Enzymes involved in arachidonic acid release in adrenal and Leydig cells

Molecular and Cellular Endocrinology 265–266 (2007) 113–120

Review

Enzymes involved in arachidonic acid release in adrenal and Leydig cells P. Maloberti, F. Cornejo Maciel, A.F. Castillo, R. Castilla, A. Duarte, M.F. Toledo, F. Meuli, P. Mele, C. Paz, E.J. Podest´a ∗ Department of Biochemistry, School of Medicine, University of Buenos Aires, Paraguay 2155, 5◦ (C1121ABG), Buenos Aires, Argentina

Abstract Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Fatty acids; Acyl-CoA thioesterase; Acyl-CoA synthetase; Mitochondria

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of AA in steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymes involved in AA release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Acyl-CoA thioesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acyl-CoA synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A model of AA release and its action on the regulation of steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Long-chain fatty acids represent a major energy source for many organs, especially for heart and skeletal muscle. Fatty acids are produced by lipolysis, transported bound to albumin in blood and taken up by tissues in a process mediated by transport proteins present in the plasma membrane. Before being directed into storage, membranes or oxidation, fatty acids are first activated to acyl-CoAs. Long-chain-acyl-CoA esters serve as important ∗

Corresponding author. Tel.: +54 11 4508 3672x36; fax: +54 11 4508 3672x31. E-mail address: [email protected] (E.J. Podest´a). 0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.12.026

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intermediate in lipid biosynthesis and fatty acid degradation; however besides this basal function, long-chain-acyl-CoA esters also have an important function in cell signaling (Kerner and Hoppel, 2000). AA is a 20-carbon fatty acid and it is a common constituent of phospholipids in cell membranes. On stimulation of a cell, free AA is released from the cell membrane by the action of phospholipases. The signals that cause the release of AA are not fully understood but appear to include a variety of G protein coupled receptors and a set of second messengers. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase pathway or via the lipoxygenase pathway. How the cells drive AA to these pathways is not fully

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understood. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways (Brash, 2001). Several reports have shown that AA and its lipoxygenasecatalyzed metabolites play an essential role in the regulation of steroidogenesis (Dix et al., 1984; Hunyady et al., 1985; Kojima et al., 1985; Didolkar and Sundaram, 1987; Solano et al., 1988; Didolkar and Sundaram, 1989; Abayasekara et al., 1990; LopezRuiz et al., 1992). This review focuses on the enzymes that regulate the release of AA involved in cholesterol transport from the outer to the inner mitochondrial membrane. 2. Role of AA in steroidogenesis AA and its lipoxygenase-catalyzed products affect steroidogenesis at a point between cAMP-dependent protein phosphorylation and the rate limiting step in the production of steroid hormones-i.e., the metabolism of cholesterol to pregnenolone. The conversion of cholesterol to pregnenolone is limited by the transport of cholesterol from the outer to the inner mitochondrial membrane (Karaboyas and Koritz, 1965; Crivello and Jefcoate, 1980; Privalle et al., 1983; Lambeth et al., 1987). This event is, in turn, controlled by the peripheral-type benzodiazepine receptor (PBR) recently renamed as translocator protein (TSPO) (Papadopoulos et al., 2006) and the steroidogenic acute regulatory (StAR) protein (Stocco, 2000; Papadopoulos et al., 2001; Stocco, 2001; Lacapere and Papadopoulos, 2003). This topic has been well covered in specialized reviews and is also described in this issue of the journal. It is very well accepted that hormonal stimulation of steroid synthesis in adrenal zona fasciculata and glomerulosa cells and testicular Leydig cells involves the release of AA. The subsequent metabolism of AA by the lipoxygenase pathway has also been involved in the regulation of adrenal and Leydig cells steroidogenesis (Kojima et al., 1985; Jones et al., 1987; Nadler et al., 1987; Tamura et al., 1987; Mikami et al., 1990; Nishikawa et al., 1994). In Leydig cells, AA is released within one minute following stimulation with lutropin, (Cooke et al., 1991; Moraga et al., 1997). AA release also occurs in a dose and time dependent manner in human chorionic gonadotropin-stimulated Leydig cells (Moraga et al., 1997) and in adrenocorticotropin-stimulated adrenal cells (Solano et al., 1988). Inhibition of AA release abrogates the effect of lutropin – and adrenocorticotropin – on the stimulation of steroid production (Kojima et al., 1985; Didolkar and Sundaram, 1987; Abayasekara et al., 1990; Mele et al., 1996). In adrenal zona fasciculata cells, it is possible to demonstrate a direct effect of 5-and 15-lipoxygenase metabolites on the regulation of pregnenolone synthesis (Nishikawa et al., 1994). In fact, adrenocortical tissue possesses the 5- and 15-lipoxygenase pathways and generate 5- and 15-hydroxyeicosatetraenoate (5-HETE and 15-HETE) from AA (Omura et al., 1990). In glomerulosa cells, angiotensin II increases 15-HETE and 12HETE levels, the 12-HETE seems to play a key role in the angiotensin II-induced aldosterone secretion but not in the ACTH signaling pathway (Nadler et al., 1987). Lipoxygenated

