Báo cáo khoa học: cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells pdf

cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells Ana Fernanda Castillo, Fabiana Cornejo Maciel, Rocı ´ o Castilla, Alejandra Duarte, Paula Maloberti, Cristina Paz and Ernesto J. Podesta ´ Department of Biochemistry, School of Medicine, University of Buenos Aires, Argentina Arachidonic acid (AA) is a fatty acid with 20 carbons and four cis double bonds that are the source of its flexibility and its reactivity with molecular oxygen. The oxidation can happen nonenzymatically or through the action of three types of oxygenases: cyclooxygenase, lipoxygenase and cytochrome P450. Most of the effects of AA are attributable to its conversion by those enzymes to prostaglandins, leukotrienes and other bio- active products [1]. AA itself also has biological activ- ity; however, the number of its described actions is reduced compared to the effects described for the AA metabolites. Moreover, it is not very well documented whether nonmetabolized AA is released and elicits spe- cial functions in a specific cellular compartment [2]. Transport of long-chain fatty acids in cells definitely occurs when they are tightly linked to CoA by esterifi- cation catalyzed by acyl-CoA synthetases [3]. In mam- malian and yeast cells [4] it appears that the acyl-CoA synthetases merely enhance uptake indirectly. Thus, formation of the polar CoA-ester effectively traps the fatty acid in the cell and functions as part of a facilita- ted distribution in different cellular compartments. The mechanisms involved in the compartmentaliza- tion of long-chain acyl-CoA esters and free fatty acids Keywords acyl-CoA synthetase; acyl-CoA thioesterase; arachidonic acid compartmentalization; Leydig cells; steroidogenesis Correspondence E. J. Podesta ´ , Department of Biochemistry, School of Medicine, University of Buenos Aires, Paraguay 2155–5th, C1121ABG, Buenos Aires, Argentina Fax: +54 11 45083672 ext. 31 Tel: +54 11 45083672 ext. 36 E-mail: biohrdc@fmed.uba.ar (Received 27 July 2006, revised 23 August 2006, accepted 12 September 2006) doi:10.1111/j.1742-4658.2006.05496.x We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces choles- terol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol trans- port in isolated mitochondria from nonstimulated cells. The effect of arach- idonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accu- mulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regu- lation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role. Abbreviations AA, arachidonic acid; AA-CoA, arachidonoyl-CoA; Acot2, mitochondrial acyl-CoA thioesterase; ACS4, acyl-CoA synthetase 4; BPB, 4-bromophenacyl bromide; 8Br-cAMP, 8-bromo-cAMP; CHX, cycloheximide; CPT1, carnitine-palmitoyl transferase 1; DBI, diazepam-binding inhibitor; NDGA, nordihydroguaiaretic acid; P450scc, cholesterol side-chain cleavage cytochrome P-450 enzyme; PBR, peripheral benzodiazepan receptor; StAR, steroidogenic acute regulatory protein. FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5011 are important unresolved issues [5]. The simple struc- ture of AA and the natural occurrence of so many close chemical analogues are, not surprisingly, associ- ated with a lack of specificity. The selective actions of free AA may be explained simply by its specific release under physiological conditions and by the absence of such mechanisms for releasing other long-chain fatty acids, compounds which might otherwise share its bio- chemical effects [2]. Thus, the accessibility of AA to a specific cellular compartment and the specificity of its action are certainly linked. The enzymes involved in the release of AA have been well characterized, with the phospholipase A2 being the most important [6]. However, it remains unclear as to how exactly AA is released in a specific compartment of the cells under physiological condi- tions [2]. Recently, using steroidogenic cells as an experimental system, we described an alternative releasing mechanism for AA as a mediator of hormone action with the participation of an acyl-CoA synthe- tase (ACS4) and a mitochondrial acyl-CoA thioest- erase (Acot2) [7,8]. ACS4 has been described as an AA-preferring acyl-CoA synthetase [9], while Acot2 is a member of a new thioesterase family with long-chain acyl-CoA thioesterase activity and it is associated with the inner mitochondrial membrane [10–13]. In the steroidogenic cells, the step that determines the rate of steroid synthesis (the rate-limiting step) is the transport of cholesterol to the inner mitochondrial membrane [14], a process in which ACS4 and Acot2 play a key role. In this mechanism, it has been sugges- ted that ACS4 and Acot2 may constitute a system to deliver AA into a specific intracellular compartment, e.g. the mitochondria [8]. AA plays a crucial role in the steroidogenic cells, mediating the induction of the steroidogenic acute reg- ulatory (StAR) protein, one of the proteins involved in cholesterol transport [15–19]. Although it is clear from previous publications that AA plays its role in