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Departments of Neuropsychiatry, Garrison Institute on Aging,
1 Cell Biology and Biochemistry,
2 Neuropsychiatry, and
3 Pathology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA
(Requests for offprints should be addressed to X Wang; Email: xingjia.wang{at}ttuhsc.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the AA-mediated signaling pathway, AA is mainly metabolized by one of the three groups of enzymes: the lipoxygenases, cyclooxygenases, or epoxygenases (Needleman et al. 1986). It was reported that AA metabolites specifically produced through the lipoxygenase pathway stimulated steroidogenesis (Dix et al. 1985, Nishikawa et al. 1994). Our previous studies indicated that cAMP stimulation of MA-10 mouse Leydig cells increased the synthesis of 5-lipoxygenase-generated metabolites of AA, namely, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE), both of which enhanced cAMP-stimulated StAR expression (Wang et al. 2003b). Also, the inhibition of 5-lipoxygenase activity reduced StAR protein and steroidogenesis (Wang et al. 2000). In contrast to the action of 5-lipoxygenase in Leydig cells, the cyclooxygenase-2 enzyme (COX2, an isoform of cyclooxygenase) appears to be responsible for a tonic inhibition of StAR gene expression and steroid production (Wang et al. 2003a). The overexpression of COX2 in MA-10 cells reduced the steroidogenic sensitivity of the cells to cAMP stimulation. Similarly, the increased expression of COX2 in Leydig cells isolated from aging rats correlated with a reduction in StAR-mediated steroidogenesis (Wang et al. 2005). Presently, there is less evidence regarding what role the epoxygenases may serve with respect to StAR gene expression and steroid production. It was reported that LH or cAMP stimulation of granulosa cells increased 5,6-epoxyeicosatrienoic acid, an epoxygenase metabolite of AA, suggesting a possible role for this AA metabolite in granulosa cell steroidogenesis (Zosmer et al. 1990, 2002). In addition, we have previously reported that inhibiting epoxygenase activity reduced StAR protein expression and steroid production in MA-10 mouse Leydig cells (Wang et al. 2000). In order to completely understand how the signaling pathways involving AA metabolism are implicated in the regulation of steroidogenesis, we have analyzed the production of epoxygenase-derived AA metabolites in MA-10 cells following stimulation with a cAMP analog. Then, we examined the effects of these metabolites on StAR gene expression and steroidogenesis. Our findings indicate that the cAMP stimulation of MA-10 cells results in the increase of several AA metabolites produced by epoxygenase activity that are each capable of significantly enhancing cAMP-stimulated StAR gene expression and steroid hormone production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N6,2-Dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) was purchased from Sigma. (±)5(6)-epoxy-8Z, 11Z,14Z-eicosatrienoic acid (5,6-EET), (±)8(9)-epoxy-5Z, 11Z,14Z-eicosatrienoic acid (8,9-EET), (±) 11(12)-epoxy-5Z, 8Z,14Z-eicosatrienoic acid (11,12-EET), (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET), 5(S)-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), and 5(S)-hydroperoxy-6E,8Z,11Z, 14Z-eicosatetraenoic acid (5-HPETE) were purchased from Cayman (Ann Arbor, MI, USA). Rabbit antiserum generated against StAR protein was a generous gift from Prof W L Miller (Department of Pediatrics and the Metabolic Research Unit, University of California San Francisco, San Francisco, CA, USA; Bose et al. 1999). Donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase was purchased from Biosource (Camarillo, CA, USA). Waymouth’s MB/752 medium was obtained from Sigma. Horse serum was purchased from Invitrogen. The Dual-Luciferase Reporter Assay System was purchased from Promega. The other common chemicals used in this study were obtained from either Sigma or Fisher Chemicals (Pittsburgh, PA, USA).
Cell culture
The MA-10 mouse Leydig tumor cell line was a generous gift from Dr Mario Ascoli (Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, IA, USA) and were cultured in 12-well culture plates in Waymouth’s MB/752 medium containing 15% horse serum as previously described (Ascoli 1981). The cells were cultured in an incubator at 37 °C and 5% CO2. Before each experiment, the medium was replaced with serum-free Waymouth’s medium.