metabolites act through induction of StAR protein. The differences in the formation and action of these metabolites depending on the cellular type and the stimulating hormone may be due to the access of AA to the different lipoxygenases and/or to the access of the lipoxygenated products to StAR gene. The involvement of epoxygenase metabolites of AA in cAMPstimulated steroidogenesis and StAR gene expression has also been described recently by Wang et al. (2006). 3. Enzymes involved in AA release For some years it was thought that the phospholipase A2 (PLA2) was the enzyme involved in the release of AA regulated by trophic hormones such as lutropin and adrenocorticotropin. This thinking was supported by the fact that the usual inhibitors of the PLA2 such as dexamethasone, 4-bromophenacil bromide (BPB) and quinacrine were able to abolish the stimulation of steroid synthesis induced by the respective trophic hormones. However, there is no direct evidence showing that AA is released by PLA2 in steroidogenic tissues nor is there evidence showing that steroidogenic hormones activate the PLA2. Moreover, a new specific inhibitor of PLA2, ATK, was not able to inhibit hormone-induced steroid synthesis in Leydig or adrenal cells (unpublished results). These observations suggested that another type of mechanism of AA release should operate during the regulation of steroid biosynthesis triggered by trophic hormones. In this regard, our laboratory showed a new mechanism that controls the level of free AA in steroidogenic cells (Maloberti et al., 2002; Maloberti et al., 2005). This hormoneregulated mechanism involves the concerted action of two enzymes, an acyl-CoA synthetase and a mitochondrial acyl-CoA thioesterase. The steroidogenic hormones regulate both enzymes: while the acyl-CoA thioesterase is activated by phosphorylation and substrate availability (Maloberti et al., 2000), a synthetase named ACS4 is rapidly induced after hormone treatment (Cornejo Maciel et al., 2005). Recent experiments show that steroid production is inhibited in Y1 and MA-10 cells by silencing the expression of either, the acyl-CoA thioesterase or the synthetase ACS4 (Cornejo Maciel et al., 2005; Maloberti et al., 2005). These results led us to propose that hormone-induced ACS4 acts by sequestering free AA forming an intracellular pool of arachidonoyl-CoA that is delivered to the mitochondrial acylCoA thioesterase which, in turn, will release AA in a specific compartment of the cell to increase StAR and steroidogenesis. 4. Mitochondrial Acyl-CoA thioesterase Searching for the phosphoprotein involved in steroid synthesis through the release of AA, we identified a 43 kDa phosphoprotein (Neher et al., 1982; Dada et al., 1991; Paz et al., 1994; Mele et al., 1997). The activity of the protein was dependent on cAMP and cAMP-dependent protein kinase. Adrenocorticotropin treatment of rat adrenal glands resulted in the appearance of multiple phosphorylated forms of the protein which were sensitive to acid phosphatase treatment. Therefore,

