Concerted regulation of free arachidonic acid and hormone-induced steroid synthesis by acyl-CoA thioesterases and acyl-CoA synthetases in adrenal cells Paula Maloberti, Rocı ´ o Castilla Lozano, Pablo G. Mele, Florencia Cano, Cecilia Colonna, Carlos F. Mendez, Cristina Paz and Ernesto J. Podesta ´ Department of Biochemistry, School of Medicine, University of Buenos Aires Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA releaseis notclear. Therefore,the aim ofthis studywas to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a K m of 2 l M . Antibodies raised against recombinant acyl-CoA thioest- erase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydro- guaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochond- rial acyl-CoA thioesterases. ACTH-stimulated steroid syn- thesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC 50 for NDGA was reduced from 50 l M to 25, 7.5 and 4.5 l M in the presence of 0.1, 0.5 and 2 l M triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells. Keywords: arachidonic acid; steroidogenesis; acyl-CoA thio- esterases; acyl-CoA synthetase; arachidonoyl-CoA. The rate-limiting step, which initiates the synthesis of all steroids, depends on the availability of cholesterol to cytochrome P450scc [1,2]. The involvement of a cAMP- dependent protein kinase (PKA) phosphorylation event is accepted as an intermediate step in the cAMP-mediated stimulation of cholesterol availability [3–9] and particularly in the transport of cholesterol from the outer to the inner mitochondrial membrane [1,2,10,11]. The latter event is, in turn, controlled by the steroidogenic acute regulatory protein (StAR protein) [12–16]. Several reports have shown that arachidonic acid (AA) and its metabolites play an essential role in the regulation of steroidogenesis and both the expression and function of StAR [17–26]. Adrenal cholesterol metabolism is inhibited by nordihydroguaiaretic acid (NDGA) [19–25], a lipoxy- genase inhibitor that also acts as phospholipase A 2 inhibitor [27]. NDGA blocks the acute response of bovine cells to ACTH particularly when cAMP release is low [24], and it also decreases the expression of StAR mRNA in rodent steroidogenic cells [23,25,26]. Those effects can be reversed by AA hydroperoxides, a fact that suggests the involvement of lipoxygenases [23,25]. Although the role of AA in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented [17–26], the mechanism responsible for the release of AA remains unknown. Previous studies have reported that phospholipase A 2 (PLA 2 ) inhibitors abrogate the effect of LH- and ACTH-stimulated steroid production thereby suggesting the involvement of PLA 2 in the mech- anism of action of trophic hormones [18,19,23]. However, no evidence has been reported demonstrating the activation of PLA 2 by steroidogenic hormones. We have previously identified a hormone-dependent phosphoprotein involved in steroid synthesis through the release of AA. The protein was identified by its capacity to increase mitochondrial steroidogenesis in a cell-free assay [28,29]. The activity of the protein was dependent on cAMP and PKA and blocked by the use of 4-bromophenacyl- bromide (BPB) and NDGA, both inhibitors of AA release. Importantly, this inhibition could be overcome by the addition of AA [29,30], an indication that this protein regulates steroid synthesis through the (direct or indirect) activation of AA release. The protein was later purified to homogeneity and identified as a 43-kDa phosphoprotein (p43) [31]. Further cloning and sequencing of a cDNA Correspondence to E. J. Podesta ´ , Depto. de Bioquı ´ mica, Facultad de Medicina, Paraguay 2155, 5° piso, C1121ABG Buenos Aires, Argentina. Fax: 5411 45083672. ext. 31 E-mail: biohrdc@fmed.uba.ar Abbreviations: PKA, cAMP-dependent protein kinase; AA, arachi- donic acid; AA-CoA, arachidonoyl-CoA; NDGA, nordihydro- guaiaretic acid; StAR protein, steroidogenic acute regulatory protein; PLA 2 , phospholipase A 2 ; ATK, arachidonoyl trifluoromethyl ketone; BPB, 4-bromophenacyl-bromide; MTE-I, mitochondrial acyl- CoA thioesterase; CTE-I, cytosolic acyl-CoA thioesterase; ACS4, arachidonic acid preferred acyl-CoA synthetase; Y1, murine adreno- cortical tumor cell line; RIA, radioimmunoassay; IPTG, isopropyl thio-b- D -galactoside; DBI, diazepam binding inhibitor; PBR, periph- eral benzodiazepine receptor; HDL, high density lipoprotein; ACBP, acyl-CoA binding protein. (Received 1 July 2002, revised 29 August 2002, accepted 18 September 2002) Eur. J. Biochem. 