Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase inhibits hormone-induced steroidogenesis ´ Paula 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 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 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 expression 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 significant 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 phospholipase A2 cascade Steroid hormones are synthesized in specialized steroidogenic cells in the adrenal gland, ovary, testis, placenta and brain and are essential for maintaining normal body homeostasis and reproductive capacity The biosynthesis of all steroid hormones begins at the mitochondria with the conversion of cholesterol into pregnenolone by the cholesterol side-chain cleavage cytochrome P-450 enzyme (P450scc) [1,2] The transport of cholesterol to the inner mitochondrial membrane 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 lipoxygenase metabolites play an essential role in the regulation of steroidogenesis In steroidogenic cells, AA participates in hormone-stimulated induction of StAR expression [10,11] However, the manner in which 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 P Maloberti et al 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 (ACS4) and mitochondrial acyl-CoA thioesterase I (MTE-I) as important regulators of AA release in the mechanism of action of trophic hormone-stimulated cholesterol metabolism [12] ACS4 has been described 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 mitochondrial 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 acylCoA thioesterase activity [16] which includes four isoforms 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 accordance with the postulated role of MTE-I in steroidogenesis, 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 arachidonoylCoA, so far, the phospholipase A2 pathway is the most commonly accepted mechanism operating to produce lipoxygenated products from plasma membrane signaling [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 adrenal 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 inhibition 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 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS Acyl-CoA thioesterase and synthetase 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 steroidogenesis, we performed experiments aimed at silencing the expression of MTE-I or at overexpressing the protein in steroidogenic cells For this purpose, we transiently 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 transfection using the protocol described in Experimental Procedures was 50–70% The effect of sense and antisense plasmid transfection 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 transfected 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-diphenyl2H-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-transfected 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-diamidino-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-bromo3¢,5¢-cAMP (8Br-cAMP)-stimulated progesterone production (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 1805 Acyl-CoA thioesterase and synthetase P Maloberti et al Fig 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 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-transfected 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 electroporation or times Progesterone inhibition followed the same pattern To investigate if the reduction in MTE-I expression produced by the antisense-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 cholesterol side-chain cleavage cytochrome P-450 enzyme (P450scc) Steroidogenesis stimulated by 22(R)-OHcholesterol was assayed under conditions of time and substrate concentration in the linear range No significant difference in 22(R)-OH-cholesterol-sustained progesterone production was detected among the different treatments (Fig 2C, inset) This result provides evidence that the reduction in MTE-I expression 1806 impaired cholesterol transport without affecting mitochondrial 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] Considering 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 h of 8Br-cAMP treatment in mock-transfected cells (Fig 3A) The effect of 8BrcAMP on StAR protein concentrations was strongly reduced in cells transfected with the plasmid containing MTE-I antisense The reduction in StAR protein concentrations in antisense-transfected cells reached 56 ± 14% as compared with mock-transfected cells FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS P Maloberti et al A Acyl-CoA thioesterase and synthetase A B B C Fig Effect of MTE-I knock down on steroidogenesis in MA-10 cells Mock-transfected (h), EGFP-transfected ( ) or MTE-I antisense-transfected (MTEas, ) MA-10 cells were incubated for 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 densitometry Bars denote relative levels of MTE-I expression (C) Determination of progesterone production by RIA Inset: MA-10-transfected cells were incubated for 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 when quantified by densitometry related to the b-tubulin 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 stimulated for 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 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS Fig Effect of MTE-I knock down on StAR expression in steroidogenic 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 h with 8Br-cAMP The membrane was blotted sequentially with anti-MTE-I, anti-StAR and antib-tubulin sera (B) Representative northern blot analysis of total RNA from MA-10 transfected cells stimulated for and h with 8Br-cAMP The membrane was developed using probes against StAR and 28S concentrations, we investigated whether treatment with MTE-I antisense had any effect on StAR mRNA concentrations Down-regulation of MTE-I produced a marked inhibition of StAR mRNA concentrations, which was already detectable after 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 h (data not shown) However, the effect of cycloheximide on steroidogenesis stimulated with 8Br-cAMP or AA produced inhibition before 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 1807 Acyl-CoA thioesterase and synthetase A B P Maloberti et al Also, we studied the effect of submaximal doses of 8Br-cAMP on steroidogenesis in MA-10 cells overexpressing 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 differences in steroid production among the different treatments (Fig 4C, inset) Effect of AA on steroid production inhibited by MTE-I knock down C Fig 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 incubated for 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 expressed 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-treated cells, respectively (C) Determination of progesterone production by RIA Inset: MA-10 transfected cells were incubated for 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 overexpression in MA-10 cells As predicted, transfection 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-transfected cells (Fig 4B) 1808 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 transfected with MTE-I antisense cDNA As shown in Fig 5, MTE-I antisense produced a significant inhibition 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 arachidic acid Moreover, oleic acid and arachidic acid did not affect the stimulated steroidogenesis in controltransfected 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 antisense experiments, we also designed siRNA duplexes directed against this acyl-CoA thioesterase Transfection 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, progesterone 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 siRNAtransfected cells is lower than the corresponding in Fig In addition, it is observed that down-regulation of MTE-I by siRNA is accompanied by a small but significant increase in ACS4 expression We have previously demonstrated a concerted action of ACS4 and MTE-I on hormone-induced steroid FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS P Maloberti et al Acyl-CoA thioesterase and synthetase Discussion Fig 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 h in the presence or absence of 8Br-cAMP (0.5 mM) and with or without 300 lM different 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 antisensetransfected 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 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 