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it was concluded that the activity of p43 and its state of phosphorylation were dependent on adrenocorticotropin. Further cloning and sequencing of a cDNA encoding p43 revealed its primary structure (Finkielstein et al., 1998). 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) (Lindquist et al., 1998; Svensson et al., 1998). CTE-I and MTE-I are members of an acyl-CoA thioesterase family with very long chain and long chain acyl-CoA thioesterase activity (Lindquist et al., 1998; Svensson et al., 1998). The family includes four isoforms with different subcellular locations and a high degree of homology (Hunt et al., 1999): a cytosolic (CTE-I), a mitochondrial (MTE-I) and peroxisomal forms (PTE-Ia and Ib) of the enzyme. Recently, this gene family was cloned and characterized in mouse showing that all isoforms are encoded by three exons spaced by two introns (Hunt et al., 1999; Hunt and Alexson, 2002). According to its mitochondrial localization, the sequence of MTE-I includes a mitochondrial leader peptide. Like StAR, MTE-I is targeted to the inner mitochondrial membrane (Stocco and Clark, 1996; Finkielstein et al., 1998; Svensson et al., 1998). The sequence of the phosphoprotein shows consensus sites for protein kinase A, protein kinase C, calcium-calmodulindependent kinase and MAP kinases. 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 (Finkielstein et al., 1998). Inhibition of pituitary-adrenal axis with dexamethasone produced a dose-dependent decrease in the abundance of rat adrenal transcript. A lipase serine motif is contained in the sequence as well as a Gly-Xaa-His motif close to the C-terminal region which has been shown to be necessary for the hydrolytic activity of the thioesterase. Antibodies raised against a synthetic peptide that includes the lipase serine motif blocked the activity of the enzyme (Finkielstein et al., 1998). 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. Acyl-CoA thioesterases (EC 3.1.2.1. and EC 3.1.2.2.) are enzymes that catalyze the hydrolysis of CoA esters of various molecules to the free acid plus coenzyme A (CoA) (Hunt and Alexson, 2002; Yamada, 2005). These enzymes have also been referred to in the literature as acyl-CoA hydrolases, acyl-CoA thioester hydrolases and palmitoyl-CoA hydrolases. Although the functions for many of the acyl-CoA thioesterases in this gene family are not fully understood, they are considered to regulate intracellular levels of CoA esters, the corresponding free acid and CoA-SH and, consequently, cellular processes involving these compounds. Over the years, several different groups have identified and cloned unrelated acyl-CoA thioesterases, which has led to many inconsistencies regarding the nomenclature in the literature. In view of this, we have put together a short article (Hunt et al., 2005), with the revised and approved nomenclature for the acyl-CoA thioesterase gene family in human, mouse and rat, to help avoid confusion in this field. This nomenclature has been carried out in co-operation with the HUGO Gene Nomenclature Committee (HGNC) and the Mouse Genomic

