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Cyclooxygenases in Rat Leydig Cells: Effects of Luteinizing Hormone and Aging

Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Haolin Chen, Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins University Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: hchen{at}jhsph.edu.

	Abstract

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Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF- or IL-1ß also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

	Introduction

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ADULT LEYDIG CELL testosterone production depends on the pulsatile secretion of LH by the pituitary gland into the peripheral circulation (1). Acting predominantly through a cAMP-dependent pathway, LH has both rapid (acute) and long-term (trophic) effects on Leydig cell testosterone production (2, 3, 4). In its trophic actions, LH maintains the steroidogenic enzymes and normal Leydig cell morphology (2, 3). Acting acutely, LH stimulates the transport of cholesterol to the inner mitochondrial membrane (4). Two proteins have been identified as having important roles in cholesterol transport: peripheral-type benzodiazepine receptor (5) and steroidogenic acute regulatory protein (StAR) (6). There is compelling evidence that the two proteins work coordinately in cholesterol transport (7, 8, 9) with peripheral-type benzodiazepine receptor serving as the "gate" for cholesterol entry into mitochondria (9).

In addition to its role in stimulating cholesterol transport and the steroidogenic enzymes via cAMP, LH stimulates arachidonic acid release from cell membrane lipids through phospholipase A2 and/or acyl-CoA synthetase (10, 11, 12). In turn, arachidonic acid and/or its metabolites have been proposed to be involved in modulating the acute effects of LH on steroidogenesis (12, 13, 14, 15, 16). Arachidonic acid can be metabolized in vivo either to prostaglandins by cyclooxygenases (COX1, COX2) or into 5-hydroxyeicosatetraenoic acid and 5-hydro-peroxyeicosatetraenoic acid by 5-lipoxygenase. Inhibition of COX2 has been shown to enhance StAR and progesterone production by dibutyryl cAMP (dbcAMP) -stimulated MA-10 Leydig tumor cells (17), suggesting that COX2 may serve to suppress steroidogenesis.

In Brown Norway rat Leydig cells, COX2 mRNA and protein levels have been shown to increase with age (18, 19), whereas testosterone production decreases. Recent studies reported that incubation of aged Leydig cells with a COX2 inhibitor resulted in significant increase in testosterone biosynthesis in vitro (19) and that the in vivo treatment of old rats with a COX2 inhibitor resulted in dose-dependent increases in serum testosterone concentration (19). Taken together, these studies suggested a role for COX2 in the reduced testosterone production that characterizes old Leydig cells (20).

The major objective of the present study was to further elucidate the relationships among LH stimulation, Leydig cell expression of the COX proteins (COX1 and 2), aging, and testosterone production so as to better understand the possible role played by the COX proteins in regulating Leydig cell steroidogenesis.

	Materials and Methods

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Reagents
dbcAMP, monoclonal ß-actin antibody, trypan blue (0.4% solution), recombinant mouse IGF-I, mouse TGF-, and mouse IL-1ß were obtained from Sigma (St. Louis, MO). M-199 medium was purchased from GIBCO (Grand Island, NY). Type III collagenase was from Worthington (Freehold, NJ). [1,2,6,7,16,17-³H(N)]testosterone (115.3 Ci/mmol) was from PerkinElmer Life Sciences, Inc. (Boston, MA). Testosterone antibody was from ICN (Costa Mesa, CA). StAR antibody was from Affinity BioReagents Inc. (Golden, CO). COX1 and COX2 antibodies, NS398, and COX activity assay kit were from Cayman Chemical (Ann Arbor, MI). Anti-p44 and p42 MAPKs (ERK1/2) and phospho-ERK1/2 antibodies were from Cell Signaling Technology, Inc. (Beverly, MA). The horseradish peroxidase-conjugated antirabbit donkey IgG and the enhanced chemiluminescence Western blot detection and [³H]cAMP assay kits were from Amersham Pharmacia Biotech (Piscataway, NJ). Bovine LH (USDA-bLH-B-6) was provided by the United States Department of Agriculture Animal Hormone Program (Beltsville, MD). 5,5-Dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl) phenyl-2((5)H)-furanone (DFU) was kindly provided by Dr. Denis Riendeau from Merck Frosst Canada Ltd. (Kirkland, Quebec, Canada).