HPLC analysis of arachidonic acid metabolites
The methods previously described were modified for extraction and HPLC separation of AA metabolites (Powell 1982, Borgeat et al. 1990). MA-10 cells in 100 mm culture plates were incubated in 5 ml Waymouth’s medium containing 1.5% horse serum and 25 µCi/ml 3H-AA for 4 h. The cells were washed twice with 5 ml Waymouth’s medium containing 0.1% fatty acid-free BSA. The cells were then incubated in 5 ml Waymouth’s medium and stimulated with 1.0 mM dbcAMP for 6 h. The stimulation was terminated by their transfer to –80 °C and the addition of 1.0 ml cold methanol containing 5 µl of 37% HCl and 15 µg non-labeled standards, including AA and a mixture of several AA metabolites (3 µg of each: 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, and 5-HPETE). The cells were collected and centrifuged at 8000 g for 20 min. The resulting supernatants were loaded onto 3 ml/500 mg Discovery DSC-18 SPE tubes (Supelco, Bellefonte, PA, USA) preconditioned with washes of 3 ml methanol followed by 3 ml water. The tubes were then eluted with 3 ml methanol, followed by 3 ml acetonitrile. The elutes from the tubes were dried under nitrogen and resuspended in 350 µl of 60% acetonitrile. The concentrated extracts were immediately analyzed by RP-HPLC with a Gradient Component System consisting of two 515 HPLC pumps (Waters, Milford, MA, USA) on a Discovery C18 column (4.6 mm x 25 cm, 5 µm particle size; Supelco), and using a stepped gradient of solvent A, containing water/acetonitrile/methanol/phosphoric acid (75/12.5/12.5/0.01), and solvent B, containing water/acetonitrile/methanol/phosphoric acid (5/63/32/0.01). The gradient steps used are as follows: 0 min, 100%A, 1.0 ml/min; 0–10 min, 44%A + 56%B, 0.98 ml/min; 10–89 min, 40%A + 60%B, 0.96 ml/min; 89–140 min, 100%B, 0.96 ml/min. The sample was analyzed spectrophotometrically at 206 and 235 nm using a Waters 2487 Dual 
Absorbance Detector (Waters). The column elute was directly collected into scintillation vials and the radioactivities of the fractions were measured using a Beckman LS 6500 scintillation counter (Beckman, Fullerton, CA, USA).
Steroid production
MA-10 cells were cultured for 30 min in serum-free Waymouth’s medium containing 5,6-EET, 8,9-EET, or 11,12-EET respectively (as described in Figures) and 0.2 mM dbcAMP was added to the culture for 6 h. The medium was collected at the end of each experiment and stored at –80 °C. Progesterone concentrations in the medium were determined by RIA (Resko et al. 1974).
Western blot analysis
StAR protein in MA-10 cells was detected by Western blot analysis as described previously (Townson et al. 1996). Western blot analysis experiments were performed at least three times and the results of one representative experiment are shown in the Figures.
Transfection
MA-10 cells were cultured in 12-well plates (0.2 x 106 cells/well) overnight. The cells in each well were transfected with 0.5 µg DNA of the StAR promoter/luciferase plasmid PGL2/StAR expressing firefly luciferase driven by the –966 bp sequence of the StAR promoter (Caron et al. 1997). Transfections also included 6.0 ng pRL-SV40 vector DNA (a plasmid, which constitutively expresses Renilla luciferase under the control of the SV40 promoter; Promega). Transfections were performed using FuGENE6 Transfection reagent (Roche) following the manufacturer’s instructions. After 48-h culture, the cells were utilized for further experiments.
Luciferase assays
Following experiments, the cells were washed thrice with ice-cold PBS and lysed with Passive Lysis Buffer (Promega). The supernatants were utilized for luciferase assays using a Dual Luciferase Reporter Assay System following the manufacturer’s instructions (Promega). The relative light units (determined by dividing the reading from the PGL2/StAR promoter by the reading from Renilla luciferase) were measured using a TD-20/20 luminometer (Turner Designers, Sunnyvale, CA, USA).
Reverse transcriptase (RT)-PCR
In experiments designed to determine StAR mRNA expression, cells were washed thrice with cold PBS and used for total RNA purification using TRIzol reagent in accordance with the manufacturer’s instructions (Invitrogen). RT-PCR was performed as previously described (Rao et al. 2003). A ribosomal protein L19 was used as internal Marker in the RT-PCR (Al-Bader & Al-Sarraf 2005).
Statistical analysis
Each experiment was repeated at least three times. Statistical analysis of the data was performed with ANOVA and Fisher’s protected least significant difference test using the Stat View SE system (Abacus Concepts, Berkeley, CA, USA). The data are shown as the mean ± S.E.M.
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Results |
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To determine how the profile of epoxygenase-derived AA metabolites changes in response to cAMP, MA-10 Leydig cells loaded with 3H-AA were incubated with or without the cAMP analog dbcAMP and the AA metabolites from these cells were resolved by HPLC. Our results indicated that MA-10 cells stimulated with dbcAMP have increased amounts of three epoxygenase-derived AA metabolites: 5,6-EET, 8,9-EET, and 11,12-EET. Total detected amounts of 8,9-EET and 11,12-EET were significantly increased by 120% and 81%, respectively, over the levels seen in controls (P < 0.05). The amount of 5,6-EETwas also increased by dbcAMP stimulation, but the increase is not statistically significant (Fig. 1
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Since EET concentrations appeared to increase in MA-10 cells following treatment with dbcAMP, we sought to determine whether EETs served in a causative role in regulating StAR expression and steroidogenesis. MA-10 cells were incubated with increasing concentrations of 5,6-EET, 8,9-EET, or 11,12-EET in medium containing 0.2 mM dbcAMP. Figures 2–4 show the steroid hormone production and StAR protein expression that followed each of these dose–response treatments. Treating MA-10 cells with 0.2 mM dbcAMPalone consistently resulted in a small detectable rise in steroidogenesis. Since EET concentrations in the medium increased from 0 to 20 µM, StAR protein increased in a dose–dependent manner. Concomitant with the increase in StAR protein, steroid hormone production also increased significantly compared with that of cells treated with 0.2 mM dbcAMP alone. To ascertain whether the increases in steroid hormone production resulted from increased activity of P450scc itself, the levels of P450scc protein were analyzed by Western blot and 22(R)hydroxycholesterol, a membrane-permeable form of cholesterol, was also included during the stimulation. Incubation of the cells with the EETs did not induce an obvious increase in the levels of the P450scc protein. There was no significant difference in steroid production among the treatments when the cells were cultured with 22(R)hydroxycholesterol for 6 h (Fig. 5).
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To understand how these epoxygenated AA metabolites might serve to enhance StAR protein expression, we investigated the effects that the EETs had on StAR gene transcription in the presence of 0.2 mM dbcAMP. The relative abundance of StAR mRNA levels in the EET-treated MA-10 cells was determined by RT-PCR, and the activity of the StAR promoter under the same conditions was analyzed by luciferase assay. The results in Fig. 6 show that each EET tested increased StAR mRNA levels in MA-10 Leydig cells relative to 0.2 mM dbcAMP. These increases were paralleled by increases in StAR promoter activity. In the cells treated with 20 µM of 5,6-EET, 8,9-EET, or 11,12-EET, StAR promoter activities increased by 3.0-, 2.6-, and 3.8-fold, respectively, over the level of the cells treated with 0.2 mM dbcAMP alone.
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Each of the EETs was added to MA-10 cell cultures with or without 0.2 mM dbcAMP to determine the role of this low level of cAMP analog in EET-enhanced StAR gene expression and steroid hormone production. As shown in Fig. 7, in the absence of dbcAMP, none of the EETs alone were able to induce a significant increase in steroidogenesis, whereas in the presence of 0.2 mM dbcAMP, all of them dramatically increased StAR protein and steroid hormone production to the levels of maximal stimulation. Also, the inhibition of PKA activity using the selective inhibitor, H89, abolished the stimulatory effects of the EETs on StAR protein expression and steroid hormone production (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The involvement of epoxygenase metabolites of AA in cAMP-stimulated steroidogenesis is supported by the results from HPLC analysis of the AA metabolites extracted from MA-10 cells stimulated with dbcAMP. Stimulation with this cAMP analog not only increased AA release, as reported previously (Maloberti et al. 2000, Wang et al. 2002, Castilla et al. 2004), but also appeared to enhance AA metabolism through the epoxygenase pathway and resulted in increases in its metabolites 5,6-EET, 8,9-EET, and 11,12-EET. All of these AA metabolites significantly stimulated steroid hormone production in MA-10 Leydig cells. These stimulatory effects can readily be seen from the concentration-dependent increases in progesterone production in MA-10 cells incubated with increasing levels of these metabolites. A similar observation was reported previously in human granulosa cells (Zosmer et al. 1990, 2002). The results from the present and previous studies suggest that LH or cAMP stimulation enhance the positive steroidogenic signals produced through the AA signaling pathway by increasing the levels of epoxygenase-generated AA metabolites, which in turn enhance steroid hormone production.
The mechanism by which the epoxygenase metabolites of AA enhance steroid hormone production is unknown. The results from the experiments with 22(R)-hydroxycholesterol-treated MA-10 cells suggest that the increase in steroid hormone production is mainly due to the increased supply of the substrate to the inner mitochondrial membrane, since there was no significant difference in steroid hormone production among the treatments when this substrate was used. This observation encouraged us to analyze StAR protein in the cells, a protein critical for cholesterol transfer from the outer to inner mitochondrial membrane. The results from Western blot analyses indicated that each of these epoxygenase-generated AA metabolites increased StAR protein expression. The increases in StAR protein expression occurred concomitantly with the increases in progesterone production in EET-treated MA-10 cells. This is in keeping with ourearlierobservations that the inhibition of epoxygenase activity in MA-10 cells reduced StAR protein expression and steroidogenesis (Wang et al. 2000). While other steroidogenic factors might be involved in EET-increased steroidogenesis, these observations suggest that the epoxygenase-generated AA metabolites enhanced steroid hormone production mainly through its regulation of StAR protein expression.
The present study further suggests that the AA metabolites produced by epoxygenase activity regulate StAR gene expression at the level of transcription. This was supported by the results from luciferase assays of StAR promoter activities and RT-PCR analysis of StAR mRNA levels. The mechanisms by which EETs influence StAR promoter activity are unknown and thus could act directly or indirectly to effect changes in promoter activity. It will be interesting to determine whether the EETs induce or activate specific transcription factors that bind to the StAR promoter and enhance StAR gene transcription.
Whereas increases in EETs were capable of enhancing StAR gene expression in the presence of cAMP, none of them alone was able to increase StAR promoter activity, StAR mRNA level, and StAR protein or steroid hormone production. Also, the inhibition of PKA activities with the selective PKA inhibitor H89 significantly reduced the EET-increased StAR protein expression and steroid hormone production. These results suggested that signaling through the cAMP–PKA-phosphorylation pathway is necessary for the EETs to elicit their steroidogenic response. This observation is consistent with results from our previous studies (Wang et al. 2000). In those studies, we determined that without PKA-phosphorylation, AA-mediated signaling alone is unable to induce StAR gene expression and steroidogenesis. On the other hand, if AA-release or its metabolism by epoxygenase and lipoxygenase is blocked by the use of specific inhibitors, even high levels of cAMP or PKA-phosphorylation could not stimulate significant increases in StAR gene expression and steroid hormone production (Wang et al. 2000). These data support the concept that in LH or cAMP-stimulated steroidogenesis, signal transduction through both the cAMP–PKA-phosphorylation pathway and the AA pathway are required to accomplish maximal steroidogenesis in response to tropic hormone stimulation.
From the results of the present and previous studies (Wang & Stocco 1999, Wang et al. 2002, 2003a,2003b), it can readily be seen that in the AA-mediated signaling pathway, both positive and negative signals are produced to regulate StAR gene expression. The positive signals are generated by 5-lipoxygenase activity that converts AA to 5-HPETE and 5-HETE as reported previously (Wang et al. 2003b) and also by epoxygenase activity that converts AA to various EETs as discussed earlier. These AA metabolites act together to stimulate StAR gene expression when minimal levels of PKA-mediated phosphorylating activity are present in the cells. The negative signals were produced through the activity of COX2. Although the mechanism for the action of COX2 has not been elucidated completely, its expression resulted in inhibitory effects on StAR gene expression and steroidogenesis in Leydig cells (Wang et al. 2005). Therefore, the levels of these three enzymes in Leydig cells regulate the intracellular levels and the ratios of the positive and negative signals, which in turn may serve to regulate the sensitivity of Leydig cells to LH or cAMP stimulation.
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Acknowledgements |
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References |
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Received 24 April 2006
Received in final form 19 June 2006
Accepted 23 June 2006
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Highly significantly different from control (P < 0.01). 
Very highly significantly different from control (P < 0.001).