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Nomenclature Committee (MGNC) and proposes the use of ACOT as the root symbol for the acyl-CoA thioesterase gene family. Therefore we will continue with this review calling Acot2 and Acot1 for the MTE-1 (ARTISt) and CTE-1, respectively (Hunt et al., 2005). We demonstrated that recombinant Acot2 and Acot1 release AA from arachidonoyl-CoA in vitro and that adrenocorticotropin increases the activity of the endogenous enzyme and promotes AA release from arachidonoyl-CoA. We also demonstrated that inhibitors of AA release usually used to inhibit the PLA2 are effective inhibitors of recombinant Acot2 and Acot1. The inhibitor of AA metabolism nordihydroguaiaretic acid (NDGA) was also an effective inhibitor of recombinant Acot2 and Acot1. A possible explanation for the effect of classical PLA2 inhibitors on thioesterase activity could be the presence of a serine-histidine-aspartic acid catalytic triad in the active site of both, the thioesterase and other ␣/␤ hydrolases, as determined by site-directed mutagenesis (Huhtinen et al., 2002). 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 (Finkielstein et al., 1998). These observations are also in agreement with previous results showing that BPB, another PLA2 inhibitor, also blocks the activity of Acot2 and Acot1 (Maloberti et al., 2000). In addition, another specific inhibitor of PLA2, ATK, at low concentration was ineffective to inhibit the thioesterase activity of Acot2 (data not shown). These results further support the concept that in steroidogenic cells, the mechanism of AA release does not operate through the activation of the PLA2 pathway. Dexamethasone, commonly used as PLA2 inhibitor and steroid synthesis inhibitor, is ineffective to inhibit the thioesterases; however dexamethasone produces a dose-dependent decrease in the abundance of Acot2 adrenal transcript and produces an inhibition in the induction of an acyl-CoA synthetase that works in a concerted fashion with the thioesterase to release AA (the role of the acyl-CoA synthetase will be discuss bellow). Reduction in the expression of the Acot2 by antisense or siRNA and of the ACS4 by siRNA produced a marked reduction in the steroid output of the cAMP stimulated Leydig cells (Maloberti et al., 2005). Knock down of the expression of Acot2 lead to a significant reduction in the expression of the StAR protein. Overexpression of Acot2 resulted in an increase of cAMP-induced steroidogenesis. On the other hand, overexpression of the cytosolic form 92% homologous to Acot2 did not affect this parameter (Castillo et al., 2006). In summary, all the studies described above are the first 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. 5. Acyl-CoA synthetase The second enzyme involved in AA release in steroidogenic tissues, the acyl-CoA synthetase, is an enzyme designed ACS4 that belongs to a five-member family. ACS4 shares 68% of its

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amino acid sequence with ACS3, another member of this family, although this sequence is poorly related to the other family members (Kang et al., 1997; Mashek et al., 2004). The purified enzyme utilizes arachidonate as substrate most preferentially among other C8–C22 saturated fatty acids and C4–C22 unsaturated fatty acids. The striking feature of ACS4 is its abundance in steroidogenic tissues, especially adrenal gland and ovary. ACS4 immunoreactivity was detected in the zona fasciculata (ZF) and reticularis of the adrenal cortex, in the corpus luteum and stromal luteinized cells of the ovary and in the Leydig cells of the testis (Kang et al., 1997). Interestingly, the activity of this protein appears to be essential for StAR induction. Hormone stimulation of AA release, StAR induction and steroid production through the cAMPdependent phosphorylation involves the new synthesis of ACS4 as an early step (Cornejo Maciel et al., 2005). The data supporting this hypothesis are: (i) the inhibition of protein synthesis rapidly decreases basal levels of ACS4, (ii) ACS4 is rapidly induced by hormones through a cAMP-dependent process in vivo and in vitro (ZF and ZG of adrenal tissue, ZF cells, Y1 adrenal cells and MA10 Leydig cells), (iii) the acute increase in protein levels seems to be due to an increase in protein synthesis rather than to a decrease in protein degradation or an increase in mRNA levels, (iv) exogenous AA is capable of restoring StAR induction and steroidogenic activity of cells treated with the ACS4 activity inhibitor triacsin C, (v) ACS4targeted siRNA reduces both ACS4 and StAR protein levels, and (vi) ACS4-targeted siRNA results also in a decreased steroid production, an effect that is reversed by exogenous AA. According to this model, ACS4 induction should be an early event in hormone action. In fact, we demonstrated a rapid action (5 min) of hormone treatment on ACS4 induction. This protein is newly synthesized since 30 min of incubation with the proteinsynthesis inhibitor prior to hormone treatment is sufficient to abolish the induction of ACS4. These results are confirmed by 35 S-methionine incorporation experiments. Moreover, in the presence of cycloheximide, both basal and stimulated protein levels decrease rapidly, suggesting that ACS4 is a high turnover protein. The acute effect of the hormone is exerted at the level of protein synthesis, since mRNA levels can be stimulated only after a longer time of stimulation (Castillo et al., 2006). The results suggest a hormone regulation of arachidonoyl-CoA (AACoA) levels through the control of ACS4 induction. The fact that ACS4 is a protein with a high turnover is in agreement with the fact that fatty acyl-CoA esters need a tight control of their intracellular concentrations since they are important intermediates in lipid metabolism and signal molecules, being strong detergents at the same time. On the other hand, the induction of the acyl-CoA thioesterase Acot2 appears not to be controlled by hormone treatment. Previous observations showed that adrenocorticotropin stimulates Acot2 activity in Y1 cells (Maloberti et al., 2002). The thioesterase activity can be regulated by PKAdependent phosphorylation (Maloberti et al., 2000), however, it also requires an acyl-CoA pool as a source of AA. Therefore we cannot rule out a possible activation of Acot2 by a hormoneincreased availability of its substrate, concept supported by the fact that ACS4 protein level is regulated by hormone treatment.