the pro- cess through the conversion to its lipoxygenated metabolites [15,17,20], a direct action of this fatty acid on cholesterol transport into the mitochondria cannot be ruled out. In this context, a positive action of nonesterified fatty acids on cholesterol metabolism has been des- cribed in the mitochondrial membrane [21]. It was also demonstrated that cholesterol binding to the enzyme that transforms it into pregnenolone (P450scc) in lipid vesicles is greatly potentiated when the local membrane is rendered more fluid by the addition of nonesterified fatty acids [22]. All the evidence described above led us to propose the hypothesis that AA might have a direct action on cholesterol transport into the mitochondria via a speci- fic release in this organelle. This knowledge would be important for the understanding of cholesterol trans- port in the classical steroidogenic as well as in neuro- logical systems, since changes in cholesterol transport in the central nervous system are part of the phenotype seen in the neuropathology and neurological disorders such as Alzheimer’s, Parkinson’s and Huntington’s dis- eases, and brain injury and inflammation, as well as in animal models of epilepsy [23]. This is also valid for cholesterol transport and metabolism in tumors such as glioma and mammary tumor cells [24,25]. For these reasons, the objective of the present work was to study the release of AA into the mitochondria and a possible direct role of fatty acids on cholesterol transport in this organelle. Results It is known that the acute response of steroidogenesis to hormonal stimulation has an absolute requirement: de novo protein synthesis [26,27]. This conclusion is based on the fact that hormone stimulated steroid syn- thesis is totally inhibited by cycloheximide (CHX), a protein synthesis inhibitor. The two proteins required in this step are ACS4 [28] and StAR [29]. ACS4 works in the release of AA, which, in turn, acts on StAR pro- tein induction. Because exogenous AA stimulates steroidogenesis in cells, the first experiment was carried out to study the direct effect of AA on cholesterol transport in the absence of newly synthesized StAR protein. For this purpose, MA-10 cells were incubated with exogenous AA either with or without submaximal concentration of 8-bromo, 5¢-cAMP (8Br-cAMP) in the presence or absence of CHX. Progesterone production as measure- ment of cholesterol transport was evaluated in the culture media after 1 h of incubation, as described in Experimental procedures (Fig. 1). Exogenous AA alone stimulated progesterone production, reaching 50–60% of the maximal value obtained with 8Br- cAMP (Fig. 1A). Submaximal doses of 8Br-cAMP in combination with AA stimulated steroid production at the same level as the stimulation produced by satur- ating doses of 8Br-cAMP. 8Br-cAMP-stimulated pro- gesterone production was completely abolished in the presence of CHX. However, AA-induced steroid pro- duction was only partially blocked by the protein syn- thesis inhibitor. Again, CHX did not totally reduce the synergistic effect of AA on steroid production, either (Fig. 1A). Protein synthesis inhibition did not affect progesterone production supported by the water- soluble derivative of cholesterol, 22(R)-OH-cholesterol, AA release in a specific compartment of the cells A. F. Castillo et al. 5012 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS which travels freely across the membranes to reach the inner mitochondrial cholesterol side-chain cleavage cytochrome P-450 enzyme (P450scc) (Fig. 1B). The widely known fact that cAMP cannot stimulate steroidogenesis in the absence of protein synthesis is due to the absence of two crucial proteins, ACS4 and StAR. ACS4 is induced by hormones and it is neces- sary for AA release [28], which participates in StAR protein induction. Therefore, in the absence of ACS4, no AA or StAR protein induction occurs, as previ- ously described [28]. This is the reason why CHX com- pletely abolished cAMP-stimulated steroidogenesis. When exogenous AA is used in the presence of CHX, the fatty acid bypasses the absence of ACS4 but not the absence of StAR. Then, the partial inhibition pro- duced by CHX on AA stimulated steroidogenesis was unexpected. The stimulatory effect of AA on steroid synthesis in the absence of protein synthesis suggests that AA can per se enhance the cholesterol transport and steroidogenesis in mitochondria of steroidogenic cells without de novo protein synthesis. In order to test this hypothesis, firstly, we tested whether AA exogen- ously added to intact cells could reach the mitochon- dria. Second, we studied the effect of exogenous AA on cholesterol transport in isolated mitochondria from nonstimulated MA-10 steroidogenic cells. For the first approach, MA-10 cells were labeled with [1- 14 C] AA during 5 h. After this period, the cells were incubated in the presence or in the absence of 8Br- cAMP. After this incubation, free AA was measured in the mitochondria, as described in the Experimental pro- cedures. Figure 2 shows the uptake of AA into the