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Endocrinology, doi:10.1210/en.2002-0081
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Endocrinology Vol. 144, No. 8 3368-3375
Copyright © 2003 by The Endocrine Society

Inhibition of Cyclooxygenase-2 Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

XingJia Wang, Matthew T. Dyson, Youngah Jo and Douglas M. Stocco

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

Address all correspondence and requests for reprints to: XingJia Wang, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: xingjia.wang{at}ttmc.ttuhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt2cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 µM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt2cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt2cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN TROPHIC HORMONE-stimulated steroidogenesis, the rate-limiting step is cholesterol transfer to the mitochondrial inner membrane where the substrate cholesterol is converted to pregnenolone by the P450 side chain cleavage (P450scc) enzyme (1, 2, 3). The steroidogenic acute regulatory (StAR) protein has been demonstrated to play a critical role at this step by facilitating this transfer (4, 5, 6, 7). However, the mechanism for the trophic hormone regulation of StAR gene expression is not completely clear. It is well known that trophic hormone stimulation of steroidogenic cells induces cAMP formation followed by activation of protein kinase A (PKA), which phosphorylates transcription factors and possibly other unknown proteins involved in StAR gene transcription (8). Trophic hormone stimulation also induces arachidonic acid (AA) release from intracellular phospholipids (9, 10, 11, 12), and studies in the last two decades have documented the essential role of AA in the regulation of steroid biosynthesis in humans and animals (13, 14, 15, 16). However, the exact mechanism whereby AA regulates steroidogenesis remains unknown. Earlier observations suggested that AA and its metabolites regulate steroid production at the rate-limiting step of cholesterol transfer raising the possibility that StAR protein might be involved (17, 18). Indeed, when we inhibited AA release using phospholipase A2 inhibitors, StAR gene expression and steroid production were inhibited. Importantly, the inhibitory effects were reversed by the addition of AA (19, 20). These studies indicated that StAR gene expression was involved in AA regulation of steroid hormone biosynthesis. To further understand the mechanism for the role of AA in steroidogenesis, we attempted to characterize the roles of the three AA metabolic pathways (as identified and named by the enzymes metabolizing AA), the cyclooxygenase (COX), lipoxygenase, and epoxygenase pathways, in StAR gene expression and steroid production. Among these, the negative effect of a COX metabolite on StAR gene expression was described in earlier studies (21, 22). Previously, we reported that inhibition of COX activity significantly increased StAR protein expression and steroid production in MA-10 mouse Leydig cells (20). We have continued these studies and the results suggest that COX2, one of the COX isoforms, is possibly involved in the regulation of StAR gene transcription and steroid hormone biosynthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dibutyryl cAMP (Bt2cAMP) and indomethacin were purchased from Sigma (St. Louis, MO). H89 was obtained from Calbiochem (La Jolla, CA). NS398, SC560, antibody against COX2, and prostaglandin (PG)E2 enzyme immunoassay (EIA) kit were purchased from Cayman (Ann Arbor, MI). Waymouth’s MB/752 medium, DMEM, fetal bovine serum (FBS), horse serum, trypsin-EDTA, antibiotics, and PBS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Rabbit antisera generated against StAR protein was a generous gift from Dr. W. L. Miller (Department of Pediatrics, University of California, San Francisco, CA). Donkey antirabbit IgG antibody conjugated with horseradish peroxidase was purchased from Amersham (Arlington Heights, IL). [{gamma}32P]-deoxy-ATP and 3H-AA were purchased from Dupont-NEN Life Science Products (Boston, MA). North2South Biotin Random Prime kits and Chemiluminescent Nucleic Acid Hybridization and Detection kits were obtained from Pierce (Rockford, IL). SF-1 (steroidogenic factor-1) expression plasmid was a generous gift of Dr. Keith Parker (University of Texas Southwestern Medical School, Dallas, TX). The Dual-Luciferase Reporter Assay System was purchased from Promega (Madison, WI). Other common chemicals used in this study were obtained from either Sigma (St. Louis, MO) or Fisher Chemicals (Pittsburgh, PA).

Cell culture
The MA-10 mouse Leydig tumor cells were a generous gift from Dr. Mario Ascoli (Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, IA) and were cultured in six-well culture plates in Waymouth’s MB/752 medium containing 15% horse serum as previously described (23). R2C cells were purchased from American Type Culture Collection (ATCC, Rockville, MD) and grown in Waymouth’s MB/752 medium containing 15% horse serum and 2.5% FBS. COS-1 cells were obtained from ATCC and cultured in DMEM containing 10% FBS. 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.

Steroid production
MA-10 cells were cultured for 30 min in serum-free Waymouth’s medium containing COX inhibitors (as described in the figure legends) and then stimulated with 0.05 mM Bt2cAMP for 6 h. The medium was collected at the end of each experiment and stored at -80 C. The cells were washed twice with cold PBS and stored at -80 C. Progesterone concentrations in the medium were determined by RIA (24).

pCR3.1/COX2 plasmid construction
The pCR3.1/COX2 expression plasmid was constructed to express the COX2 gene in mammalian cells. Mouse COX2 cDNA (1.9 kb) was cut at the BamHI/XholI sites from the pBS/COX2 plasmid (a generous gift from Dr. Donald Young, Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, NY), and inserted into the pCR3.1 vector pre-cut with the BamHI/XholI restriction enzymes. The plasmid thus constructed was tested by transfection into COS-1 cells and detection of the COX2 protein by Western blot analysis.

Transfections
MA-10 cells (0.5 x 106 per well) were cultured in six-well plates overnight. The cells in each well were transfected with 1.0 µg DNA of the StAR promoter/luciferase plasmid PGL2/StAR expressing firefly luciferase driven by the -966-bp sequence of the StAR promoter (25). Transfections also included 12.5 ng of the pRL-simian virus 40 (SV40) vector DNA (a plasmid that constitutively expresses Renilla luciferase, a control reporter under the control of the SV40 promoter, Promega). Transfections were performed using FuGENE6 Transfection Reagent (Roche, Indianapolis, IN) following the manufacturer’s instructions. After 48 h in culture, the cells were used for experiments. COS-1 cells were transfected with 1.0 µg StAR promoter/luciferase DNA, 0.5 ng pRL-SV40 DNA, 1.0 µg PCR3.1/COX2 plasmid, or 0.5 µg of SF-1 expression plasmid using the same method for MA-10 cell transfection.

Luciferase assays
Following experiments, the cells were washed three times with ice-cold PBS and lysed with Passive Lysis Buffer (Promega). The supernatants were used for luciferase assays using a Dual Luciferase Reporter Assay System following the manufacturer’s instructions (Promega). The relative light units (expressed as the reading from the StAR promoter/luciferase divided by the reading from Renilla luciferase) were measured using a TD-20/20 luminometer (Turner Designers, Sunnyvale, CA). Data were expressed as fold increases over the relative light units of the control.

Protein kinase A (PKA) activity assays
PKA activity was measured as described previously (20), using the SignaTECT cAMP-Dependent Protein Kinase Assay System (Promega). PKA activity was expressed as picomoles 32P incorporated per minute per microgram protein.

PGE2 production
MA-10 cells were incubated with the COX2 inhibitor NS398 with or without 0.05 mM Bt2cAMP for 6 h. The culture medium was collected and stored at -80 C. PGE2 in the culture medium was assayed using an EIA kit (Cayman) following the manufacturer’s instructions.

Northern blot analysis
In experiments designed to determine StAR expression at the mRNA level, cells were washed three times with cold PBS and used for total RNA purification using TRIzol reagent following the manufacturer’s instructions (Gibco-BRL, Grand Island, NY). The RNA was separated by electrophoresis in an agarose/formaldehyde gel (1%/6%) and blotted onto a Hybond-N+ membrane (Amersham). StAR mRNA on the membrane was probed with biotin-labeled mouse StAR cDNA and detected using the North2South Chemiluminescent Nucleic Acid Hybridization and Detection kit (Pierce) following the manufacturer’s instructions. The membrane was stripped with a buffer containing 15 mM NaCl, 15 mM sodium citrate and 1% SDS (pH 7.0) for 30 min at 55 C. 18S rRNA on the membrane was probed to adjust for the RNA loading in each lane.

Western blot analysis
StAR protein and COX2 protein in MA-10 cells and R2C cells were detected by Western blot analysis as described previously (26). Western blot analysis experiments were performed at least three times and the results of one representative experiment are shown.

Statistical analysis
Each experiment was repeated at least three times. Statistical analysis of the data were performed with ANOVA using the StatView SE System (Abacus Concepts, Berkeley, CA). The data are shown as mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
StAR protein expression and steroid production
COX activity in MA-10 cells was inhibited by indomethacin to determine the effect of COX on cAMP-stimulated steroidogenesis. The addition of indomethacin to MA-10 cells treated with 0.05 mM Bt2cAMP significantly increased StAR protein levels in a concentration-dependent manner with StAR protein mass increasing by 5.7-fold as indomethacin concentrations were increased from 0 to 1 µM (Fig. 1Go). A concomitant increase in progesterone production was detected, increasing 34.3-fold and paralleling the increase in StAR protein. 22(R)-Hydroxycholesterol, a compound that can readily diffuse to the cytochrome P450scc, was added to the cell cultures to test if indomethacin affected the activities of steroidogenic enzymes, such as P450scc and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Following addition of 22(R)hydroxycholesterol, all the cells produced high levels of steroid and there was no significant difference in steroid production among the treatments. Also, as shown in Fig. 2Go, indomethacin alone did not induce significant increases in StAR protein or progesterone production. However, increases in Bt2cAMP concentrations did enhance the effect of indomethacin on steroidogenesis. As Bt2cAMP concentrations were increased from 0 to 0.1 mM, indomethacin-enhanced StAR protein expression increased from 1.1- to 13.0-fold of that seen in the paired group stimulated with Bt2cAMP alone. Whereas Bt2cAMP alone stimulated steroidogenesis with 1 mM Bt2cAMP, resulting in the maximal levels of StAR protein expression and steroid production, 1 µM indomethacin in the culture medium allowed both StAR protein expression and stimulation of steroid production to reach a maximum at 0.25 mM Bt2cAMP.



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  		FIG. 1. Inhibition of COX activity enhances cAMP-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 increasing concentrations of the COX inhibitor indomethacin (Indo) as shown and were then stimulated with 0.05 mM Bt2cAMP for 6 h. (A), the cells were collected and 25 µg of cell lysate 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 OD (IOD). B, Progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production. To test the effect of indomethacin on the activities of P450scc and 3ß-HSD, 25 µM of 22(R)-hydroxycholesterol was added to each treatment for 6 h and the medium was collected for progesterone assay. *, Significantly different from stimulation with Bt2cAMP alone (P < 0.05). **, Highly significantly different from stimulation with Bt2cAMP alone (P < 0.01). ***, Very highly significantly different from stimulation with Bt2cAMP alone (P < 0.001).

 


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  		FIG. 2. Effects of cAMP and a COX inhibitor on StAR protein expression and progesterone production in MA-10 cells. MA-10 cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium with or without 1 µM of the COX inhibitor indomethacin (Indo) and then stimulated for 6 h with increasing concentrations of Bt2cAMP as indicated in the figure. A, Cells were collected and 25 µg of cell lysate protein was used to analyze StAR protein by Western blot. Each StAR-specific band was quantitated and expressed as integrated OD (IOD). B, Progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production. Progesterone production in the groups with addition of 1 µM Indo was significantly higher (P < 0.05) than those of the paired groups treated with Bt2cAMP alone except that of the control group without Bt2cAMP.

 
StAR gene transcription
Northern blot analyses and promoter activity assays were performed to study the role of COX in StAR gene transcription. Similar to the results seen with StAR protein expression, inhibition of COX activity by indomethacin significantly increased StAR mRNA levels and StAR promoter activity (P < 0.001). Stimulation with 0.05 mM Bt2cAMP alone did not increase StAR mRNA or promoter activity in MA-10 cells. However, StAR mRNA and StAR promoter activity increased by 2.8- and 2.9-fold, respectively, when indomethacin concentrations in the culture were increased from 0.1 to 10 µM (Fig. 3Go). Bt2cAMP is critical for the stimulatory effect of indomethacin. As shown by the control (Fig. 4Go), in the absence of Bt2cAMP, indomethacin did not increase StAR mRNA or StAR promoter activity. The presence of Bt2cAMP also enhanced the stimulatory effect on StAR gene transcription that was obtained using the COX inhibitor. As Bt2cAMP concentrations were increased from 0 to 0.25 mM, indomethacin-enhanced StAR promoter activity was elevated from 1.1- to 4.3-fold of that seen in the paired group without indomethacin. Surprisingly, both StAR promoter activity and mRNA expression increased beyond that observed in the groups stimulated with 1 mM Bt2cAMP alone (Fig. 4Go).



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  		FIG. 3. Inhibition of COX activity increased cAMP-stimulated StAR mRNA levels and StAR promoter activity in MA-10 Leydig cells. A, MA-10 cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium with increasing concentrations of indomethacin (Indo) as indicated in the figure and then stimulated for 6 h with 0.05 mM Bt2cAMP. Cells were collected for total RNA purification. StAR mRNA was analyzed by Northern blot and StAR specific bands were quantitated and expressed as integrated OD (IOD). B, MA-10 cells were transfected with a StAR promoter/luciferase plasmid (PGL2/StAR) expressing firefly luciferase driven by -966 bp of the StAR promoter and pRL-SV40 vector DNA, a plasmid that constitutively expresses Renilla luciferase. After 48 h in culture, the cells were treated as described above. The cell lysate was used for the luciferase assay using a Dual Luciferase Reporter Assay System as described in Materials and Methods. Data were expressed as fold increases over the promoter activity of the control. *, Significantly different from the stimulation with Bt2cAMP alone (P < 0.05). **, Highly significantly different from the stimulation with Bt2cAMP alone (P < 0.01).

 


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  		FIG. 4. Effects of cAMP and a COX inhibitor on StAR gene transcription in MA-10 Leydig cells. A, MA-10 cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium with or without 1 µM indomethacin (Indo) and then stimulated for 6 h with increasing concentrations of Bt2cAMP as indicated in the figure. Cells were collected for total RNA purification. StAR mRNA was analyzed by Northern blot and StAR-specific bands were quantitated and expressed as integrated OD (IOD). B, MA-10 cells were transfected with a StAR promoter/luciferase plasmid (PGL2/StAR) and a pRL-SV40 vector DNA, a plasmid that constitutively expresses Renilla luciferase. The cell lysate was used for the luciferase assay using a Dual Luciferase Reporter Assay System as described in Materials and Methods. Data were expressed as fold increases over the promoter activity of the control. *, Significantly different from the paired group stimulated with Bt2cAMP alone (P < 0.05). **, Highly significantly different from paired group stimulated with Bt2cAMP alone (P < 0.01).

 
Different effects of COX1 and COX2 activities on steroidogenesis
The selective COX1 inhibitor SC560 or the selective COX2 inhibitor NS398 was used to determine which COX isoform was involved in StAR protein expression and steroid production in MA-10 cells. As shown in Fig. 5Go, inhibition of COX1 activity using SC560 did not increase StAR protein expression or steroid production, there being no significant difference between control and SC560-treated cells. However, inhibition of COX2 activity using NS398 significantly increased StAR protein expression. As StAR protein increased, progesterone production increased 81.4-fold over that seen in cells stimulated with 0.05 mM Bt2cAMP alone. Because COX2 is an inducible isoform that is not constitutively expressed in most cells, these results led us to examine the levels of COX2 protein in MA-10 mouse Leydig cells and R2C rat Leydig cells in an effort to confirm these results. Interestingly, whereas COX2 protein in R2C cells was almost undetectable by Western blot, high levels of COX2 protein were detected in nonstimulated MA-10 cells (Fig. 6Go). The levels of COX2 protein in these two cell lines are inversely correlated with the levels of StAR protein and steroid production.



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  		FIG. 5. Different effects of COX1 and COX2 inhibitors on 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 1 µM of the COX inhibitor indomethacin, 5 µM of the COX1 inhibitor SC560 or 35 µM of the COX2 inhibitor NS398, and then stimulated with 0.05 mM Bt2cAMP for 6 h. A, The cells were collected and 25 µg of cell lysate protein was used to analyze StAR protein by Western blot. B, Progesterone production in the medium was analyzed by RIA and expressed as percentages of the highest production. *, Significantly different from the stimulation with Bt2cAMP alone (P < 0.05). **, Highly significantly different from the stimulation with Bt2cAMP alone (P < 0.01).

 


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  		FIG. 6. Basal levels of COX2 protein, StAR protein and progesterone production in nonstimulated MA-10 cells and R2C cells. MA-10 cells and R2C cells were cultured in serum-free Waymouth’s MB/752 medium for 6 h. The cells were collected and 25 µg of cell lysate protein was used to analyze COX2 and StAR protein levels by Western blot. Progesterone production in the medium was analyzed by RIA and expressed as picogram progesterone per microgram cell lysate protein. *, Very highly significantly different from that of MA-10 cells (P < 0.001).

 
Inhibition of StAR promoter activities by COX2 gene expression
To directly determine the inhibitory effect of COX2 expression on StAR promoter activity, StAR promoter DNA and the COX2 gene were cotransfected into COS-1 cells. Transfection of COS-1 cells with DNA containing the StAR promoter alone resulted in an 8.8-fold increase in StAR promoter activity over levels seen with empty plasmid (Fig. 7Go). However, this StAR promoter activity was significantly reduced by coexpression of the COX2 gene in the same cells. Also, cotransfection of SF-1-containing DNA with StAR promoter-containing DNA enhanced StAR promoter activity to a much higher level than that seen in cells without SF-1. However, the SF-1-enhanced StAR promoter activity was abolished by expression of COX2 protein in the cells.



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  		FIG. 7. Inhibition of StAR promoter activity by over-expression of COX2 in COS-1 cells. COS-1 cells were cotransfected with a StAR promoter/luciferase plasmid (PGL2/StAR), pRL-SV40 vector that constitutively expresses Renilla luciferase, pCR3.1/COX2 or SF-1 plasmid. After 48 h in culture, the cell lysate was used for the luciferase assay using a Dual Luciferase Reporter Assay System as described in Materials and Methods. Data were expressed as fold increases over the promoter activity of control. *, Significantly different from control (P < 0.05). **, Highly significantly different from control (P < 0.01). ***, Very highly significantly different from control (P < 0.001).

 
PKA activity
PKA activity was assayed in NS398-treated cells to determine if inhibition of COX2 activity affected PKA phosphorylating capacity. As shown in Fig. 8Go, stimulation of MA-10 cells with 0.5 mM Bt2cAMP significantly increased PKA activity and StAR protein expression. Stimulation with 0.05 mM Bt2cAMP induced a low level of PKA activity, but did not increase StAR protein as detected by Western blot. NS398 alone increased neither PKA activity nor StAR protein expression significantly. Also, whereas NS398 did not affect 0.05 mM Bt2cAMP-stimulated PKA activity, it consistently increased StAR expression to much higher levels. Addition of 25 µM H89 to the cells treated with NS398 and 0.05 mM Bt2cAMP highly significantly (P < 0.01) decreased PKA activity by 0.24 pmol 32P incorporated per minute per microgram protein (Fig. 9CGo). This decrease represents 92% of the increase in PKA activity (0.26 pmol) that resulted from treatment of the cells with 0.05 mM Bt2cAMP as seen in Fig. 8Go. When this low level of PKA activity in 0.05 mM Bt2cAMP-stimulated MA-10 cells was inhibited by H89, NS398-enhanced StAR protein and steroid production were significantly reduced (Fig. 9Go).



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  		FIG. 8. PKA activity and StAR protein expression in NS398-treated MA-10 Leydig cells. The cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium with or without the COX2 inhibitor NS398 and then stimulated with Bt2cAMP as indicated in the figure. A, Cells were collected and 25 µg of cell lysate protein was used to analyze StAR protein by Western blot. B, Cells were collected in extraction buffer, homogenized and centrifuged at 13,000 x g for 10 min. The supernatant was used for PKA activity assay as described in Materials and Methods. PKA activity was expressed as picomoles of 32P incorporated per minute per microgram protein. *, Significantly different from control (P < 0.05). **, Highly significantly different from control (P < 0.01).

 


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  		FIG. 9. Inhibition of PKA activity reduced NS398-enhanced StAR protein expression and steroid production in MA-10 Leydig cells. MA-10 cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium with the COX2 inhibitor NS398 or the PKA inhibitor H89 as indicated in the figure and then stimulated for 6 h with 0.05 mM Bt2cAMP. A, The cells were collected and 25 µg of cell lysate protein were used to analyze StAR protein by Western blot. B, Progesterone production in the medium was analyzed by RIA and expressed as a percentage of the highest production. C, PKA activities in the cells treated with NS398 or H89 were assayed and expressed as picomoles of 32P incorporated per minute per microgram protein. *, Significantly different from control (P < 0.05). **, Highly significantly different from control (P < 0.01).

 
PGE2 production
Because PGE2 is one of the main products of COX2-catalyzed AA metabolism, PGE2 production in the NS398-treated MA-10 cells was assayed to ensure that COX2 activity was inhibited by this inhibitor. As shown in Fig. 10Go, 0.05 mM Bt2cAMP alone slightly reduced PGE2 production, but, the reduction was not significant. PGE2 levels in all groups treated with NS398 were significantly lower than those of the controls and the 0.05 mM Bt2cAMP-stimulated cells, indicating that COX2 activities were inhibited in the NS398-treated MA-10 cells.



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  		FIG. 10. PGE2 production in NS398-treated MA-10 Leydig cells. MA-10 cells were cultured for 30 min in serum-free Waymouth’s MB/752 medium containing the COX2 inhibitor NS398 or the PKA inhibitor H89 as indicated in the figure and then stimulated for 6 h with 0.05 mM Bt2cAMP. The culture medium was collected and PGE2 production in the medium was assayed using an EIA kit following the manufacturer’s instructions (Cayman). The PGE2 production was expressed as picograms per milliliter culture medium. *, Significantly different from NS398-treated groups (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AA is metabolized through three principle pathways, as named by the AA metabolizing enzymes, lipoxygenase, COX, and epoxygenase. AA metabolites synthesized through the COX pathway include various PGs and thromboxanes (27, 28). In addition to its well-known function in the course of inflammation, studies in recent years have demonstrated important roles for the COX enzyme in steroidogenesis and steroid hormone-regulated physiological functions in both human and animals (29, 30, 31, 32). However, the manner in which COX regulates trophic hormone-stimulated steroidogenesis is unknown.

The present study demonstrated that inhibition of COX activity dramatically increased Bt2cAMP-stimulated StAR protein expression and concomitantly increased steroid hormone production in MA-10 Leydig cells. The increase in steroid output could not be attributed to an increase in the activity of the steroidogenic enzymes P450scc or 3ß-HSD because there was no significant difference in progesterone production among the experimental groups of MA-10 cells coincubated with 22(R)-hydroxycholesterol. The data in the present study suggest that the increase in StAR protein expression is involved in the enhanced steroid production in COX inhibitor-treated cells.

The results presented here indicate that inhibition of COX activity significantly increases the sensitivity of steroidogenesis to cAMP stimulation. Normally, a low level of Bt2cAMP, such as 0.05 mM, is unable to induce detectable amounts of StAR protein or a significant increase in steroids in MA-10 mouse Leydig cells. However, in the presence of the COX inhibitor, indomethacin, the effectiveness of such low levels of Bt2cAMP-stimulation was greatly enhanced and resulted in increases in StAR protein and progesterone to levels comparable to that seen with 0.5 mM Bt2cAMP alone. In addition, progesterone production plateaued at much lower concentrations of Bt2cAMP indicating that a reduction in COX activity lowers the concentration of cAMP necessary for maximal steroid production. Despite the impressive increase in steroidogenesis attributed to the reduction in COX activity, a minimal level of cAMP always appears to be necessary for enhanced steroidogenesis. In the absence of Bt2cAMP, indomethacin alone could induce neither detectable StAR protein nor a significant increase in progesterone production. These observations suggested that the inhibitor itself did not have a direct stimulatory effect on steroid production, but rather, lowered the threshold of cAMP-stimulated steroidogenesis.

The molecular mechanism for the observed effect is unknown. The results from Northern blot analysis of StAR mRNA suggested that inhibition of COX activity enhanced StAR gene transcription. This was confirmed by similar results using StAR promoter assays. Although the COX inhibitor greatly enhanced cAMP-induced StAR gene transcription, the inhibitor itself was not able to increase StAR mRNA or StAR promoter activity indicating that the presence of cAMP is essential for enhanced StAR gene transcription in indomethacin-treated MA-10 cells. Previously, the inhibitory effect of prostaglandin F2{alpha}, an AA metabolite derived through the COX pathway, on StAR gene transcription was described (21, 22). It is possible that blocking the metabolism of AA through the COX pathway reduced an inhibitory metabolite(s) and/or forced more AA to proceed through an alternative metabolic pathway, thus producing a metabolite(s) that enhanced StAR gene transcription when in the presence of cAMP. Obviously, further studies are needed to elucidate the mechanism for the role of COX activity in steroidogenesis.

Two isoforms of the COX enzyme, COX1 and COX2 have been characterized in humans and other animals. It is generally considered that COX1 is constitutively expressed in tissues, whereas COX2 is typically inducible in most cells (33). However, COX2 is constitutively present in non-stimulated MA-10 mouse Leydig cells as detected by Western blot analysis (Fig. 6Go). Although both COX1 and COX2 are capable of catalyzing the conversion of AA to PGH2, a precursor for PGs and thromboxanes (33), their effects on cAMP-stimulated steroidogenesis are quite different. Whereas the selective COX1 inhibitor SC560 was unable to enhance Bt2cAMP-stimulated steroidogenesis, the selective COX2 inhibitor NS398 dramatically increased Bt2cAMP-stimulated StAR protein and steroid production in MA-10 cells, suggesting that COX2 activity is involved in the observed effect on steroidogenesis. Also, cotransfection of the COX2 gene and StAR promoter DNA into COS-1 cells resulted in significant inhibition of both basal and SF-1-enhanced StAR promoter activities. Although evidence explaining why only COX2 is inhibitory is unavailable, the results from the present study suggest that COX2 activity is possibly involved in inhibition of StAR gene expression in MA-10 Leydig cells.

In previous studies, we have discussed the requirement for two signaling pathways (the AA-mediated pathway and the cAMP-PKA phosphorylation pathway), and their concerted involvement in trophic hormone-stimulated steroidogenesis (12, 20, 34). Of the two, cAMP-PKA phosphorylation is the better-known signaling pathway in trophic hormone stimulation and acts by phosphorylating transcription factors as well as perhaps other unknown proteins regulating StAR gene expression (8). It also phosphorylates StAR itself, an event required for maximal StAR activity (35). However, three lines of evidence from the present study indicate that low levels of cAMP-PKA phosphorylation is sufficient for maximal StAR gene expression and steroid production if COX2 activity is inhibited by these COX2 inhibitors. First is the observation that with inhibition of COX2 activity by either NS398 or indomethacin, a low level of cAMP analog was able to induce maximal levels of StAR protein and progesterone in MA-10 cells. Second, in the presence of the inhibitor indomethacin, a low level of Bt2cAMP induced maximal levels of StAR mRNA and StAR promoter activity. Third, the increase of StAR protein expression and steroid production in NS398-treated cells was not due to an increase in PKA phosphorylation because NS398 was unable to enhance PKA activity in MA-10 cells. Although NS398 increased StAR protein expression and steroid production to maximal levels, PKA phosphorylation still remained at the low level observed following stimulation with 0.05 mM-Bt2cAMP. Therefore, it can be readily seen that AA metabolism plays a critical role in the regulation of StAR gene expression and steroidogenesis.

In the absence of a COX2 inhibitor, a low level of PKA activity was unable to induce steroidogenesis; however, this low level is critical for StAR protein expression and steroid biosynthesis because their induction was completely abolished by a PKA inhibitor. Thus, these results corroborate our earlier observations, in which both the AA-mediated pathway and the cAMP-PKA-phosphorylation pathway are required with neither one alone being sufficient for the trophic hormone-stimulated StAR gene expression and steroid production.


   Footnotes
 
The authors would like to acknowledge the support of NIH Grants HD-39308 (to X.J.W.) and HD-17481 and funds from the Robert A. Welch Foundation (to D.M.S.).

Abbreviations: AA, Arachidonic acid; Bt2cAMP, dibutyryl cAMP; COX, cyclooxygenase; EIA, enzyme immunoassay; FBS, fetal bovine serum; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; P450scc, P450 side chain cleavage enzyme; PG, prostaglandin; PKA, protein kinase A; SF, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein; SV40, simian virus 40.

Received November 26, 2002.

Accepted for publication May 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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