Since StAR protein was demonstrated to play a critical role in the cholesterol transfer to the mitochondrial inner membrane, our results are in agreement with earlier experiments suggesting that AA regulates steroidogenesis at the rate-limiting step of mitochondrial cholesterol transfer (Wang et al., 2000). During the first six hours of stimulation, the steroidogenesis is supported by two different mechanisms. One of them does not need transcription but is dependent on protein synthesis where ACS4 and StAR are two key proteins. In this process, AA plays a role on the regulation of StAR translation. In the second mechanism, there is a necessity of ACS4 and StAR transcription. Very recently, we demonstrated that 20–30% of total steroid production can be elicited without the necessity of StAR synthesis. 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 cholesterol transport. The sustained phase of the acute response will then need the induction of StAR protein and then the regulation of StAR transcription. It has been suggested that the intracellular concentration of unbound acyl-CoA esters is tightly controlled by the presence of specific acyl-CoA binding proteins (ACBP) and acyl-CoA thioesterases (Knudsen et al., 1989; Faergeman and Knudsen, 1997). In such a cellular environment, transport by diffusion of unbound acyl-CoA esters is very unlikely, suggesting that supply of substrate to acyl-CoA-consuming enzymes depends on direct transfer from acyl-CoA synthetases, or relies on ACBPs or carrier proteins. In this regard, it is known that an ACBP known also as DBI (diazepam binding inhibitor) is expressed in high concentration in specialized cells such as steroid producing cells of the adrenal cortex and testis (Papadopoulos, 1993). Thus, it is possible that after the hormone induction of the acyl-CoA synthetase, the AA-CoA binds to DBI, which in turn binds to the TSPO located in the outer mitochondrial membrane (Papadopoulos, 1993). This will possibly lead to a facilitated transfer of AACoA to the mitochondria, with the consequent availability of the substrate for the thioesterase at its site of action. Regarding other acyl-CoA synthetases, a gonadotropinregulated long chain acyl-CoA synthetase (GR-LACS) was described (Li et al., 2006). This is a 79 kDa protein and its mode of regulation suggests that it participates in testicular steroidogenesis. GR-LACS is constituitively expressed in rat testicular Leydig cells of pubertal and adult rats and is down regulated during desensitization of steroidogenic enzymes by gonadotropins (Tang et al., 2001). Then, the authors proposed that GR-LACS contributes to the provision of energy requirements and biosynthesis of steroid precursors in the male gonad. Since it is expressed in other steroidogenic tissues (rodent gonads, ovary and brain, and only in the mouse in the adrenal cortex), this enzyme could exert tissue-specific functions by generating specific fatty acyl-CoA thioesters for participation in anabolic and catabolic reactions. GR-LACS may modulate cellular metabolism in a tissue- and species-specific manner to regulate steroidogenesis of the testis and adrenal gland, and participate in follicular atresia and as yet unidentified functions in the brain (Li et al., 2006). Another important issue is the origin of cytosolic free AA to be esterified into AA-CoA. As already mentioned, AA could