269, 5599–5607 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03267.x encoding p43 revealed its primary structure [32]. The protein resulted 100% homologous to a mitochondrial-peroxisome- proliferator-induced acyl-CoA thioesterase (MTE-I) and 92.5% homologous to a cytosolic thioesterase (CTE-I) [33,34]. CTE-I and MTE-I are members of an acyl-CoA thioesterase family with very long chain and long chain acyl- CoA thioesterase activity [33,34] that includes four isoforms with different subcellular locations and a high degree of homology [35]. The family includes a cytosolic (CTE-I), a mitochondrial (MTE-I) and peroxisomal forms (PTE-Ia and Ib) of the enzyme [35]. Recently, this gene family was cloned and characterized in mouse showing that all isoforms are encoded by three exons spaced by two introns [35,36]. Like StAR, p43 is targeted to the inner mitochondrial membrane [15,32,33]. In accordance with the postulated obligatory role of the protein in steroidogenesis, we detected the protein and its mRNA in all steroidogenic tissues including placenta and brain [32]. Inhibition of ACTH release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenal with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min and returned to basal levels at 30 min. The effect of ACTH on the transcript was inhibited by actinomycin D and enhanced by cycloheximide [32]. Given the obligatory role of the protein in the activation of steroidogenesis through the release of AA, we proposed the name arachidonic acid- related thioesterase involved in steroidogenesis (ARTISt) for p43 [32]. Recently, the expression and the activity of different isoforms of acyl-CoA synthetases was described in the rat liver [37,38]. In addition, a report by Kang [39] implicates the participation of an AA-preferring acyl-CoA synthetase named ACS4, in steroidogenic tissues. The expression of ACS4 was observed in adrenal cortex cells, luteal and stromal cells of the ovary and Leydig cells of the testis [39]. Moreover, it was demonstrated that ACS4 expression in the murine adrenocortical tumor cell line Y1 (Y1) is induced by ACTH and suppressed by glucocorticoids [40]. Here, we address the question of whether AA-CoA, acyl- CoA synthetase and thioesterase are, indeed, essential for AA release and adrenal cholesterol metabolism. We dem- onstrate that recombinant MTE-I and CTE-I release AA from arachidonoyl-CoA in vitro and that ACTH increases the activity of the endogenous enzyme and promotes AA release from AA-CoA. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C, an inhibitor of the AA-CoA synthetase. Our results are consistent with the involvement of an acyl-CoA synthetase and an acyl-CoA thioesterase as important regulators of AA release in the mecha- nism of action of trophic hormone-stimulated cholesterol metabolism. EXPERIMENTAL PROCEDURES Materials The Bulk GST Purification Module and pGEX-4T3 vector were purchased from Amersham Pharmacia Biotech, Little Chalfont, UK. Restriction enzymes were obtained from Promega Corp. Madison, WI, USA and [1- 14 C]arachido- noyl-CoA from NEN Life Science Products, Inc (Boston, MA, USA). Arachidonoyl trifluoromethyl ketone (ATK) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). NDGA and 22R-OH-cholesterol were purchased from Sigma Chemicals Co., St Louis, MO, USA. Triacsin C was from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA) and Ham-F10 cell culture medium was from Life Technologies Inc. (Gaithersburg, MD, USA). All other reagents were of highest grade available. Tissue culture Murine Y1 adrenocortical tumor cells, generously provided by B. Schimmer (University of Toronto, Toronto, Canada), were maintained in Ham-F10 medium, supplemented with 12.5% heat-inactivated horse serum and 2.5% heat-inac- tivated fetal bovine serum, 1.2 gÆL )1 NaHCO 3 , 200 IUÆmL )1 penicillin and 200 lgÆmL )1 streptomycin sulfate [41]. ACTH stimulation was performed in culture medium containing 0.1% bovine serum albumin. Steroids produced were measured by radioimmunoassay (RIA). Determin- ation of progesterone production by RIA and of steroids by fluorometry showed comparable results. Therefore, data are shown as progesterone production (ngÆmL )1 )intheincu- bation medium. Expression and purification of CTE-I in E. coli The full-length CTE-I cDNA, generously provided by S. Alexson (Karolinska Institute, Stockholm, Sweden), was amplified and modified by PCR to include restriction sites for BamHI and EcoRI enzymes. The subsequent amplified cDNA was cloned into the BamHI and EcoRI sites of vector pGEX-4T3 and used for transformation of XL1 cells. Cloning into the BamHI and EcoRI sites creates an open reading frame for the expression of the mouse CTE-I as a GST-tagged fusion protein. Plasmids were isolated, partially sequenced, and subsequently used for the trans- formation of BL21 cells. For the expression of the fusion protein, bacteria were cultured at 30 °C in YTA medium until an D 600 of 0.6–2.0 was reached. Induction of protein expression was carried out for 2 h by adding 0.1 m M isopropyl thio-b- D -galactoside (IPTG). After recovery by centrifugation, cells were resuspended and lysed by sonica- tion (4 · 15 s) in NaCl/P i , containing 0.1% 2-mercaptoeth- anol, 1 mgÆmL )1 lysozyme, 10 l M leupeptin, 1 l M pepstatin Aand1m M EGTA. The sonicate was incubated for 30 min with 1% Triton X-100, and subsequently clarified by centrifugation at 12 000 g for 15 min at 4 °C. The purifi- cation of recombinant CTE-I was performed by the GST Purification Module according to manufacturer’s instruc- tions (Amersham Pharmacia), by thrombin cleavage and DEAE ionic exchange chromatography. Protein content was determined according to Bradford [42]. Expression and Purification of MTE-I in E. coli The recombinant MTE-I was first expressed in E. coli using the GST tag system. The yield of protein expression obtained by this procedure was extremely low. Therefore, we made use of the HIS tag system for the expression and purification of the recombinant protein. pET22b (+) bearing MTE-I sequence was transformed into BL21 cells 5600 P. Maloberti et al. (Eur. J. Biochem. 269) Ó FEBS 2002 according to standard procedures. Transformants were grown overnight at 37 °C in 3 mL of LB medium contain- ing 50 lgÆmL )1 ampicillin. These precultures were then used to inoculate 100 mL of fresh LB/ampicillin medium. Expression of the MTE-I was induced by the addition of 1m M IPTG. After a 3-h incubation period, cells were harvested and used for protein extraction following Nova- gen’s recommendations. The His-tag domain adjacent to the cloning site borne by the pET22b(+) vector, allowed purification of the expressed fusion proteins using a Ni 2+ chelation resin. Purification of the recombinant His-tagged MTE-I enzyme was performed according to the manufac- turer’s instructions. Even though the yield of purified protein was again low, the amount of protein obtained was sufficient to perform the experiments. Production of polyclonal antibodies against recombinant CTE-I Rabbits were injected once with 500 lg of recombinant CTE-I as antigen dissolved in 0.5 mL of distilled water and mixed with equal volumes of Freund’s complete adjuvant, andthreetimeswith500lg of antigen mixed with equal volumes of Freund’s incomplete adjuvant. Antibody titre against recombinant CTE-I was determined by ELISA. Western blot Proteins were separated on 12% SDS/PAGE and electro- phoretically transferred to nitrocellulose membranes in a buffer containing 25 m M Tris, 192 m M glycine, pH 8.3 and 20% methanol at a constant voltage of 15 mA for 30 min. Membranes were then incubated with 5% fat-free powdered milk in NaCl/Tris/Tween (500 m M NaCl, 20 m M Tris/HCl pH 7.5; 0.5% Tween-20) for 30 min at room temperature with gentle shaking. The membranes were then rinsed twice in NaCl/Tris/Tween and incubated overnight with the appropriate dilutions of primary antibody at 4 °C. Bound antibodies were detected by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech). Immunohistochemistry and immunocytochemistry Rat adrenals were dissected out and fixed by immersion in 4% paraformaldehyde in 0.01 M phosphate buffered saline (NaCl/P i ) for 2 h at room temperature and left overnight at 4 °C. The tissue was stored in NaCl/P i containing 12% sucrose at 4 °C, sectioned at 16 lm in a cryostat and thaw- mounted onto gelatinized glass slides. Y1 cells grown on poly- L -lysine glass cover slips were washed once with NaCl/P i and then fixed overnight at 4 °C with 4% (w/v) paraformaldehyde in NaCl/P i . Both cover slips and glass slides were processed for indirect immuno- cytochemistry and immunohistochemistry, respectively. Briefly, sections were rinsed in NaCl/P i and incubated with blocking solution (1.5% goat serum in 0.3% Triton-X100 NaCl/P i ) for 1 h at room temperature and incubated with rabbit polyclonal antibodies against recombinant acyl-CoA thioesterase (1/100), or vehicle (control) in a humidified chamber for 24 h at 4 °C. Detection of the primary antibody was done by means of a cy3-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). After rinsing with NaCl/P i , the sections were mounted in FluorSave reagent (Calbiochem, CA, USA) and examined in an Olympus BX 50 epifluorescence Microscope. Kodak T-400 C41 film was used for photography. Preparation of postmitochondrial and mitochondrial fractions Isolation of mitochondria was carried out as described [28,43]. Briefly, Y1 cell cultures were washed with NaCl/P i , scraped in 10 m M Tris/HCl, pH 7.4, 250 m M sucrose, 0.1 m M EDTA, 10 l M leupeptin, 1 l M pepstatin A and 1m M EGTA. (buffer A), homogenized with a Pellet pestle motor homogeniser (Kontes), and centrifuged at 600 g during 15 min. A second centrifugation at 10 000 g during 15 min rendered a mitochondrial pellet and a supernatant (postmitochondrial fraction). The mitochondrial pellet was washed once with buffer A, and resuspended in 10 m M Tris/ HCl pH 7.4, 10 l M leupeptin, 1 l M pepstatin A and 1 m M EGTA. Thioesterase activity determination Acyl-CoA thioesterase activity was determined using [1- 14 C]arachidonoyl-CoA (specific activity: 51.6 mCiÆ mmol )1 , concentration: 0.02 mCiÆmL )1 ) as substrate. The reaction was carried out at 37 °C and under vigorous shaking, using 0.1 lg of the recombinant protein in a buffer containing 10 m M Hepes, 50 m M KCl, pH 7.4 and 15 l M of the substrate. Arachidonic acid released during the reaction was extracted from the aqueous phase with n-hexane and quantified by scintillation counting. Statistical analysis The results of the studies of different inhibitors on enzyme activity are expressed as progesterone produced (ngÆmL )1 ). Comparison of mean values was performed using either the analysis of variance ( ANOVA ) or two-way analysis of variance (two-way ANOVA ) followed by the Student-New- man-Kuels test. RESULTS Detection of recombinant CTE-I and MTE-I by antibodies The recombinant cytosolic and mitochondrial acyl-CoA thioesterases were produced in E. coli and purified to homogeneity as described in Experimental procedures. In order to identify the recombinant proteins we used antibodies raised against a synthetic peptide corresponding to the catalytic domain of ARTISt (G11K antibody) [31]. The antibody recognized the protein from adrenal zona fasciculata (Fig. 1A, lane 3) as it was previously described [31] and also CTE-I and MTE-I recombinant proteins (Fig. 1A, lanes 1 and 2). In another set of experiments we obtained an antibody using the purified recombinant CTE-I protein as described in Experimental procedures. The immune rabbit serum recognized both recombinant proteins (Fig. 1A, lanes 4 and 5) and the 43-kDa protein from mitochondrial and postmitochondrial fractions of Y1 tumor cells (Fig. 1A, lanes 6 and 7). Preadsorbed antibody with the purified Ó FEBS 2002 Acyl-CoA thioesterases and synthetase on steroidogenesis (Eur. J. Biochem. 269) 5601 recombinant CTE-I, did not recognize the 43-kDa protein in both fractions (Fig. 1A, lanes 8 and 9). The expression of the thioesterase protein in Y1 cells was also studied by immunocytochemistry. Cells were uniformly stained with the exception of nuclei as observed in Fig. 1B, panel A. No signal was detected when the cells were incubated in the presence of secondary antibody alone (Fig. 1B, panel C). Taken together, these results clearly indicate that the endogenous protein is indeed similar to the recombinant proteins. The expression of the thioesterase was also studied by immunohistochemistry in rat adrenals. Intense fluorescence was detected throughout the adrenal cortex, while the medulla was devoid of signal (Fig. 2, panel A). The label was most intense in the zona fasciculata as compared with that shown by both zona glomerulosa and reticularis (Fig. 2, panels C and D). The specificity of the label was corroborated when the tissue sections were incubated with preadsorbed primary antibody (Fig. 2, panel B). CTE-I and MTE-I thioesterase activity To further characterize the recombinant CTE-I and MTE-I, we studied the kinetic parameters of the enzymes by measuring thioesterase activity using [1- 14 C]AA-CoA as described in Experimental procedures. Expectedly, incuba- tion of [1- 14 C]AA-CoA in the presence of the purified enzyme resulted in the release of AA. K m and Vmax values obtained for the reactions were 4.1 and 2 l M and 948 and 193 nmolÆmin )1 Æmg )1 for CTE-I and a MTE-I, respectively. Next, we measured the effect of ACTH on mitochondrial thioesterase activity in Y1 adrenal cells. For that purpose, mitochondria were isolated from cultures of confluent Y1 cells incubated in the presence or absence of 5 mIUÆmL )1 ACTH and enzyme activity was determined as AA released from AA-CoA. ACTH significantly stimulated enzyme activity in the mitochondrial fraction from 1.59 ± 0.22 to 3.0 ± 0.25 and from 1.67 ± 0.28 to 4.21 ± 0.29 pmol AAÆmin )1 for control and ACTH at 5 and 30 min, respectively. A similar effect in enzyme activity was observed when the postmitochondrial fraction was used as a source of enzyme (data not shown). Effect of NDGA on acyl-CoA thioesterase activity Although NDGA is commonly used as an inhibitor of the lipoxygenase pathway, it is also known to inhibit PLA 2 activity [27]. Since NDGA strongly inhibited both steroid production and StAR protein expression [23,25,26], we tested here whether NDGA inhibits acyl-CoA thioesterase Fig. 2. Immunohistochemical analysis of acyl-CoA thioesterases in the adrenal gland. Tissue sections from rat adrenal gland were incubated with anti-(CTE-I) (A, C and D) or antibody preadsorbed with purified CTE-I (B). Specific binding was detected using a cy3-conjugated goat anti-rabbit IgG as secondary antibody and observed under standard fluorescence microscopy. Original magnifications are: A and B, · 100; CandD· 200. Acyl-CoA thioesterase immunoreactivity was found in the zona fasciculata (f), zona glomerulosa (g) and zona reticularis (r). No signal was detected in the medulla (m). Fig. 1. Immunodetection of CTE-I and MTE-I. (A) Samples of purified recombinant CTE-I (CTE-I) and MTE-I (MTE-I), homogenates from adrenal zona fasciculata (Fasciculata), mitochondrial (MF) or post- mitochondrial fractions (PMF) obtained from Y1 cells, as indicated, were resolved on SDS/PAGE and immunoblotted as described in Experimental procedures. Antibodies used were: anti-G11K (lanes 1–3), anti-(CTE-I) (lanes 4–7) or antibody preadsorbed with purified recombinant CTE-I (control) (lanes 8 and 9). (B) Immunocytochemi- cal detection of acyl-CoA thioesterase in Y1 cells. Cells were fixed and stained using anti-(CTE-I) serum as described in Experimental pro- cedures. Specific binding was detected by means of a cy3-conjugated secondary antibody and fluorescence observed under a standard epifluorescence microscope (A). (C) Cells stained only with secondary antibody. (B) and (D), phase contrast micrographs of cells observed in (A) and (C), respectively. Original magnification · 400. 5602 P. Maloberti et al. (Eur. J. Biochem. 269) Ó FEBS 2002 activity in vitro. Figure 3 shows that a 5-min preincubation of recombinant protein with NDGA inhibited thioesterase activity. Interestingly, NDGA produced only a 20% inhibition of the mitochondrial enzyme activity when measured in mitochondria isolated from ACTH-stimulated Y1 cells (data not shown). This later result is in agreement with the previous observation that NDGA inhibitory effect is manifested only when the inhibitor is added prior to ACTH [21]. Role of arachidonoyl-CoA and arachidonoyl-CoA synthetase on ACTH-stimulated cholesterol metabolism Next, we tested the effect of NDGA on ACTH-induced steroid production in Y1 cells. According with the effect observed in isolated rat zona fasciculata cells [44], NDGA significantly (P < 0.001) inhibited ACTH-induced steroid production in a dose-dependent manner (Fig. 4), with an apparent IC 50 of 50 l M . The expression of an acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4 (ACS4), in steroidogenic tissues has been reported [39]. Triacsin C has been described as an inhibitor of acyl-CoA synthetases with a preferential effect for AA-CoA synthetases in intact cells [45,46]. Thus, we investigated a possible concerted regulatory role of acyl- CoA synthetases and thioesterases in the regulation of steroidogenesis by using triacsin C and NDGA on ACTH- stimulated steroid synthesis in Y1 cells. For this purpose, ACTH-stimulated Y1 cells were treated with ineffective concentrations of NDGA (5–25 l M ,Fig.4) alone or in combination with increasing concentrations of triacsin C (0.1–2 l M ) and steroids measured as indicated in Experimental procedures. Expectedly, NDGA alone had no effect on ACTH-induced steroid production, while triac- sin C alone produced a slight but significant (P ¼ 0.0445) inhibition of steroid output (Fig. 5B). However, NDGA significantly inhibited steroid biosynthesis when combined with 0.1, 0.5 and 2 l M triacsin C (Fig. 5A). A two-way analysis of variance rendered a highly significant (P ¼ 0.0026) value for the combination of NDGA and triacsin C, thereby indicating a synergistic effect on steroid production. Moreover, the synergistic effect is evidenced when the IC 50 for the inhibitors are analysed. Thus, the apparent IC 50 for NDGA is reduced from 50 l M to 25, 7.5 and 4.5 l M in the presence of 0.1, 0.5 and 2 l M triacsin C, respectively. The apparent IC 50 for triacsin C is also reduced from 5.5 l M to 1.75, 0.275 and 0.1 l M in the presence of 5, 10 and 25 l M of NDGA, respectively (Fig. 5B). The inhibitory effect of both triacsin C and NDGA was clearly not due to an inhibition of P450 scc activity since no significant inhibition of steroid production was observed when 22R-OH-cholesterol was added to the Y1 cell culture (data not shown). These results confirm the participation of ACS4 and of AA-CoA in the regulation of steroidogenesis in Y1 cells. DISCUSSION AA is present in the plasma membrane of most mammalian cells esterified to phospholipids. Free cytosolic AA can be produced by the action of phospholipase A 2 or cholesterol esterase which release the fatty acid by cleavage from membrane phospholipids or cholesterol esters, respectively [47]. Previous studies have demonstrated that inhibitors of PLA 2 activity affect hormone-induced steroidogenesis [17–19,25]. Those studies have raised the possibility that PLA 2 could be involved in the mechanism of action of hormones that control steroid production. However, there is no direct evidence showing that AA is released by PLA 2 in steroidogenic tissues. The present study is the first one to provide evidence for an alternative pathway of AA generation. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of an acyl-CoA thioesterase activity. We show here that the mitochondrial acyl-CoA thioesterase activity hydrolyses AA-CoA to release free AA Fig. 3. Effect of NDGA on recombinant thioesterase activity. Activity of recombinant CTE-I (open bars) and MTE-I (filled bars) was measured in the presence of increasing doses of NDGA. Acyl-CoA thioesterase activity was determined using [1- 14 C]AA-CoA as sub- strate. Results are expressed as the mean ± SD of one representative experiment performed in triplicate. Fig. 4. Effect of NDGA on steroid hormone synthesis. Y1 cells were preincubated with variable concentrations (5–100 l M )ofNDGAfor 15 min at 37 °C and further incubated in the absence or the presence of 2mIUÆmL )1 of ACTH for 60 min. Determination of progesterone production by RIA and of steroids by fluorometry showed comparable results. Therefore, data are shown as progesterone production (ngÆmL )1 ) in the incubation medium. Basal steroid production is denoted by the dotted line. The effect of NDGA was highly significant (P < 0.001) by ANOVA , and indicated values (*) significantly different (P < 0.05) by Student-Newman-Keuls post test. Ó FEBS 2002 Acyl-CoA thioesterases and synthetase on steroidogenesis (Eur. J. Biochem. 269) 5603 and that the activity of the mitochondrial acyl-CoA thioesterase increases significantly after ACTH stimulation in Y1 cells. We also demonstrate that inhibitors of AA release and metabolism such as NDGA, are effective inhibitors of recombinant CTE-I and MTE-I. The experi- ment using inhibitors of acyl-CoA synthetases in combina- tion with NDGA demonstrates that the two enzymes, the synthetase and the acyl-CoA thioesterase act in concert and are essential for steroidogenesis. A possible explanation for the effect of classical PLA 2 inhibitors on thioesterase activity could be the presence of a serine-histidine-aspartic acid catalytic triad containing ab hydrolases as determined by site-directed mutagenesis [48]. This possibility is supported by our previous results showing that antibodies raised against a synthetic peptide matching a sequence that contains the serine included in the catalytic triad inhibit steroid synthesis in a recombinant cell-free assay [32]. These observations are also in agree- ment with previous results showing that BPB, another PLA 2 inhibitor, also blocks the activity of CTE-I [44]. In addition, another specific inhibitor of PLA 2 (100 l M ATK) was also effective to inhibit the activity of the enzyme (data not shown). NDGA is known to inhibit the activity of lipoxygenase by binding to the reduced form (Fe 2+ )oftheenzyme thereby keeping it inactive [49]. In our experiments NDGA was effective in inhibiting the activity of purified recombin- ant MTE-I in an in vitro assay performed in a Hepes-based buffer containing only KCl. Information about the tertiary structure and of ion requirements of acyl-CoA thioesterases is still lacking and the mechanism of inhibition of acyl-CoA thioesterases by NDGA is unknown. Nevertheless, we cannot exclude an inhibitory mechanism for the thioesterase involving electron transfer. Noteworthy, the thioesterase displays a decreased sensitivity for NDGA after ACTH stimulation. This latter effect could result from a confor- mational change of the enzyme induced by protein phos- phorylation. Blocking lipoxygenase-mediated AA metabolism by NDGA, greatly decreased Bt 2 cAMP-induced StAR protein expression and reduced cholesterol metabolism [25]. How- ever, in view of our present results, the use of NDGA only as lipoxygenase inhibitor has to be reviewed since it can also act as inhibitor of acyl-CoA thioesterases. Recombinant CTE-I and MTE-I expressed in E. coli exhibited the expected molecular mass, showed high acyl- CoA thioesterase activity using AA-CoA as substrate and were detected by antipeptide antibodies that recognize the catalytic domain of the purified enzyme from adrenal. In addition, antibodies raised against recombinant CTE-I recognize both recombinant proteins and the protein from cytosol and mitochondria from adrenal cells. Using this antibody, immunohistochemical experiments show that the protein is exclusively located in steroidogenic cells of adrenal gland. The expression of a cytosolic and a mitochondrial thioesterase activity with different subcellular localizations suggests that these enzymes have different functions in vivo, although the role of CTE-I in steroidogenesis is currently not known. The high degree of sequence similarity between the CTE-I and MTE-I genes suggests that they diverged relatively recently by gene duplication, possibly by duplica- tion of an MTE-I gene with loss of the 5¢-end encoding the mitochondrial targeting signal, resulting in a cytosolic enzyme [35]. In the present study we demonstrate that AA-CoA is an important intermediate in steroidogenesis. The observation that triacsin C, an inhibitor of AA-CoA synthetase [44,45], affects hormone-induced steroid synthesis in Y1 cells supports this hypothesis. The participation of the AA-CoA-mediated pathway for AA release in steroidogen- esis was further demonstrated by the combined inhibitory effect of triacsin C and NDGA. Our results clearly show that addition of triacsin C in combination with ineffective doses of NDGA produced a marked reduction of the IC 50 for NDGA. Therefore, the regulatory role of AA in Fig. 5. Combined effect of triacsin C and NDGA on steroid hormone synthesis. Y1 cells were preincubated in the presence or the absence of increasing concentrations of triacsin C (4 h, 37 °C) and of NDGA (15 min, 37 °C).Cellswerethenincubatedfor60minwith2mIUÆmL )1 of ACTH or its vehicle. Progesterone produced was determined in the incubation medium by RIA. Data are expressed as the mean ± SD (n ¼ 3) of different concentrations of triacsin C, 0.1 (d), 0.5 (m), 2 (j) lMorcontrol(r) with indicated concentrations of NDGA (panel A), or variable concentrations of NDGA, 5 (s), 10 (n), 25 (h) l M or control (e) in the presence of triacsin C (panel B). Basal steroid production by Y1 cells is denoted by the dotted line. 5604 P. Maloberti et al. (Eur. J. Biochem. 269) Ó FEBS 2002 steroidogenesis needs the concerted action of the acyl-CoA synthetase and thioesterase. Although we cannot rule out a possible nonspecific effect of both triacsin C and NDGA, our observation of a synergistic effect on steroid production suggests this is unlikely. This, along with the fact that 22R-OH-cholesterol by-passes the effect of the inhibitors strongly indicates that the thioesterase and the acyl CoA-synthetase act in the same signalling pathway in a step prior to the rate-limiting passage of cholesterol from the outer to the inner mito- chondrial membrane. The thioesterase activity requires an acyl-CoA pool as a source of AA. The presence of an acyl-CoA synthetase specific for arachidonate described in steroidogenic tissues suggests that such a mechanism is operable in steroid biosynthesis. The concept that long chain fatty acyl-CoA esters are regulatory ligands as well as intermediates in cellular metabolism is now well appreciated from results of a number of investigations in a variety of organism and tissues [50,51]. The question then arises as to why free cytosolic AA has to be re-esterified in order to stimulate steroidogenesis. One possible explanation is the need for AA in a special compartment of the cell (e.g. mitochondria). The compart- mentalization of long-chain acyl-CoA esters is an important unsolved problem, and the actual cytosolic concentration of free long-chain acyl-CoA esters is not known for any tissue [51]. The high degree of sequestration of CoA into long chain acyl-CoA suggests that AA is likely to become limiting for diverse roles in specific compartments of the cell [51]. Here we show that ACTH stimulates mitochondrial thioesterase activity in Y1 cells. This points to a direct effect of ACTH upon enzyme activity. However, we cannot rule out a possible activation of the enzyme by an ACTH- mediated increased availability of its substrate. It is known that an acyl-CoA binding protein (ACBP) known also as DBI (diazepam binding inhibitor) is expressed in high concentrations in specialized cells such as steroid producing cells of the adrenal cortex and testis [52,53]. Thus it can be proposed that AA-CoA binds to DBI, which in turns binds to the peripheral benzodiazepine receptor (PBR) located in the outer mitochondrial mem- brane [53,54]. This will possibly lead to facilitated transfer of AA-CoA into the mitochondria. Another important issue is the origin of cytosolic free AA to be esterified into AA-CoA. As already mentioned, AA could derive from plasma membrane phospholipids or from cholesterol esters. The major source of cholesterol in the rat adrenal is the cholesterol esterified in high-density lipopro- teins (HDL) [55,56]. In adrenocortical cells, HDL enhances steroid production and increases cellular cholesterol con- tent. Rat HDL contains a high amount of arachidonate in its cholesterol esters fatty acids. This is an agreement with the suggestion that free AA, which will be esterified to acyl- CoA may come from the hydrolysis of cholesterol ester [34]. Nevertheless, since it has been shown that dexamethasone inhibits cholesterol metabolism and that this effect is reverted by free AA, we can not rule out the possibility that the free AA that is esterified into acyl-CoA may come from membrane phospholipids by the action of PLA 2 . However, in none of those studies, there is a demonstration that dexamethasone is in fact working through the inhibi- tion of PLA 2 . Our current data indicate the presence of a new pathway that regulates intracellular levels of AA, in which ACS4 could act by sequestering free AA by esterification into AA-CoA. CoA-esterified AA may bind ACBP/DBI thus forming an intracellular pool that could then be delivered to an acyl-CoA thioesterase, which will, in turn, release AA in a specific compartment of the cell upon hormone treatment. 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