mito- chondria in basal and stimulated conditions. As can -CHX + CHX AA Control 8Br-cAMP 0.2 m M + AA 8Br-cAMP 0.2 m M 8Br-cAMP 0.5 m M A b b a a a a b,c b,c 0 2 4 6 8 10 12 14 B 22(R)OH- cholesterol 0 10 20 30 40 50 60 Progesterone (ng/ml) Progesterone (ng/ml) Fig. 1. Effect of cAMP, AA and CHX on progesterone production by MA-10 cells. MA-10 cells were incubated in the presence or absence of 10 lgÆmL )1 CHX for 30 min and then stimulated for 1 h with 8Br-cAMP (0.2 m M or 0.5 mM) and ⁄ or 300 lM AA (A), or 5 lM 22(R)-OH-cholesterol (B) in serum-free culture medium containing 0.1% fatty acid-free bovine serum albumin. Progesterone concen- trations were measured by RIA and data are shown as progester- one production (ngÆmL )1 ) in the incubation medium. Results are expressed as the mean ± SD from five independent experiments. (a) P < 0.001 versus control cells without CHX treatment; (b) P < 0.001 versus respective treated cells in absence of CHX; and (c) P < 0.01 versus control cells treated with CHX. B 17 16 15 4 3 2 1 0 Control 8Br-cAMP A AA Nuclei Mitochondria Control 8Br-cAMP *** a i r d n o h c o t i m ) s t i n u y r a r t i b r a ( A i e l c u n a i r d n o h c o t i m i e l c u n Fig. 2. Effect of cAMP on mitochondrial and nuclear AA content. MA-10 cells were labeled for 5 h at 37 °C with [1- 14 C] AA (1 lCiÆmL )1 per 2 · 10 6 cells) in serum-free media containing 0.5% fatty acid-free bovine serum albumin. Then, cells were incubated in either the presence or absence of 1 m M 8Br-cAMP for 30 min. After washing the cells, they were scraped and nuclear and mitoch- ondrial fractions were obtained as described in the Experimental procedures. The fractions were sonicated and lipids were extracted with ethyl acetate. The organic phase was collected and dried under nitrogen. The dried extracts were dissolved in chloro- form:methanol (9 : 1, v ⁄ v) and analyzed by thin-layer chromato- graphy on silica gel plates. (A) Representative autoradiography showing AA spots in nuclear and mitochondrial fractions. (B) Auto- radiography spots quantification by densitometry. The autoradio- graphies were quantified by densitometry and the data were normalized against the intensity of the signal of unlabeled AA stained with iodine. Bars denote levels (in arbitrary units) of AA in mitochondria and nuclei. Results are expressed as the mean ± SD from three independent experiments. *** P < 0.001 versus control mitochondria. A. F. Castillo et al. AA release in a specific compartment of the cells FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5013 clearly be seen, 8Br-cAMP increased the uptake of AA 3 times compared with nonstimulated conditions, with- out changing the content in the nuclear fraction. For the second approach, mitochondria from non- stimulated MA-10 cells were isolated and incubated in the presence of AA. Figure 3A shows that AA elicited a stimulatory effect on cholesterol transport measured as progesterone synthesis. As expected, this effect was not affected by the addition of CHX previous to the addition of AA. Nordihydroguaiaretic acid (NDGA), an inhibitor of AA metabolism, had no effect on AA action. Although NDGA is commonly used as lip- oxygenase inhibitor, it is also able to inhibit Acot2 [11]. However, in this case, we only evaluated its action on AA metabolism since the stimulation was per- formed with AA. Other fatty acids, such as oleic and arachidic acids produced a small, but not significant, effect. The total steroidogenic capacity of the isolated mitochondria was determined by the incubation with the water-soluble derivative of cholesterol, 22(R)-OH- cholesterol. Neither CHX nor NDGA affected this total steroidogenic capacity. As we have proposed [8] that the conversion of AA- CoA to AA within the mitochondria may constitute a mechanism to deliver AA into specific compartment of the cells, the next experiment was carried out to deter- mine the effect of AA-CoA, the substrate of Acot2, on steroid synthesis in isolated mitochondria. Figure 3B shows that AA-CoA not only stimulated cholesterol transport in isolated mitochondria but also had a higher effect than free AA action. When another acyl- CoA, such as oleoyl-CoA, was tested, it was also proved capable of increasing progesterone production in mitochondria to a similar extent. To determine if the effect of AA-CoA on cholesterol transport was due to its conversion to AA by the action of Acot2, we studied the effect of the blockage of Acot2 expression or activity on AA-CoA-stimulated steroid synthesis. Our next experiment was conducted as described in Fig. 3B in the presence and absence of BPB or NDGA, both inhibitors of Acot2 activity [11]. Figure 3C shows that blockage of Acot2 activity pro- duces a significant inhibition of progesterone synthesis stimulated by AA-CoA. To silence the expression of Acot2, we transiently transfected MA-10 cells with pRc ⁄ CMVi plasmid con- taining an antisense Acot2 cDNA (accession number Y09333). The effect of antisense plasmid transfection on Acot2 protein concentrations was studied by west- ern blot, by means of a specific antibody against the Acot2 and b-tubulin as control. As expected [8], anti- sense-transfected cells showed a strong reduction in Acot2 protein levels compared with cells transfected with vector alone (Fig. 4A,B). The stimulatory effect of AA-CoA on steroid synthesis in mitochondria isola- ted from non stimulated MA-10 cells where Acot2 was a b 0.15 0.05 0.00 Control AA Oleoyl-CoA AA-CoA 0.25 0.20 0.15 0.10 0.05 0.00 B )nietorpgm/gn(enoretsegorP a a,b a a b a 0.25 0.20 0.15 0.10 0.05 0.00 Malonyl-CoA BPB NDGA -AA-CoA + AA-CoA Control C )nietorpg m/gn (enoretsegorP b bb 0.70 0.20 0.10 0.00 )nietorpgm/gn(enoretsegorP 0.85 Arachidonic acid Arachidic acid Oleic acid A Control + NDGA + CHX None 22(R)OH- cholesterol Fig. 3. Effect of fatty acids, CoA derivatives of fatty acids, BPB, NDGA and malonyl-CoA on progesterone production in isolated mitochondria. Mitochondria were isolated from MA-10 cells and preincubated for 5 min at 37 °C in the absence or in the presence of 10 lgÆmL )1 CHX or 100 lM NDGA (A) or 0.1 mM BPB, 100 lM NDGA or 100 lM malonyl-CoA (C). Mitochondria were then incuba- ted for 20 min with 200 l M AA, oleic acid or arachidic acid or with 5 l M 22(R)-OH-cholesterol (A); 200 lM AA, AA-CoA or oleoyl-CoA (B); or 200 l M AA-CoA (C). Mitochondria were pelleted by centri- fugation and progesterone concentrations were measured in the supernatants by RIA. Data are shown as progesterone production (ngÆmg )1 mitochondrial protein) in the incubation media. (A) Results are expressed as the mean ± SD from six independent experi- ments. *** P < 0.001 versus control. (B) Results are expressed as the mean ± SD from three independent experiments. (a) P < 0.001 versus control mitochondria; (b) P < 0.01 versus AA treated mito- chondria. (C) Results are expressed as the mean ± SD from three independent experiments. (a) P < 0.001 versus control mitochon- dria; and (b) P < 0.01 versus AA-CoA treated mitochondria. AA release in a specific compartment of the cells A. F. Castillo et al. 5014 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS knocked down was significantly reduced compared with mitochondria isolated from mock-transfected cells (Fig. 4C). Acot2 knockdown did not produce any effect on progesterone synthesis in mitochondria trea- ted with 22(R)-OH-cholesterol (Fig. 4D). This provides evidence that the reduction in Acot2 expression does not affect mitochondrial integrity. The results described above indicate the necessity of Acot2 in AA-CoA-stimulated steroidogenesis, indica- ting also that the effect of AA-CoA is due to its con- version to AA into the mitochondria. If this is the case, inhibition of AA-CoA uptake into the mitochon- dria should inhibit steroid synthesis. Indeed, we inhib- ited the carnitine-dependent acyl-CoA transport with malonyl-CoA and the stimulatory effect of AA-CoA on mitochondrial steroid synthesis was significantly reduced (Fig. 3C). The requirement of Acot2 for the action of AA- CoA on steroid synthesis suggests the participation of this enzyme in the mitochondrial cAMP-induced AA accumulation. Then, we next tested the effect of Acot2 on mitochondrial [1- 14 C]-AA accumulation induced by 8Br-cAMP, using the same strategy described in Fig. 3C and Fig. 4: inhibition of Acot2 activity and expression, respectively. As shown in Fig. 5, BPB inhibited cAMP induced accumulation of labeled AA into the mitochondria (Fig. 5A,B). In accordance with this effect produced by BPB on AA mitochondrial content, there is an increase in AA-CoA retained in the postmitochondrial fraction of cells treated with this compound (Fig. 5C). The same effect was observed in cells where Acot2 expression was blunted (Fig. 6). Discussion In the present paper, we show for the first time that cAMP can regulate the release of AA in a specialized compartment of the cells, e.g. the mitochondria, and that the fatty acid induces steroid synthesis through a mechanism different from the classical pathway invol- ving the stimulation of StAR protein expression. This biological effect can be seen by the addition of exogen- ous AA to intact MA-10 Leydig cells (300 lm)or isolated mitochondria (200 lm). In determining the Acot2 β -tubulin *** D c R p / iVMC cRp / - i VMC sa 2 to cA AB 22(R)OH-cholesterol Control pRc/CMVi pRc/CMVi- pRc/CMVi pRc/CMVi- Acot2 antisense C a b,c 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 8 4 0 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Acot2 antisense *** *** AA-CoA Control cRp / i V MC c Rp / - i VMC sa 2 t o cA /2 toc A β ni l u b u t - )stinuyrartibra( / )nietorpgm/gn(enoretsegorPenoretsegorP )nie torpgm/gn( Fig. 4. Effect of AA-CoA on progesterone production in isolated mitochondria from Acot2 knockdown MA-10 cells. MA-10 cells were transfected with pRc ⁄ CMVi or pRc ⁄ CMVi-Acot2 antisense cDNA plasmids. After 72 h, mitochondria were isolated from MA- 10 transfected cells. (A) Representative western blot of mitochon- dria from MA-10 transfected cells. The membrane was blotted sequentially with anti-Acot2 and anti-b-tubulin sera. (B) Western blot quantification by densitometry. Bars denote relative levels of Acot2 expression in arbitrary units. *** P < 0.001 versus pRc ⁄ CMVi transfected cells. (C) Mitochondria from MA-10 trans- fected cells were incubated for 20 min at 37 °C in the absence or in the presence of 200 l M AA-CoA. (D) Mitochondria from MA-10 transfected cells were incubated for 20 min at 37 °C in the absence or in the presence of 5 l M 22(R)OH-cholesterol. In (C) and (D), mitochondria were pelleted by centrifugation and progesterone concentrations were measured in the supernatants by RIA. Data are shown as progesterone production (ng ⁄ mg mitochondrial pro- tein) in the incubation media. Results are expressed as the mean ± SD from three independent experiments. In (C), (a) P < 0.001 versus control mitochondria from pRc ⁄ CMVi-transfected cells; (b) P < 0.01 versus control mitochondria from pRc ⁄ CMVi-Acot2 antisense-transfected cells; and (c) P<0.01 versus AA-CoA treated mitochondria from pRc ⁄ CMVi-transfected cells. In (D), *** P < 0.001 versus mitochondria from respective-transfected cells without AA-CoA treatment. A. F. Castillo et al. AA release in a specific compartment of the cells FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5015 effective concentration of AA, we also assess AA’s bio- activity. Although in some cases concentrations between 1 and 10 lm AA are sufficient to detect a bio- logical effect [2], other effects require 100–300 lm [2]. However, at this high concentration, AA may not even be in solution. Previous studies performed with con- centrations of AA of the same magnitude as ours (200 lm) observed either none [30] or a small but signi- ficant [31,32] effect of exogenously added AA on basal steroid synthesis in MA-10 and rat Leydig cells, respectively. In the present paper, we show the stimu- lation of steroidogenesis using 300 lm AA in the pres- ence of albumin. These different results can be explained by the fact that protein binding can increase the overall concentration of fatty acids present in an aqueous environment by effectively decreasing the insoluble fraction [2]. Albumin, in particular, binds specifically to fatty acids [33]. In our experiments, we added albumin in the preparation of the AA solution, which allowed us to detect a significant stimulatory effect of exogenous AA on steroid production of non- stimulated steroidogenic cells (50–60% compared with cAMP). The stimulation of steroid synthesis by exog- enously added AA was lower than the stimulation reached by cAMP or 22(R)-OH-cholesterol. These results are similar to those obtained by us in rat Ley- dig cells and by other authors in the same or other steroidogenic tissues [20,32,34]. When exogenous AA is added together with sub- maximal doses of 8Br-cAMP, there is a synergistic a b Acot2 antisense B AA 8Br-cAMP Control pRc/CMVi pRc/CMVi- pRc/CMVi pRc/CMVi- Acot2 antisense A a b 0 1 2 3 4 5 6 7 control 8Br-cAMP control 8Br-cAMP )stinuyrartibra(A Fig. 6. Effect of Acot2 knockdown on AA accumulation into mito- chondria. MA-10 cells were transfected with pRc ⁄ CMVi or pRc ⁄ CMVi-Acot2 antisense cDNA plasmids as described in Fig. 5. After 72 h, MA-10 transfected cells were labeled and stimulated as described in Fig. 2. (A) Representative autoradiography showing AA spots in mitochondrial fractions. (B) Autoradiography spots quantifi- cation by densitometry. Bars denote levels (in arbitrary units) of AA in mitochondria from MA-10 transfected cells treated with or with- out 8Br-cAMP. Results are expressed as the mean ± SD from three independent experiments. (a) P < 0.01 versus mitochondria from control pRc ⁄ CMVi-transfected cells; and (b) P < 0.001 versus mitochondria from 8Br-cAMP treated pRc ⁄ CMVi antisense trans- fected cells. b a 0 4 8 12 16 20 A Control 8Br-cAMP 8Br-cAMP + BPB B Control 8Br-cAMP 8Br-cAMP + BPB AA b a )stinuyrartibra(A 8Br-cAMP + BPB 0 25 Control 8Br-cAMP [ 14 C]AA-CoA (cpm x10 –3 /mg protein) C 50 *** Fig. 5. Effect of Acot2 activity inhibition on AA accumulation into mitochondria and AA-CoA accumulation in the postmitochondrial fraction. MA-10 cells were labeled as described in Fig. 2. When indicated, cells were incubated with 0.1 m M BPB for 30 min prior to the stimulation with 8Br-cAMP. (A) Representative autoradiogra- phy showing AA spots in mitochondrial fractions. (B) Autoradiogra- phy spots quantification by densitometry. The autoradiographies were quantified by densitometry and the data were normalized against the intensity of the signal of unlabeled AA stained with iod- ine. Bars denote levels (in arbitrary units) of AA in mitochondria. Results are expressed as the mean ± SD from three independent experiments. (a) P < 0.001 versus mitochondria from control cells; (b) P < 0.05 versus mitochondria from 8Br-cAMP-treated cells. (C) AA-CoA content in the postmitochondrial fraction. Data are shown as 14 C-AA-CoA in cpm ⁄ mg protein in the postmitochondrial fraction. Results are expressed as the mean ± SD from three independent experiments. ***P < 0.001 versus control. AA release in a specific compartment of the cells A. F. Castillo et al. 5016 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS effect in which steroid synthesis reaches maximal activation (Fig. 1). This result agrees with that obtained by other authors [35], who suggested that a critical threshold of cAMP or cAMP-dependent pro- tein kinase activation is required for the synergistic effect of AA on cAMP-stimulated StAR protein expression and steroidogenesis. The fact that the cells treated with exogenous AA can be stimulated with 8Br-cAMP indicates that mitochondrial function regarding the regulation of cholesterol metabolism remains intact. Moreover, it is known that any distur- bance or swelling of the mitochondria produces a total loss of regulation, rendering mitochondria that pro- duce full steroidogenesis. In our case, treatment with AA also does not cause loss of the regulation of ster- oid biosynthesis supported by 22(R)-OH-cholesterol (data not shown). This explanation rules out the possi- bility that AA or bovine serum albumin would have effect on mitochondrial integrity. This consideration is also valid when fatty acids or AA-CoA are used in iso- lated mitochondria, as both treatments can still be increased by stimulation with 22(R)-OH-cholesterol (data not shown). As is already known [26,27], the cAMP-dependent transport of cholesterol from the mitochondrial outer to inner membrane can be blocked by a protein syn- thesis inhibitor such as CHX. However, this protein synthesis inhibitor is not totally able to abolish the sti- mulation produced by exogenously added AA. These results strongly suggest that AA can exert a role on cholesterol transport without the induction of StAR protein. The demonstration that AA and ⁄ or AA-CoA stimu- late cholesterol transport in isolated mitochondria sug- gests that the accumulation of AA can occur by direct uptake of AA itself inside the mitochondria or by the previous esterification to AA-CoA by ACS4 and subse- quent action of Acot2 to render free AA in the mito- chondria. The fact that cAMP increases AA uptake into the mitochondria and that this effect on AA accu- mulation is reduced when Acot2 activity or expression are blocked strongly indicates that the operating mechanism is dependent on the concerted action of ACS4 ⁄ Acot2. In this mechanism, cAMP acts to increase AA-CoA formation in the cytosol. The CoA derivative enters the mitochondria through the CPT1- dependent pathway. The specificity of this mechanism to release AA inside the mitochondria is shown by the fact that the content of labeled AA in another organ- elle such as the nucleus is neither increased by cAMP nor reduced by the inhibition of Acot2 (Fig. 2). This is the first time that AA incorporation into a specific subcellular compartment (the mitochondria) has been shown as a consequence of the action of this second messenger. Steroidogenic cells express Acot2 and also a cytosolic isoform, Acot1, which is 92.5% homolog- ous to the mitochondrial enzyme. We have ruled out the possibility that mitochondria would uptake AA produced by the action of Acot1 outside the mitochon- dria, as part of our experiments were performed with isolated mitochondria where Acot1 was not present. Moreover, while the overexpression of Acot2 results in an increase of hormone induced steroid synthesis [8], overexpression of Acot1 does not produce this effect; conversely, it produced a slight inhibition of the pro- cess (data not shown). This last result supports the notion that Acot2 is the thioesterase involved in the release of AA inside the mitochondria. Our model explaining how AA is released into the mitochondria also supports the concept that the select- ive actions of free AA may be explained simply by its specific release under physiological conditions and by the absence of such mechanisms for releasing other long-chain fatty acids, compounds that might otherwise share its biochemical effects. This is demonstrated by the fact that when the mitochondria are stimulated with other fatty acids, the response is lower than with AA; however, there is a significant response of steroid- ogenesis to a different Acyl-CoA (oleoyl-CoA, Fig. 4). Thus, the specificity of the action is not due to the fatty acid itself but to the acyl-CoA available to the mito- chondrial Acot2. In our case, AA-CoA is formed pref- erentially because of the specificity of ACS4 on AA [9]. The mitochondrial inner membrane is not permeable to acyl-CoAs [3]; we wanted to know how AA-CoA reaches the mitochondrial Acot2. The experiment using malonyl-CoA (Fig. 5) indicates that AA-CoA follows the usual pathway involving carnitine-palmitoyl transf- erase 1 (CPT1) [3]. This enzyme plays a central role in mitochondrial fatty acid oxidation. However, in our case, it seems that CPT1 directs AA to another func- tion. In this context, it has been proposed that a potential route for long-chain acyl-CoAs to cross the mitochondrial outer membrane could be the voltage- dependent anion selective channel, also called mitoch- ondrial porin and located in the contact sites [36]. It is very interesting that a protein obligatory for choles- terol transport in steroidogenic cells, the peripheral benzodiazepine receptor (PBR), is also located in the mitochondrial contact sites and includes the voltage- dependent anion selective channel in its structure together with the adenine nucleotide carrier [37]. PBR is involved in cholesterol transport to the cytochrome P450 side chain cleaving enzyme localized on the outer surface of the mitochondrial inner membrane [37]. The endogenous ligand of this receptor is an acyl-CoA A. F. Castillo et al. AA release in a specific compartment of the cells FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS 5017 binding protein known also as diazepam-binding inhib- itor (DBI) [37,38]. It can be postulated that the role of DBI is to facilitate the transport of fatty acids through the mitochondrial outer membrane. The homology between the DBI and an acyl-CoA binding protein cer- tainly enhances this possibility. The specific interaction between DBI and its endogenous receptor, the PBR located on the outer ⁄ inner mitochondrial membrane contact sites [37,38], may direct the AA-CoA to this organelle. Contact sites between the mitochondrial outer and inner membrane could represent the microenviron- ment for bringing the machinery together to transport AA-CoA [37] into the mitochondria and facilitate AA release, which in turn facilitates cholesterol transport. How intramitochondrial AA could stimulate cholesterol transfer from the outer to the inner mitochondrial mem- brane can also be explained by the action of AA on the membrane permeability in the contact sites. This sugges- tion is also in line with experiments showing that AA induces the specific membrane permeability in heart and liver mitochondria by opening the mitochondrial per- meability transition pore [39,40]. The pore is a multiple protein complex located in the mitochondrial contact sites [41] and, in mitochondria of steroidogenic cells, it participates in cholesterol transport. In the present paper, we demonstrate that 20–30% of total steroid production can be elicited without the necessity for StAR synthesis. This is in accordance with the pathological situation where deletions or mutations of StAR are detected in humans born with the steroid deficiency disease, lipoid adrenal congenital hyperplasia [17,20,42–44]. Disruptions of the StAR gene in mice produce similar phenotypes [18,45]. The effect of these deletions establishes that StAR is necessary for 80–90% of adrenal cholesterol metabolism [19,46]. In other words, our results may explain the mechanism by which in these situations there is a remaining 20% of steroid synthesis, due to the direct effect of AA ⁄ AA-CoA produced within the mitochondria by the action of ACS4 ⁄ Acot2 together with DBI ⁄ PBR. Thus, it can be postulated that in the acute phase (early response) of steroid synthesis, the release of AA into the mitochondria is the first stimulator of choles- terol transport. The sustained phase of the acute response will then need the induction of StAR. We cannot exclude that an extraordinarily small amount of intramitochondrial StAR present in resting condi- tions and not detectable by current techniques can contribute to the effect of AA on cholesterol transport in mitochondria. The absence of hormone ⁄ cAMP-induced steroid synthesis when protein synthesis is inhibited can be explained now by the inhibition in the induction of ACS4 [28] during the early response and the inhibi- tion of ACS4 and StAR inductions during the sus- tained phase. In both phases, the presence of DBI ⁄ PBR may be necessary. This new feature in the regulation of cholesterol transport by AA and the release of AA in a specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis, but would also be important in other situations such as the neurosteroid biosynthesis or oncology disorders, where cholesterol transport, ACS4 and PBR play an important role [23–25,47]. Experimental procedures Materials Fatty acid-free bovine serum albumin, AA, arachidic and oleic acids, 4-bromophenacyl bromide (BPB), oleoyl-CoA, malonyl-CoA, 8Br-cAMP, 22(R)-OH-cholesterol, cyclohexi- mide (CHX) and Waymouth MB752 ⁄ 1 cell culture media were purchased from Sigma Chemical Co. (St Louis, MO, USA). Nordihydroguaiaretic acid (NDGA) and AA-CoA were from Fluka (Buchs, Switzerland). Sera, antibiotics and trypsin-EDTA were from Gibco-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 testoster- one as the major steroid. The cells were generously provi- ded by M. Ascoli, University of Iowa, College of Medicine (Iowa City, IA, USA) and were handled as originally des- cribed [48]. Cells were incubated in the presence or absence of 10 lgÆmL )1 CHX for 30 min and then stimulated with 8Br- cAMP (0.2 mm or 0.5 mm), 300 lm AA or 5 lm 22(R)-OH- cholesterol in the culture medium containing 0.1% fatty acid-free bovine serum albumin. Progesterone production was measured by radioimmunoanalysis (RIA) [7], and data are shown as progesterone production (ngÆmL )1 ) in the incubation medium. Preparation of mitochondrial fraction Mitochondria were obtained as previously