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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 lipoproteins (HDL) (Gwynne and Hess, 1980; Andersen and Dietschy, 1981). In adrenocortical cells, HDL enhances steroid production and increases cellular cholesterol content. Rat HDL contains a high amount of arachidonate esterifying its cholesterol (Kraemer et al., 2004). In addition, using several different experimental approaches, there is evidence that a hormone sensitive lipase could play a critical role in maintaining cholesterol homeostasis in the adrenal gland. Moreover, evidence is presented to suggest that the hormone sensitive lipase is functionally linked to the selective pathway of cholesterol delivery and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis. Given that the selective delivery of cholesteryl esters from lipoproteins and stores of cellular cholesteryl esters constitute the major sources of cholesterol for steroid production, it is possible to speculate that cholesteryl esters could be the source of cholesterol and also for the source of AA to be esterified by the ACS4 enzyme. Taken together, the current data indicate the presence of a new hormone-dependent labile protein in steroidogenic tissues (ACS4) essential for AA release, StAR induction and steroidogenesis. 6. A model of AA release and its action on the regulation of steroidogenesis Although it was known that AA and its metabolites work at a point between cAMP-dependent protein phosphorylation and the metabolism of cholesterol to pregnenolone (Solano et al., 1988), the mechanism for its role was recently described. AA and its metabolites are involved in the regulation of the StAR protein expression (Wang et al., 2000). In fact, the results indicate that 5-lipoxygenase metabolites of AA are involved in trophic hormone-induced signaling and are stimulatory in StAR gene expression and that AA-response element resides in the −67/−96 region on the StAR promoter (Wang et al., 2000; Wang et al., 2003). With this knowledge and the recent evidence that steroidogenenesis activating pathway involves the release of AA into the mitochondria, two questions arise:

in specific compartments of the cell (Faergeman and Knudsen, 1997). The simple structure of AA and the natural occurrence of so many close chemical analogues are, not surprisingly, associated 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 biochemical effects (Brash, 2001). Then, the accessibility of AA to a specific cellular compartment and the specificity of its action are certainly linked. Therefore, the questions mentioned above can be answered by the fact that both, AA and AA-CoA have to be compartmentalized to exert their specific functions. Indeed, when isolated mitochondria are stimulated with several fatty acids different from AA, the response in cholesterol transport is lower than with AA; however the steroidogenic response is similar when the mitochondria are stimulated with different Acyl-CoAs (AA-CoA and oleoyl-CoA) (Castillo et al., 2006). Then, the specificity of the action is not due to the fatty acid itself but to the acyl-CoA available to the mitochondrial Acot2. In the case of steroidogenesis, AA-CoA is preferentially formed due to the specificity of ACS4 on AA (Kang et al., 1997). This is in agreement with the fact that very recently, we showed for the first time that cAMP can regulate the release of AA in specialized compartment of the cells, the mitochondria. cAMP induced-AA release into the mitochondria is reduced when the mitochondrial thioesterase Acot2 activity or expression is blocked (Castillo et al., 2006). These results provide evidence to suggest that AA is needed in a special compartment of the cell (e.g. mitochondria). It is known that the acyl-CoA binding protein DBI is expressed in high concentrations in specialized cells such as steroid producing cells of the adrenal cortex and testis (Gwynne and Hess, 1980; Knudsen et al., 1989). Thus, it can be proposed that arachidonoyl-CoA binds to DBI, which in turn binds to the TSPO located in the outer mitochondrial membrane (Gwynne and Hess, 1980; Papadopoulos, 1998). This would possibly lead to facilitate transfer of arachidonoyl-CoA into the mitochondria. Our current data indicate the presence of a new pathway that regulates intracellular levels of AA (Fig. 1), in which there should be an acyl-CoA synthetase that could act by sequestering free AA by esterification into arachidonoyl-CoA. Esterified AA may bind ACBP/DBI thus forming an intracellular pool

