Y, Fujino T

& Yamamoto TT (2000) Regulation by adrenocortico-

tropic hormone and arachidonate of the expression of

acyl-CoA synthetase 4, an arachidonate-preferring

enzyme expressed in steroidogenic tissues. Biochem

Biophys Res Commun 274, 741–745.

15 Paz C, Dada LA, Cornejo Maciel MF, Mele PG,

Cymeryng CB, Neuman I, Mendez CF, Finkielstein CV,

Solano AR, Park M et al. (1994) Purification of a novel

43-kDa protein (p43) intermediary in the activation of

steroidogenesis from rat adrenal gland. Eur J Biochem

224, 709–716.

16 Finkielstein C, Maloberti P, Mendez CF, Paz C,

Cornejo Maciel F, Cymeryng C, Neuman I, Dada L,

Mele PG et al. (1998) An adrenocorticotropin-regulated

phosphoprotein intermediary in steroid synthesis is

similar to an acyl-CoA thioesterase enzyme. Eur J

Biochem 256, 60–66.

17 Svensson LT, Engberg ST, Aoyama T, Usuda N,

Alexson SE & Hashimoto T (1998) Molecular cloning

and characterization of a mitochondrial peroxisome

proliferator-induced acyl-CoA thioesterase from rat

liver. Biochem J 329, 601–608.

18 Hunt MC, Nousiainen SE, Huttunen MK, Orii KE,

Svensson LT & Alexson SE (1999) Peroxisome prolif-

erator-induced long chain acyl-CoA thioesterases

comprise a highly conserved novel multi-gene family

involved in lipid metabolism. J Biol Chem 274, 34317–

34326.

19 Hunt MC & Alexson SE (2002) The role acyl-CoA

thioesterases play in mediating intracellular lipid meta-

bolism. Prog Lipid Res 41, 99–130.

20 Mosmann T (1983) Rapid colorimetric assay for cellular

growth and survival: application to proliferation and

cytotoxicity assays. J Immunol Methods 65, 55–63.

21 Piccini M, Vitelli F, Bruttini M, Pober BR, Jonsson JJ,

Villanova M, Zollo M, Borsani G, Ballabio A & Renieri

A (1998) FACL4, a new gene encoding long-chain acyl-

CoA synthetase 4, is deleted in a family with Alport

syndrome, elliptocytosis, and mental retardation. Geno-

mics 47, 350–358.

22 Cho YY, Kang MJ, Sone H, Suzuki T, Abe M, Igarashi

M, Tokunaga T, Ogawa S, Takei YA, Miyazawa T

et al. (2001) Abnormal uterus with polycysts, accumula-

tion of uterine prostaglandins, and reduced fertility in

mice heterozygous for acyl-CoA synthetase 4 deficiency.

Biochem Biophys Res Commun 284, 993–997.

23 Fujino T, Kang MJ, Suzuki H, Iijima H & Yamamoto T

(1996) Molecular characterization and expression of rat

acyl-CoA synthetase 3. J Biol Chem. 271, 16748–16752.

24 Wang X, Walsh LP, Reinhart AJ & Stocco DM (2000)

The role of arachidonic acid in steroidogenesis and ster-

oidogenic acute regulatory (StAR) gene and protein

expression. J Biol Chem 275, 20204–20209.

25 Faergeman NJ & Knudsen J (1997) Role of long-chain

fatty acyl-CoA esters in the regulation of metabolism

and in cell signalling. Biochem J 323, 1–12.

26 Knudsen J, Mandrup S, Rasmussen JT, Andreasen PH,

Poulsen F & Kristiansen K (1993) The function of acyl-

CoA-binding protein (ACBP) ⁄ diazepam binding inhib-

itor (DBI). Mol Cell Biochem 123, 129–138.

27 Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty

M, Garnier M, Hardwick M, Li H, Vidic B, Brown AS

et al. (1997) Peripheral benzodiazepine receptor in

cholesterol transport and steroidogenesis. Steroids 62,

21–28.

28 Boujrad N, Hudson JR Jr & Papadopoulos V (1993)

Inhibition of hormone-stimulated steroidogenesis in

cultured Leydig tumor cells by a cholesterol-linked

phosphorothioate oligodeoxynucleotide antisense to

diazepam-binding inhibitor. Proc Natl Acad Sci USA

90, 5728–5731.

29 Papadopoulos V, Amri H, Li H, Boujrad N, Vidic B &

Garnier M (1997) Targeted disruption of the peripheral-

type benzodiazepine receptor gene inhibits steroidogen-

esis in the R2C Leydig tumor cell line. J Biol Chem 272,

32129–32135.

30 Jefcoate C (2002) High-flux mitochondrial cholesterol

trafficking, a specialized function of the adrenal cortex.

J Clin Invest 110, 881–890.

31 Dhariwal MS & Jefcoate CR (1989) Cholesterol meta-

bolism by purified cytochrome P-450scc is highly stimu-

lated by octyl glucoside and stearic acid exclusively in

large unilamellar phospholipid vesicles. Biochemistry 28,

8397–8402.

32 Ascoli M (1981) Characterization of several clonal lines

of cultured Leydig tumor cells: gonadotropin receptors

and steroidogenic responses. Endocrinology 108, 88–95.

33 Leibiger B, Moede T, Schwarz T, Brown GR, Kohler

M, Leibiger IB & Berggren PO (1998) Short-term regu-

lation of insulin gene transcription by glucose. Proc

Natl Acad Sci USA 95, 9307–9312.

Acyl-CoA thioesterase and synthetase P. Maloberti et al.

1814 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS