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J. Biol. Chem., Vol. 275, Issue 26, 20204-20209, June 30, 2000

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The Role of Arachidonic Acid in Steroidogenesis and Steroidogenic Acute Regulatory (StAR) Gene and Protein Expression^*

XingJia Wang, Lance P. Walsh, Adam J. Reinhart, and Douglas M. Stocco

From the Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Received for publication, April 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study was conducted to examine the mechanism for arachidonic acid (AA) regulation of steroidogenic acute regulatory (StAR) protein expression and the relationship between AA and cAMP in hormone-induced steroidogenesis. Dibutyryl cyclic AMP (Bt₂cAMP)-stimulated MA-10 Leydig cells were treated with AA and/or the phospholipase A₂ inhibitor, dexamethasone. Dexamethasone significantly reduced Bt₂cAMP-stimulated progesterone production, StAR promoter activity, StAR mRNA, and StAR protein. The inhibitory effects of dexamethasone were reversed by the addition of 150 µM AA to MA-10 cells. In addition, MA-10 cells were treated with the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the 5-lipoxygenase inhibitor, AA861, the epoxygenase inhibitor, miconazole, and the cyclooxygenase inhibitor, indomethacin. Both NDGA and AA861 inhibited progesterone production and StAR protein expression. AA861-inhibited progesterone synthesis and StAR protein were partially reversed by addition of the 5- lipoxygenase metabolite, 5(S)-hydroperoxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid. Inhibition of epoxygenase activity inhibited progesterone production significantly, but StAR protein was only slightly reduced. Indomethacin enhanced StAR protein expression and significantly increased progesterone production. Inhibition of AA release or lipoxygenase activities did not affect protein kinase A activity, whereas inhibition of protein kinase A activity using H89 reduced Bt₂cAMP-induced StAR protein. AA alone did not induce StAR protein expression nor steroid production. These results demonstrate the essential role of AA in steroid biosynthesis and StAR gene transcription and suggest the possible involvement of the lipoxygenase pathway in steroidogenesis. This study further indicates that AA and cAMP transduce signals from trophic hormone receptors to the nucleus through two separate pathways and act to co-regulate steroid production and StAR gene expression and indicates that both pathways are required for trophic hormone-stimulated steroidogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The steroidogenic acute regulatory protein (StAR)¹ plays a critical role in trophic hormone-stimulated steroid biosynthesis by facilitating the transfer of cholesterol, the substrate for all steroid hormones, to the inner mitochondrial membrane where it is converted to pregnenolone by the P450 side chain cleavage enzyme (P450scc) (1-4). However, the manner in which trophic hormones regulate StAR gene expression is not entirely clear. It is generally accepted that trophic hormones, such as luteinizing hormone (LH) or adrenal corticotrophic hormone stimulate cyclic AMP (cAMP) formation followed by activation of protein kinase A (PKA) which in turn phosphorylates known transcription factors and possibly additional unknown factors which regulate StAR gene transcription (5). However, it is also known that trophic hormones induce arachidonic acid (AA) release in steroidogenic cells and that this release is a requisite for steroid biosynthesis. Following LH stimulation, AA was released within 1 min in rat testicular Leydig cells (6). Also, AA release occurred in a dose- and time-dependent manner in human chorionic gonadotrophin-stimulated Leydig cells, with the amount of AA released being dependent on hormone-receptor interaction and the concentration of LH/human chorionic gonadotrophin-binding sites on the cell surface (7). Recently, an adrenal corticotrophic hormone-induced 43-kDa protein, named arachidonic acid-related thioesterase involved in steroidogenesis (ARTISt), was identified and cloned from rat adrenal cells. ARTISt demonstrated the highest substrate specificity when assayed with arachidonyl-CoA and was reported to play a role in steroid synthesis by regulating AA release from arachidonyl-CoA (8).

The role of AA in trophic hormone-stimulated steroid production in various steroidogenic cells has been well documented over the last 20 years (9-13); however, the mechanism responsible for the role of AA remains unknown. Since previous studies suggested that AA and its metabolites act at the rate-limiting step of steroid biosynthesis, cholesterol transfer to the inner mitochondrial membrane (14, 15), we reasoned that the StAR protein might be involved in the role of AA in the regulation of steroidogenesis. This hypothesis was proven correct by our recent studies, which indicated that AA release is essential for StAR protein expression (16). Inhibition of AA release inhibited LH- or dibutyryl cyclic AMP (Bt₂cAMP)-induced progesterone production and StAR protein expression in MA-10 mouse Leydig tumor cells (16). Importantly, the inhibitory effects were reversed by the addition of AA into the cell culture with progesterone synthesis and StAR protein being increased as AA in the culture medium was increased. These studies suggest that AA regulated steroid hormone production through its regulation of StAR protein expression. However, the manner in which AA regulates StAR protein expression is not clear. Also, the relationship between AA and cAMP in steroid production and StAR protein expression is unknown. In the present study, we demonstrate that AA regulates StAR gene expression at the level of transcription and that the AA-mediated signal transduction pathway is different from the reported cAMP-PKA-phosphorylation pathway. We also once again demonstrate that both pathways are required for trophic hormone-stimulated steroid production and StAR gene expression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- Arachidonic acid, dexamethasone, nordihydroguaiaretic acid (NDGA), AA861, 5(S)-hydroperoxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HPETE), miconazole, indomethacin, Bt₂cAMP, and bovine serum albumin were purchased from Sigma. H89 was obtained from Calbiochem. Waymouth's MB/752 medium, horse serum, trypsin-EDTA, antibiotics, and PBS were purchased from Life Technologies, Inc. Rabbit antisera generated against a StAR fusion protein was a generous gift from Dr. D. B. Hales (Dept. of Physiology and Biophysics, University of Illinois, Chicago). Donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase was purchased from Amersham Pharmacia Biotech. [³²P]dCTP and [-³²P]dATP were purchased from NEN Life Science Products. Prime-It II kit for ³²P labeling was obtained from Stratagene (La Jolla, CA). The Dual Luciferase Reporter Assay System was purchased from Promega (Madison, WI). Other common chemicals used in these studies were obtained from either Sigma or Fisher.

Cell Culture-- The MA-10 mouse Leydig tumor cells were a generous gift from Dr. Mario Ascoli (Dept. of Pharmacology, University of Iowa, College of Medicine, Iowa City) and were grown in 100-mm tissue culture dishes in Waymouth's MB/752 medium containing 15% horse serum as described previously (17). The cells were cultured in incubators at 37 °C and 5% CO₂. Before each experiment, the medium was replaced with serum-free Waymouth's medium.

Steroid Production-- MA-10 cells were cultured for 30 min in serum-free Waymouth's medium containing AA, and/or its metabolic inhibitors (as described in the figure legends), and then stimulated with 0.5 mM Bt₂cAMP for 6 h. The medium was collected at the end of each experiment and stored at 80 °C. The cells were washed with cold PBS and stored at 80 °C. Progesterone concentrations in the medium were determined by RIA (18).

MA-10 Cell Transfection-- MA-10 cells (0.5 × 10⁶ per well) were cultured in 6-well plates overnight. The cells were transfected with 12 µg of DNA of the StAR promoter/luciferase plasmid expressing firefly luciferase driven by the 966-bp sequence of the StAR promoter (19). Transfections also included 75 ng of the pRL-SV40 vector DNA (a plasmid which constitutively expresses Renilla luciferase, a control reporter under the control of the SV40 promoter, Promega, Madison, WI). Transfections were performed using SuperFect Transfection Reagent, (Qiagen, Valencia, CA), following the manufacturer's instructions. After 48 h in culture the cells were used for experiments.

Luciferase Assay-- Following experiments, the cells were washed three times with ice-cold PBS, and 300 µl of reporter lysis buffer (Promega, Madison, WI) was added to each well. The cells were scraped into a tube and centrifuged for 30 s at 14,000 × g at 4 °C. The supernatants were used for luciferase assays using a Dual Luciferase Reporter Assay System following the manufacturer's instructions (Promega, Madison, WI). The relative light units (expressed as the reading from the StAR promoter/luciferase divided by the reading from Renilla luciferase) were measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).

Protein Kinase A Activity Assay-- Protein kinase A (PKA) activity was measured using the SignaTECT^TM cAMP-dependent Protein Kinase Assay System (Promega, Madison, WI). This assay measures the transfer of ³²P to the biotinylated PKA peptide substrate, Kemptide (LRRASLG). Following treatment, cells were collected in extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin), homogenized at 1000 rpm for 25 passes using a Potter-Elvehjem homogenizer fitted with a Teflon pestle, and centrifuged at 13,000 × g for 10 min. The supernatant was used for both the PKA assay and protein measurement by the Bradford method (20). Approximately 3 µg of protein was incubated for 10 min at 30 °C in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl, 0.1 mg/ml bovine serum albumin, 0.1 mM biotinylated Kemptide, 0.1 mM ATP and 0.02 µCi/µl [-³²P]dATP (specific activity, 3000 Ci/mmol; NEN Life Science Products). Reactions were stopped by the addition of 7.5 M guanidine HCl. Ten microliters of the reaction mix were spotted onto a streptavidin-coated membrane that specifically binds biotinylated Kemptide. Unincorporated [-³²P]dATP was then removed by extensive washing. The incorporation of ³²P into biotinylated Kemptide bound to the membrane was determined by liquid scintillation counting. PKA activity was expressed as picomoles of ³²P incorporated per min/mg of protein.

Northern Analyses-- Cells were washed three times with cold PBS and used for total RNA purification using TRIzol reagent following the manufacturer's instructions (Life Technologies, Inc.). The RNA was separated by electrophoresis in an agarose-formaldehyde gel (1:6%) and blotted onto a Hybond-N⁺ membrane (Amersham Pharmacia Biotech). StAR mRNA on the membrane was probed with ³²P-labeled mouse StAR cDNA. Autoradiography was performed using Hyperfilm (Amersham Pharmacia Biotech). The membrane was stripped with a buffer containing 15 mM NaCl, 1.5 mM sodium citrate, and 1% SDS, pH 7.0, for 30 min at 55 °C, and 18 S rRNA on the membrane was probed to adjust for the RNA loading in each lane.

Western Blot Analysis-- StAR protein expression in MA-10 cells was detected by Western blot analysis as described previously (21). Western analysis experiments were performed at least three times, and the results of one representative experiment is shown.