Animals and treatment
Young (4-month-old) and old (21-month-old) male Brown Norway rats were obtained from Harlan Sprague Dawley (Indianapolis, IN) through the National Institute on Aging (Bethesda, MD). Rats were housed in the animal facilities of the Johns Hopkins School of Public Health at 22 C with a 14-h light/10-h dark light cycle and free access to feed and water.

To inhibit pituitary LH secretion, rats were implanted with 4-cm testosterone-containing capsules for periods of 3 d to 2 wk (21). Testosterone implants were prepared from Dow Corning Medical SILASTIC tubing (1.98 mm internal diameter; 3.18 mm outer diameter; Dow Corning, Midland, MI), filled with testosterone (Steraloids, Newport, RI), and sealed with SILASTIC Medical Adhesive A as previously described (22). Control animals were implanted with 4-cm empty implants. All procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals according to protocols approved by the Johns Hopkins Animal Care and Use Committee.

Leydig cell isolation and treatment in vitro
Leydig cells were isolated from rat testes as previously described (23). In brief, the testicular artery was cannulated and testes were perfused with type III collagenase (1 mg/ml) in dissociation buffer (M-199 medium with 2.2 g/liter HEPES, 1.0 g/liter BSA, 25 mg/liter trypsin inhibitor, 0.7 g/liter sodium bicarbonate, pH 7.4) to clear testicular blood. Testes then were decapsulated and digested in collagenase (0.25 mg/ml, 34 C) with slow shaking (90 cycles/min, 30 min). The dissociated cells were purified by centrifugal elutriation and Percoll gradient centrifugation. The final purity of the Leydig cells, determined by staining for 3ß-hydroxysteroid dehydrogenase, consistently was approximately 95%. Cell viability, assessed by trypan blue exclusion, was over 95%.

To assess the effect of LH on COX protein levels in vitro, freshly isolated Leydig cells (5 x 10⁵) were incubated with LH (0.1–100 ng/ml) in 500 µl of M-199 medium (containing 2.2 g/liter HEPES, 0.1% BSA, 2.2 g/liter sodium bicarbonate, pH 7.4) at 34 C. Control cells were incubated in the absence of LH. In some experiments, H89 (50 µM) was added to the culture medium 30 min before LH to block protein kinase A (PKA) activation. In other experiments, dbcAMP (2 mM) rather than LH was included in the medium to activate PKA directly. To examine the effects of growth factors and cytokines on COX protein levels, the cells were treated with IGF-I (100 ng/ml), TGF- (100 ng/ml), or IL-1ß (IL-1, 100 ng/ml) for 2 h. After treatment, the media were collected for assays of testosterone by RIA. The cells were lysed for colorimetric assay of COX activity or for assay of COX and StAR protein levels by Western blots (see Western blot analysis section).

To examine the effect of COX2 inhibition on the age-related changes in steroidogenesis, Leydig cells (1 x 10⁵) isolated from 4-month-old (young) or 21-month-old (aged) rats were preincubated with one of two COX2 inhibitors, NS398 (5–80 µM) and DFU (0.1–200 µM) for 30 min. The cells then were treated with LH (0.1 ng/ml) for 3 h with COX2 inhibitors still in the medium. The medium was collected for testosterone assay by RIA.

Testosterone and COX activity assays
Testosterone in the medium was assayed by RIA. The sensitivity and intraassay and interassay coefficients of variation of the RIA were 13 pg/tube, 8.9%, and 13.6%, respectively. COX activity was assayed using kits obtained from Cayman Chemical according to the manufacturer’s instructions. In brief, the peroxidase component of the enzymes was assayed colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine at 590 nm (24) in whole-cell lysates. The two COX isoforms, COX1 and COX2, were distinguished by including specific COX1 and COX2 inhibitors (SC-560 for COX1 and DuP-697 for COX2) in the assay.

Western blot analysis
After treatment with LH, growth factors or cytokines, the cells were lysed with Tris-sodium dodecyl sulfate (SDS) buffer (62.5 mM Tris, 2% SDS, 50 mM dithiothreitol, pH 6.8) and sonicated on ice. Lysates were mixed with 3 x SDS loading buffer (New England BioLabs, Ipswich, MA). Total proteins from equal numbers of cells (2 x 10⁵) were separated on a 10% polyacrylamide SDS gel and then transferred onto a nitrocellulose membrane. After incubation with primary antibody (1:1000) and horseradish peroxidase-conjugated secondary antibody (1:5000), the signals were detected by the enhanced chemiluminescence Western blot kit from Amersham Pharmacia Biotech (Piscataway, NJ). In most experiments, bound antibodies in the membranes were stripped by Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) and reblotted with new antibodies in the sequence of COX2, COX1, StAR, and ß-actin. The proteins were quantified by densitometry scanning of the film and processed by NIH Image software. The total density of the proteins in each membrane was first corrected by ß-actin and then normalized to the control sample, usually young untreated cells, in the same membrane. At least three pools of cells that came from different animals were analyzed for each treatment.

Statistical analyses
Data are expressed as the mean ± SEM of three to eight different pools of cells that came from different animals. Statistical differences were determined by one-way ANOVA. If group differences were revealed by ANOVA (P < 0.05), differences between individual groups were determined with the Student-Newman-Keuls test using SigmaStat software. Values were considered significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vitro regulation of leydig cell COX by LH
The effect of LH on COX proteins was examined by incubating purified Leydig cells from young adult rats with maximally stimulating LH (100 ng/ml) for 15 min to 24 h in vitro (Fig. 1A). Increased intracellular COX2 protein content was first noted by 1 h of incubation with LH and further increases were seen at 2 and 24 h. In contrast, COX1 protein increased little with LH stimulation. StAR, which is highly responsive to acute LH stimulation, was used as a positive control in this study. As is evident from the Western blots, very substantial increase in Leydig cell StAR protein was noted by 24 h of LH incubation but not at 2 h. Figure 1B compares the activities of COX1 and COX2 in response to LH treatment of the Leydig cells. Without LH (basal), Leydig cells had approximately equal COX1 and COX2 activities. With 2 h of LH treatment, however, COX2 activity increased significantly (up to 240% of the basal level), whereas COX1 activity did not change. This is consistent with the Western blot results seen in Fig. 1A.

View larger version (15K): [in this window] [in a new window]   FIG. 1. LH regulation of COX proteins (A) and activities (B). A, Adult Leydig cells were incubated with maximally stimulating LH (100 ng/ml) for 0.25–24 h. Membranes containing total proteins from equal numbers of cells (2 x 105) were sequentially blotted with COX2, COX1, StAR, and ß-actin antibodies. Above, Western blots. Below, Plots of the relative densities (ratio to ß-actin) of COX2 in response to LH from four experiments. B, COX1 and COX2 activities were assayed in adult Leydig cells after their maximal stimulation by LH for 2 h. Each bar represents the mean ± SEM of four experiments. *, Significantly different from controls (designated "0" in A and "basal" in B); P < 0.05.

View larger version (15K): [in this window] [in a new window]   FIG. 2. PKA and LH stimulation of COX2 (A) and testosterone (B) production. Adult Leydig cells were incubated with LH (100 ng/ml) or dbcAMP (2 mM) for 2 h. Cells in a third group were incubated with LH and the PKA inhibitor H89 (50 µM). A, Membranes containing total proteins from equal numbers of cells (2 x 105) were sequentially blotted with antibodies to COX2, COX1, and ß-actin. Above, Western blots. Below, Plots of relative densities (ratio to ß-actin) of COX2 protein from four separate experiments. B, Testosterone in the medium of the incubated cells. Each bar represents the mean ± SEM of four experiments. *, Significantly different from control (C); P < 0.05.