described [17]. Briefly, all MA-10 cell cultures were washed with phos- phate-buffered saline, scraped in 10 mm Tris ⁄ HCl (pH 7.4), 250 mm sucrose, 0.1 mm EDTA (TSE buffer), homogenized with a Pellet pestle motor homogenizer (Kontes) and centri- fuged at 800 g during 15 min. A second centrifugation at 16 000 g during 15 min rendered a mitochondrial pellet and AA release in a specific compartment of the cells A. F. Castillo et al. 5018 FEBS Journal 273 (2006) 5011–5021 ª 2006 The Authors Journal compilation ª 2006 FEBS a supernatant (postmitochondrial fraction). The mitochond- rial pellet was resuspended in TSE buffer. Progesterone production in isolated mitochondria Thirty microliters of mitochondrial fraction (200 lgof protein) were added to 165 lL of medium consisting of 34 mm Tris ⁄ HCl (pH 7.4), 20 mm KCl, 4 mm MgCl 2 and 108 mm mannitol, containing 0.3% fatty acid-free bovine serum albumin. When indicated, 200 lm AA, 200 lm AA- CoA or 5 lm 22(R)-OH-cholesterol were added. The mix- ture was completed by adding TSE buffer to complete a final reaction volume of 500 lL (fatty acid-free bovine serum albumin final concentration 0.1%). The incubations were carried out at 37 °C for 20 min with gently shaking and were stopped by cooling the tubes in an ice ⁄ water bath. As indicated in each figure, inhibitors such as 10 lgÆmL )1 CHX, 100 lm NDGA, 0.1 mm BPB or 100 lm malonyl- CoA were added to the reaction mixture and preincubated 5 min prior to the addition of AA, AA-CoA or 22(R)-OH- cholesterol. After the incubation time, mitochondria were pelleted by centrifugation 16 000 g for 15 min and progesterone con- centrations were measured in the supernatants by RIA. Data are shown as progesterone production per mg of mitochondrial protein (ngÆmg )1 protein). [1- 14 C]Arachidonic acid incorporation in MA-10 cells MA-10 cells were labeled following a previously described methodology [17], with minor modifications. [ 14 C]-AA (New England Nuclear, Boston, MA, USA; specific activity 53.0 mCiÆmmol )1 ) was added to the cultures in a concentra- tion of 1 lCiÆmL )1 per well (2 · 10 6 cells) in serum-free Waymouth MB752 ⁄ 1 containing 0.5% fatty acid-free bovine serum albumin [17]. After 5 h of incubation at 37 °C in a humidified atmosphere containing 5% CO 2 , the cells were incubated in the presence or absence of 1 mm 8Br-cAMP for 30 min. When indicated, cells were incuba- ted with 0.1 mm BPB for 30 min prior to the stimulation with 8Br-cAMP. After these treatments, the cells were washed with serum- free Waymouth medium containing 0.5% fatty acid-free bovine serum albumin. Nuclear and mitochondrial pellets were obtained as previously described [17] and resuspended in 20 mm Hepes ⁄ KOH (pH 7.4), 250 mm sucrose, 1 mm EDTA, 10 mm KCl and 1.5 mm MgCl 2 containing 500 ng of unlabeled AA, and were then sonicated. Protein concen- tration was measured and lipids were extracted from equal amounts of nuclear or mitochondrial proteins (500 lgin both cases) from each treatment. Lipid extraction was per- formed twice with ethyl acetate (six volumes per one vol- ume of nuclear or mitochondrial fraction). The organic phase was then collected and dried under nitrogen at 25 °C and analyzed by two successive thin-layer chromatographies on silica gel. Radioactive spots were developed using a Storm Phosphorimager (Amersham Biosciences, Sweden) after 1 week of exposition. The postmitochondrial fraction was treated as described for the mitochondria and the [ 14 C]-AA-CoA formation was evaluated by extraction from the aqueous phase according to the literature [49]. Plasmid transfection MA-10 cells were transiently transfected by electroporation as previously described [8,50]. Transfection efficiency varied from 40 to 50% and was estimated by counting fluorescent cells transfected with pRc ⁄ CMVi plasmid [51] containing the enhanced form of green fluorescent protein (EGFP) [8]. MA-10 cells were transfected either with pRc ⁄ CMVi plas- mid containing the Acot2 antisense cDNA [8] or with the empty vector as control. Approximately 72 h after transfec- tion, cells were used as described in the respective figures. SDS/PAGE and immunoblot assay Mitochondrial proteins were separated by SDS ⁄ PAGE (10% acrylamide gels) and electrophoretically transferred to poly(vinylidene difluoride) membranes (Bio-Rad Laborator- ies Inc., Hercules, CA, USA) as described previously [8]. Acot2 protein was detected using anti-Acot2 antibodies [11] and immunoreactive bands were detected by chemilumines- cence using enhanced chemiluminescence reagents (GE Healthcare, Chalfont St Giles, UK). Protein quantification and statistical analysis Protein was quantitated by the method of Bradford [52] using bovine serum albumin as a standard. Statistical ana- lysis was performed by t-test or anova followed by the Student–Newman–Kuels test. Acknowledgements This work was supported in part by National Research Council (CONICET), University of Buenos Aires (UBA) and National Agency of Scientific and Techno- logical Promotion (ANPCyT). 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