Statistical Analyses-- Each experiment was repeated at least three times. Statistical analysis of the data was performed with analysis of variance using Stat View SE system (Abacus Concepts, Berkeley, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of Arachidonic Acid on StAR Protein Expression and Steroid Production-- To determine the effects of AA release on StAR protein expression and steroid hormone production, Bt₂cAMP-stimulated MA-10 mouse Leydig cells were treated with the PLA₂ inhibitor, dexamethasone, which inhibits AA release from phospholipids. As shown in Fig. 1, increasing levels of dexamethasone from 0 to 1.0 µM resulted in a dose-dependent decrease in StAR protein expression and a concomitant decrease in progesterone production. To test if dexamethasone could also act by inhibiting the activities of P450scc and/or 3-hydroxysteroid dehydrogenase, 25 µM 22(R)-hydroxycholesterol was added to each treatment. There was no significant difference in progesterone production among the treatments when 22(R)-hydroxycholesterol was added to the culture medium. In order to confirm that the inhibitory effects of dexamethasone were due to the inhibition of AA release, 150 µM AA was added to the Bt₂cAMP-stimulated MA-10 cells treated with 1.0 µM dexamethasone. Whereas Bt₂cAMP-stimulated StAR protein expression and steroid production were significantly inhibited by dexamethasone (p < 0.01), addition of AA into the culture medium reversed the inhibitory effects (Fig. 2). AA in the absence of Bt₂cAMP did not increase either StAR protein or progesterone production significantly.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Inhibitory effects of the PLA2 inhibitor, dexamethasone, on Bt2cAMP-stimulated StAR protein expression and progesterone production in MA-10 Leydig cells. Cells were cultured for 30 min in serum-free Waymouth's MB/752 medium containing various concentrations of dexamethasone as indicated in the figure and stimulated with 0.5 mM Bt2cAMP for 6 h. A, the cells were collected for mitochondria isolation, and 25 µg of mitochondrial protein was used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated using the BioImage Visage 2000 and expressed as integrated optical density (IOD). B, progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production among the treatments. To test the effect of dexamethasone on activities of P450scc and 3-hydroxysteroid dehydrogenase, 25 µM of 22(R)hydroxycholesterol was added to the each treatment for 6 h, and the medium was collected for progesterone assay. *, significantly different from Bt2cAMP (dbcAMP) stimulation (p < 0.05); **, highly significantly different from Bt2cAMP stimulation (p < 0.01). ***, very highly significantly different from Bt2cAMP stimulation (p < 0.001).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Arachidonic acid reversal of the inhibitory effects of the PLA2 inhibitor, dexamethasone, on Bt2cAMP-stimulated StAR protein expression and progesterone production in MA-10 Leydig cells. Cells were cultured for 30 min in serum-free Waymouth's MB/752 medium with or without 1.0 µM dexamethasone or 150 µM AA as indicated in the figure and stimulated with 0.5 mM Bt2cAMP (dbcAMP) for 6 h. A, the cells were collected for mitochondria isolation, and 25 µg of mitochondrial protein were used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated using the BioImage Visage 2000 and expressed as integrated optical density (IOD). B, progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production. **, highly significantly different from Bt2cAMP stimulation or from arachidonic acid reversal (p < 0.01).

Effect of Arachidonic Acid on StAR Gene Transcription-- To study the possible mechanism for the effects of inhibition of AA release, total RNA was isolated from Bt₂cAMP-stimulated MA-10 cells treated with dexamethasone as described above, and StAR mRNA levels were determined by Northern analysis. Two major bands of StAR mRNA of 1.6 and 3.4 kilobase pairs were detected and are shown in Fig. 3A. Addition of 0 to 1.0 µM dexamethasone to the MA-10 cells reduced Bt₂cAMP-induced StAR mRNA in a dose-dependent manner. The observation of the inhibitory effect of dexamethasone on StAR transcription was enhanced when StAR promoter activities were examined using Bt₂cAMP-stimulated MA-10 cells incubated in medium containing increasing levels of dexamethasone. Similar to the reduction in StAR mRNA, StAR promoter activities decreased as the dosage of dexamethasone increased (Fig. 3B). The reduction of StAR mRNA was reversed by the addition of AA to the cells (Fig. 4A). Although 1.0 µM dexamethasone significantly reduced Bt₂cAMP-stimulated StAR mRNA (p < 0.01), 150 µM AA significantly reversed this inhibition (p < 0.01). A similar reversal was observed in dexamethasone-inhibited StAR promoter activity (Fig. 4B). Importantly, AA alone did not increase StAR mRNA and slightly, but not significantly, increased StAR promoter activity.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Inhibitory effects of the PLA2 inhibitor, dexamethasone, on Bt2cAMP-stimulated StAR mRNA levels and StAR promoter activities in MA-10 Leydig cells. A, cells were cultured for 30 min in serum-free Waymouth's MB/752 medium containing various concentrations of dexamethasone as indicated in the figure and stimulated with 0.5 mM Bt2cAMP for 6 h. Cells were collected for total RNA purification. StAR mRNA was analyzed by Northern blot, and StAR-specific bands were quantitated using the BioImage Visage 2000 and expressed as total integrated optical density (IOD). B, cells were co-transfected with DNA containing the promoter/luciferase plasmid expressing firefly luciferase driven by 966 bp of StAR promoter and pRL-SV40 vector DNA, a plasmid which constitutively expresses Renilla luciferase. After 48 h culture, the cells were treated as described above. The cell lysate was used for luciferase assay using a Dual Luciferase Reporter Assay System (Promega, Madison, WI). The relative light units (expressed as the reading from StAR promoter/luciferase divided by the reading from Renilla luciferase) were measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). *, significantly different from Bt2cAMP (dbcAMP) stimulation (p < 0.05); **, highly significantly different from Bt2cAMP stimulation (p < 0.01); ***, very highly significantly different from Bt2cAMP stimulation (p < 0.001).

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Arachidonic acid reversal of the inhibitory effects of the PLA2 inhibitor, dexamethasone, on Bt2cAMP-stimulated StAR mRNA and StAR promoter activity in MA-10 Leydig cells. A, cells were cultured for 30 min in serum-free Waymouth's MB/752 medium with or without 1.0 µM dexamethasone or 150 µM AA as indicated in the figure and stimulated with 0.5 mM Bt2cAMP for 6 h. Cells were collected for total RNA purification. StAR mRNA was analyzed by Northern blot, and StAR-specific bands were quantitated using the BioImage Visage 2000 and expressed as total integrated optical density (IOD). B, cells were transfected with a StAR promoter/luciferase plasmid expressing firefly luciferase driven by 966 bp of the StAR promoter and pRL-SV40 vector DNA, a plasmid which constitutively expresses Renilla luciferase. After 48 h culture, the cells were treated as described above. The cell lysate was used for the luciferase assay using a Dual Luciferase Reporter Assay System (Promega, Madison, WI). The relative light units (expressed as the reading from StAR promoter/luciferase divided by the reading from Renilla luciferase) were measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). **, highly significantly different from Bt2cAMP (dbcAMP) stimulation or from arachidonic acid reversal (p < 0.01).

Roles of Arachidonic Acid Metabolites in StAR Protein Expression and Steroid Production-- In order to examine which metabolic pathway of AA might be involved in StAR protein expression, Bt₂cAMP-stimulated MA-10 cells were treated with the lipoxygenase inhibitors NDGA or AA861, the epoxygenase inhibitor miconazole, and the cyclooxygenase inhibitor indomethacin, respectively. StAR protein levels and progesterone production were determined. As shown in Fig. 5, both NDGA and AA861 inhibited both StAR protein expression and progesterone production (p < 0.01). Miconazole slightly inhibited StAR protein expression but significantly inhibited progesterone production (p < 0.01). Indomethacin caused an increase in StAR protein and also resulted in a significant increase in progesterone production (p < 0.01). Based on these results, we continued the studies on the lipoxygenase pathway. AA861-treated MA-10 cells were incubated in a medium containing 1.5 µM 5-HPETE, a metabolite of the 5-lipoxygenase pathway. The results are shown in Fig. 6. Addition of 5-HPETE to the cell cultures partially reversed AA861-inhibited StAR protein and also partially reversed the inhibition in progesterone production (p < 0.01).