transfection of MA-10 cells siRNA transfection resulted in a marked reduction (56 ± 6%) in ACS4 protein concentrations 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 progesterone production As was the case in previous experiments, 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 inhibition 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 production and cell viability (data not shown) FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS 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 regulators in the mechanism of action of trophic hormonestimulated 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 transfected with a vector containing an MTE-I antisense cDNA Importantly, the transfection procedure developed and used in this study did not affect cell viability and ⁄ or morphology of sense-transfected or antisensetransfected cells as compared with mock-transfected cells Moreover, a possible negative effect of the treatments on mitochondrial activity was ruled out by using the MTT assay Finally, a possible apoptotic effect produced by the manipulation of MTE-I protein concentrations was also discarded after staining of the cells with DAPI showed no changes in nuclear morphology Knocking down MTE-I expression levels leads to a strong reduction in 8Br-cAMP-stimulated steroidogenesis The reduction in protein expression does not affect steroid biosynthesis when it is induced by a permeable analog of cholesterol, 22(R)-OH-cholesterol, which bypasses the rate-limiting step of steroidogenesis 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 expression could affect steroid hormone synthesis by affecting 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 without affecting mitochondrial integrity and are in line with previous reports that AA regulates the rate-limiting step in steroid biosynthesis [10] Importantly, the reduction in steroid biosynthesis was restored by addition of exogenous AA, clearly demonstrating that the effect produced by MTE-I protein reduction is based on decreased AA concentrations The 1809 Acyl-CoA thioesterase and synthetase A P Maloberti et al C B D E Fig 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 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 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 8BrcAMP-treated cells; c, P < 0.05 vs ACS4 siRNA-transfected 8Br-cAMP-treated cells; d, P < 0.01 vs MTE-I siRNA-transfected 8Br-cAMPtreated cells (D) MA-10 cells transfected with increasing concentrations of ACS4 ( ) or MTE-I ( ) or without (j) siRNA were incubated for 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 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 overexpression of this acyl-CoA thioesterase leads to a significant increase in steroidogenesis stimulated by 8Br-cAMP whereas it did not affect basal steroidogenesis Steroidogenic cells express MTE-I but also a cytosolic isoform 1810 (CTE-I) which is 92.5% homologous to the mitochondrial 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 participation of CTE-I in our antisense approach, the results obtained with overexpression of MTE-I strongly support the participation of this enzyme in AA release and hormone-induced steroid synthesis Our results are in line with reports showing the hormonal 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 Transfection with either siRNA duplex directed against ACS4 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS P Maloberti et al or MTE-I produced a significant inhibition in 8-BrcAMP-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 accompanied by an increase in ACS4 expression We 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 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 alleles with a high frequency of uterus embryonic death ACS4+ females showed marked accumulation of prostaglandin in the uteruses of the heterozygous females, suggesting that ACS4 modulates female fertility and uterus prostaglandin production ACS4 - ⁄ Y hemizygous males presented an apparently normal phenotype The authors suggest that ACS3, an isoform of the enzyme that also prefers arachidonate [23], may compensate 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 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS Acyl-CoA thioesterase and synthetase form of arachidonoyl-CoA to bring this substrate to a special compartment of the cells The compartmentalization 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 signaltransduction 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 acylCoA 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 suggestion, a positive action on cholesterol metabolism of nonesterified fatty acids in the mitochondrial membrane has been suggested [30] It was demonstrated that cholesterol binding to P450scc in lipid vesicles is greatly potentiated when the local membrane is rendered 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 membrane Cholesterol interacts strongly with the fatty acid chains of many phospholipids and is thereby constrained 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 cholesterol 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 evidence for the pivotal role played by ACS4 and MTE-I 1811 Acyl-CoA thioesterase and synthetase in AA release, StAR protein expression, and steroidogenesis 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 ⁄ cell culture media were from Life Technologies Inc (Gaithersburg, MD, USA) All other reagents 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 testosterone 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 originally described [30] The growth medium consisted of Waymouth MB752 ⁄ containing 1.1 gỈL)1 NaHCO3, 20 mm Hepes, 50 lgỈmL)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 PulserÒ 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 density of 1.6 · 107 cellsỈmL)1 pRc ⁄ CMVi plasmid [33] containing 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) 1812 P Maloberti et al 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 transfection, MA-10 cells (5 · 105 cells per well) were grown up to 80% confluence on 24-well plates Transfection was performed using siRNA (800 nm) in Opti-MEM medium and lL Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions of the manufacturer Cells were placed in normal culture medium 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 production (ngỈmL)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 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 mcm)2 for 90 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 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 °C 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 homogenization 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 FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS P Maloberti et al 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 confirm its identity After prehybridization for 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 lgỈmL)1 denatured salmon sperm DNA Blots were subsequently washed twice with 2· SSPE (150 mm NaCl, 10 mm NaH2PO4, 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 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 h at room temperature and incubated with rabbit polyclonal antibody against recombinant acyl-CoA thioesterase I [12] and mouse monoclonal antibody against b-tubulin in a humidified chamber for 24 h at °C Primary antibodies were detected by cy2conjugated 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 microscope Statistical analysis Data from the progesterone assay were analyzed for statistical significance using analysis of variance followed by the Student–Newman–Kuels test Acknowledgements Thanks are due to Douglas Stocco for the StAR antibody (Department of Cell Biology and Biochemistry, Texas Tech University, Lubbock, TX, USA) We also thank Ingo Leibiger (Department of Molecular Medicine, 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.), FEBS Journal 272 (2005) 1804–1814 ª 2005 FEBS Acyl-CoA thioesterase and synthetase Fundacio´n Antorchas to E.J.P and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (PEI02535, to C.P.) 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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 1807 Acyl-CoA thioesterase and synthetase. .. 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. .. 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