(1) Why free cytosolic AA has to be re-esterified by the action of ACS4 to provide arachidonoyl-CoA to be substrate of the mitochondrial thioesterase to release AA into the mitochondria? Why do not the cells use directly the cytosolic pool of free AA? (2) Why AA has to be released into the mitochondria by the mitochondrial thioesterase to stimulate steroidogenesis? The compartmentalization 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 (Faergeman and Knudsen, 1997). The high degree of sequestration of AA into long chain acyl-CoA suggests that this long chain fatty acid is likely to become limiting for diverse roles

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Fig. 1. The proposed model for AA release into the mitochondria.

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that could then be delivered to the mitochondrial acyl-CoA thioesterase, which will, in turn, release AA in a specific compartment of the cell upon hormone treatment. Another question then arises as to how free mitochondrial AA stimulates steroidogenesis. There are two answers to this question. The first one is the demonstration that the release of AA into the mitochondria is necessary to trigger the initial phase in cholesterol transport without the necessity of StAR induction (mentioned above in the acyl-CoA section and recently published, (Castillo et al., 2006)). The second answer is based on the necessity of transformation of AA in lipoxygenated products for the induction of the StAR protein. These two options are also included in Fig. 1. It is known that the lipoxygenase enzyme is located in the membranes of the endoplasmic reticulum and nucleus and it is also associated with the mitochondria. The contacts between mitochondria and endoplasmic reticulum play an important function in cell metabolism; for example, they secure a direct calcium transmission from the endoplasmic reticulum to the mitochondria (Rizzuto et al., 1998). This could be also the case to secure a direct transmission of AA to the site of action of the lipoxygenase and may be an explanation to the question as how the cells drive AA via the cycloxygenase pathway or via the lipoxygenase pathway. In this context, the existence of a long chain fatty acid generation and export system has been described in mitochondria from rat heart in which Acot2 and the adenine nucleotide translocase (ANT) are involved (Gerber et al., 2006). Mitochondrial cholesterol transport requires the choreographed expression of diverse proteins such as TSPO, StAR, ACS4 and Acot2. Mitochondria have been shown to be in constant movement within the cells and it was demonstrated that mitochondrial movement can be induced after steroidogenic hormone action. It is very interesting that TSPO, a protein obligatory for cholesterol transport, is located in the mitochondrial contact sites and in the interaction between the mitochondria and the endoplasmic reticulum. The TSPO includes the voltage dependent anion selective channel (VDAC), also called mitochondrial porin (Papadopoulos et al., 2006) and ANT. Porin occurs at high density in the mitochondrial outer membrane and regulates the permeability of this membrane to ions and metabolites. As it was mentioned, it has been suggested that the endogenous ligand of TSPO, DBI, may facilitate the transport of fatty acids through the mitochondrial outer membrane (Kerner and Hoppel, 2000). Mitochondria are important dynamic organelles for cell survival and functions. Mitochondrial dynamic and biogenesis may be involved in the control of cell metabolism and signaling transduction. Identification of the topography of the outer surface of the contact site, the organization and orientation of the putative components of the contact sites and the relation with the endoplasmic reticulum and the nucleus will establish whether contact sites provide essential structural organization for the action of AA, the activity of the lipoxygenase enzyme and the action of TSPO and StAR on cholesterol transport. What is the nature of the contact between the mitochondria and the endoplasmic reticulum? Is the mitochondrial permeability transition pore in the proximity of these contacts? What is

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