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Effects of AA metabolic inhibitors on Bt2cAMP-stimulated StAR protein expression and progesterone production in MA-10 Leydig cells. Cells were cultured for 30 min in serum-free Waymouth's MB/752 medium containing 70 µM of the lipoxygenase inhibitor NDGA, 65 µM of the 5-lipoxygenase inhibitor AA861, 20 µM of the epoxygenase inhibitor miconazole (Mico.), or 10 µM of the cyclooxygenase inhibitor indomethacin (Indo.) and stimulated with 0.5 mM Bt2cAMP (dbcAMP) for 6 h. A, cells were collected for mitochondria isolation, and 25 µg of mitochondrial protein was used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated using the BioImage Visage 2000 and expressed as integrated optical density (IOD). B, progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production among the treatments. ** denotes values that are highly significantly lower than Bt2cAMP stimulation (p < 0.01); denotes values that are highly significantly higher than Bt2cAMP stimulation (p < 0.01).

View larger version (56K): [in this window] [in a new window]   Fig. 6.   5-HPETE reversal of the inhibitory effects of the 5-lipoxygenase inhibitor, AA861, on Bt2cAMP-stimulated StAR protein expression and progesterone production in MA-10 Leydig cells. MA-10 cells were cultured for 30 min in serum-free Waymouth's MB/752 medium with or without 65 µM AA861 or 1.5 µM 5-HPETE as indicated in the figure, and then stimulated with 0.5 mM of Bt2cAMP for 6 h. A, cells were collected for mitochondria isolation, and 25 µg of mitochondrial protein was used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated using the BioImage Visage 2000 and expressed as integrated optical density (IOD). B, progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production among the treatments. *, significantly different from Bt2cAMP stimulation (p < 0.05); **, highly significantly different from Bt2cAMP (dbcAMP) stimulation (p < 0.01).

PKA Activity and Arachidonic Acid in StAR Protein Expression-- Bt₂cAMP-stimulated MA-10 cells were treated with the PKA inhibitor H89, the PLA₂ inhibitor dexamethasone, or the lipoxygenase inhibitor NDGA. PKA activities and StAR protein expression were determined and are shown in Fig. 7. Stimulation with Bt₂cAMP increased PKA activity by 20-fold over control. 30 µM H89 significantly inhibited PKA activity (p < 0.01) and also inhibited StAR protein expression. Addition of 150 µM AA to the H89-treated cells did not reverse the PKA activity or StAR protein. AA alone had no effect on PKA activity. Inhibition of AA release by dexamethasone or its metabolism by NDGA inhibited StAR protein expression but did not inhibit PKA activity. There was no significant difference in PKA activities among the groups treated with Bt₂cAMP alone, Bt₂cAMP + dexamethasone, and Bt₂cAMP + NDGA.

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Inhibitory effects of PKA inhibitor and AA metabolic inhibitors on Bt2cAMP-stimulated PKA activities and StAR protein expression in MA-10 Leydig cells. Cells were cultured for 30 min in serum-free Waymouth's MB/752 medium containing 30 µM of the PKA inhibitor H89, 1.0 µM of the PLA2 inhibitor, dexamethasone (Dex.), or 70 µM of the lipoxygenase inhibitor NDGA as indicated in the figure and stimulated with 0.5 mM Bt2cAMP (dbcAMP) for 6 h. A, cells were collected for mitochondria isolation, and 25 µg of mitochondrial protein was used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated using the BioImage Visage 2000 and expressed as integrated optical density (IOD). B, cells were collected in extraction buffer, homogenized, and centrifuged at 13,000 × g for 10 min. The supernatant was used for PKA activity assay as described under "Experimental Procedures." PKA activity was expressed as picomoles of 32P incorporated per min/mg of protein. **, highly significantly different from Bt2cAMP stimulation (p < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Arachidonic acid is well known as a component of phospholipids in cell membranes, and cytosolic free AA is mainly generated through the activation of PLA₂ that catalyzes its release from phospholipids (22, 23). Metabolism of the released AA through one of the three enzyme systems, namely the lipoxygenase, the cyclooxygenase, or the cytochrome P450-dependent epoxygenase pathways, produces various metabolites (24, 25). In recent years, the regulatory effects of AA and its metabolites on steroid hormone biosynthesis have been demonstrated (26-28), but the mechanisms for the effects of AA are not clear.

The results of the present study indicate that AA release is essential for steroid production and StAR protein expression. Inhibition of AA release by the PLA₂ inhibitor, dexamethasone, reduced Bt₂cAMP-stimulated StAR protein to a remarkably low level. As StAR protein decreased, progesterone production was concomitantly decreased (p < 0.01). The inhibitory effect of dexamethasone was clearly not due to an inhibitory effect on the activities of the P450scc and/or 3-hydroxysteroid dehydrogenase enzymes. There was no significant difference in steroid production in any of the treatments when 22(R)-hydroxycholesterol was added to the MA-10 cell cultures, indicating that these enzymes were unaffected by dexamethasone. The necessity for AA release in steroidogenesis was further demonstrated when the inhibitory effects of dexamethasone were reversed by addition of exogenous AA to the cell cultures, with steroid production and StAR protein expression being increased. We have previously reported the regulatory effects of AA on LH- or Bt₂cAMP-stimulated steroid production and StAR protein expression using quinacrine as a PLA₂ inhibitor (16). Dexamethasone, which is used in the present study, inhibits PLA₂ activity through a mechanism different from that of the effects of quinacrine (29, 30). However, the results from these two inhibitors were essentially identical and thus corroborated our earlier observations. Since StAR protein was demonstrated to play a critical role in cholesterol transfer to the mitochondrial inner membrane (1-4), these results are in agreement with the earlier experiments that suggested that AA regulates steroidogenesis at the rate-limiting step of mitochondrial cholesterol transfer (14, 15).

The manner in which AA regulates StAR protein expression is currently unknown. The present study demonstrated that this fatty acid regulates StAR gene transcription. Whereas Bt₂cAMP stimulation induced StAR mRNA synthesis, inhibition of AA release by dexamethasone reduced Bt₂cAMP-stimulated StAR mRNA expression. Importantly, the reduction in StAR mRNA was restored by co-incubation of dexamethasone-treated MA-10 cells with 150 µM exogenous AA. AA regulation of StAR gene transcription was further demonstrated by utilizing a StAR promoter activity assay which showed that inhibition of AA release by dexamethasone resulted in a dose-dependent decrease in Bt₂cAMP-stimulated StAR promoter activity. The inhibitory effect of dexamethasone on promoter activity was also reversed by the addition of AA to the culture medium clearly indicating that AA release is essential for StAR promoter activity. The specific details concerning the AA regulation of StAR gene transcription is currently under investigation in our laboratory. It is possible that AA or its metabolites might regulate transcription factors controlling StAR promoter activity.

As mentioned earlier, after AA is released, it is metabolized mainly through three pathways. Our results suggested that among the three, the lipoxygenase pathway may be involved in steroid production and StAR protein expression. Blocking AA metabolism through this pathway by using the inhibitor NDGA greatly decreased Bt₂cAMP-induced StAR protein expression and reduced progesterone production significantly (p < 0.01). Although the specificity of NDGA might be questionable, similar results were also obtained when the more specific 5-lipoxygenase inhibitor, AA861, was used. Moreover, the inhibitory effects of AA861 were reversed when the 5-lipoxygenase metabolite of AA, 5-HPETE, was added to the culture suggesting a possible involvement of the 5-lipoxygenase pathway in steroidogenesis and StAR protein expression. The roles of the lipoxygenase metabolites in steroid production were previously demonstrated (31-33) and are essentially in agreement with the results obtained in this study. Besides 5-HPETE, the effects of other lipoxygenase metabolites, such as 12-hydroxyeicosatetraenoic acid and 15-HPETE, in steroid biosynthesis were described (34, 35). Further studies are needed to determine if they are involved in StAR protein expression. Stimulatory effects of epoxygenase metabolites on steroidogenesis were reported previously (36, 37). As shown in Fig. 5, inhibition of AA metabolism through this pathway inhibited progesterone production significantly (p < 0.01); however, StAR protein expression was only slightly inhibited. The reasons for this apparent discrepancy are not clear at this time but perhaps the absence of epoxygenase pathway metabolites interfered with another required component or event in supporting steroidogenesis. Blocking the cyclooxygenase pathway using indomethacin resulted in an increase in Bt₂cAMP-stimulated StAR protein expression and also in a significant increase in progesterone production in MA-10 cells (p < 0.01). The reason for the indomethacin-induced increase of StAR protein is unknown. One of the cyclooxygenase metabolites, prostaglandin F2 was reported to be able to induce a rapid decline in StAR protein expression (38). It is possible that the inhibition of cyclooxygenase activity reduced the production of inhibitory metabolites, resulting in an increase in StAR protein expression. Alternatively, perhaps after blocking the cyclooxygenase pathway more AA was available to produce lipoxygenase metabolites that enhanced StAR protein expression.

The mechanism responsible for the actions of AA and its metabolites on StAR gene expression and steroid production probably involves the signal transduction pathway from trophic hormone receptors to the nucleus (39, 40). The role of the cAMP-PKA-phosphorylation pathway is well known in trophic hormone stimulation and is important in StAR gene expression (5). However, results obtained earlier and those provided in this study indicate that the PKA pathway is not sufficient to transduce all the signals from the trophic hormone receptor to the nucleus in order to accomplish the physiological functions of hormone stimulation. As shown in Fig. 7, inhibition of either AA release or lipoxygenase activity did not affect Bt₂cAMP-induced PKA activities, but this high level of PKA activity was unable to induce StAR protein expression, suggesting that PKA phosphorylation alone does not represent the entire signal pathway involved in steroid biosynthesis. Reversal of StAR protein expression and steroid production by addition of AA indicated that the blocked pathway is an AA-mediated pathway.

Although PKA activity remained at a high level, inhibition of AA release reduced the Bt₂cAMP-stimulated StAR promoter activity, StAR mRNA, StAR protein, and steroid production (Figs. 1-4). On the other hand, inhibition of the PKA activity using H89 also inhibited StAR protein expression, but addition of AA did not reverse the H89-reduction in StAR protein. Also, whereas exogenous AA reversed the PLA₂ inhibitor-reduced StAR promoter activity, StAR mRNA, StAR protein, and steroid production, in the absence of an elevated PKA activity (resulting from an increase in cAMP), AA alone did not have a significant effect on StAR gene expression. These results indicated that 1) AA transduces signals from trophic hormones and cAMP to the nucleus through a pathway different from the cAMP-PKA-phosphorylation pathway, and 2) both pathways are essential with neither pathway alone being sufficient for trophic hormone-induced steroid production and StAR gene expression.

Collectively, the results shown here demonstrate the critical role of arachidonic acid in the transcriptional regulation of StAR gene expression and steroid production and suggest a possible involvement of the lipoxygenase pathway in steroidogenesis. These studies further suggest a new concept in signal transduction in cAMP-dependent steroidogenesis, in which trophic hormones not only induce cAMP formation but also AA release, and then these second messenger systems transduce signals to the nucleus through two separate pathways to co-regulate StAR gene expression and steroid production.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 806-743-2505; Fax: 806-743-2990; E-mail: Doug.Stocco@ttmc.ttuhsc.edu.

Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M003113200

	ABBREVIATIONS

The abbreviations used are: StAR, steroidogenic acute regulatory protein; Bt₂cAMP, dibutyryl cyclic AMP; AA, arachidonic acid; NDGA, nordihydroguaiaretic acid; PKA, protein kinase A; PLA₂, phospholipase A₂; LH, luteinizing hormone; bp, base pair; 5-HPETE, (6E,8Z,11Z,14Z)-eicosatetraenoic acid; PBS, phosphate-buffered saline; RIA, radioimmunoassay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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