View larger version (15K): [in this window] [in a new window]   FIG. 3. LH regulation of testosterone production (A) and COX2 protein (B) in vivo. Rats received 4-cm testosterone implants for 3 (+3d) or 14 (+14d) d. For some animals, the implants were removed after 14 d of implantation, and animals were allowed to recover for 14 d (±14d). Leydig cells isolated from controls (C) and from each of the three experimental groups (+3d, +14d, ±14d) were incubated in the absence (–) or presence (+) of LH (100 ng/ml) in vitro for 2 h. A, Testosterone in the medium of the incubated cells. B, Membranes containing total proteins from equal numbers of cells (2 x 105) were sequentially blotted with antibodies to COX2, COX1, StAR, and ß-actin. Above, Western blots. Below, Plots of relative densities (ratio to ß-actin) of COX2 protein from three separate experiments. Bars represent mean ± SEM. a, Significantly different from control (C); P < 0.05. b, Significantly different from values derived from cells in the absence of LH in vitro; P < 0.05.

Regulation of Leydig cell COX proteins by growth factors and cytokines
To examine whether factors in addition to LH can affect Leydig cell COX proteins, cells were incubated with IGF-I, TGF-, or IL-1ß (Fig. 4A), all of which have been reported to be involved in the regulation of Leydig cell function (25). With 2 h of treatment, both TGF- and IL-1ß increased COX2 protein levels significantly above control levels, whereas IGF-I had no effect. The effect of IL-1ß was comparable to that of LH. In contrast to LH, TGF- and IL-1 incubations also resulted in increased phosphorylation of MAPK (P-ERK1/2). Total ERK1/2 protein (T-ERK1/2) was not affected by any of these factors. With respect to testosterone production (Fig. 4B), incubation of the cells with LH increased production, but IL-1ß and TGF-, although they up-regulated COX2 protein, had little effect on testosterone production. To examine whether TGF-, IL-1ß, and LH stimulate COX2 protein through the same mechanism/pathway, cells were treated with maximal concentrations of the three proteins separately or in combination. As seen in Fig. 5, the stimulatory effect of maximally stimulating LH on COX2 protein was enhanced by TGF- and IL-1ß, suggesting that LH, TGF-, and IL-1ß may not use the same mechanism/pathway to up-regulate COX2 protein concentration in Leydig cells.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Effects of IGF-I, TGF-, and IL-1ß on COX2 (A) and testosterone (B) production. Leydig cells were incubated with LH (100 ng/ml), IGF-I (100 ng/ml), TGF- (100 ng/ml), or IL-1ß (100 ng/ml) for 2 h. A, Membranes containing total proteins from equal number of cells (2 x 105) were sequentially blotted with antibodies to COX2, COX1, ß-actin, phospho-MAP kinase (P-ERK1/2), and total MAP kinase (T-ERK1/2). Above, Western blots. Below, Plots of relative density (ratio to ß-actin) of COX2 protein from four experiments. B, Testosterone in the medium of the incubated cells. Each bar represents the mean ± SEM from four experiments. *, Significantly different from control (C); P < 0.05.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Effects of combinations of LH, growth factors, and cytokines on COX2 protein. Leydig cells were incubated with combinations of LH (100 ng/ml), IGF-I (100 ng/ml), TGF- (100 ng/ml), and IL-1ß (100 ng/ml) for 2 h. Membranes containing total proteins from equal numbers of cells (2 x 105) were sequentially blotted with antibodies to COX2 and ß-actin antibodies. Above, Western blots. Below, Plots of the relative densities (ratio to ß-actin) of COX2 protein from three experiments. Each bar represents the mean ± SEM. a–d, Differing letters indicate significant differences; P < 0.05.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Effect of age on the regulation of COX proteins. A, Leydig cells were isolated separately from old rats with normal ("big") testes (OB; testis weight 1.5 g) or with regressed or partially regressed ("small") testes (OS; testis weight < 1.5 g) to distinguish between age-related effects and those that were secondary to germ cell loss. Above, Western blots. Below, Relative COX2 protein content in Leydig cells isolated from young (Y) and old (OB and OS) testes. B, Effect of LH on COX2 protein in young and old Leydig cells. Each bar represents the mean ± SEM of three experiments. *, Significantly different from controls ("Y" in A and "0" in B); P < 0.05.

To further examine the involvement of COX2 protein in the age-related decrease in Leydig cell testosterone production, COX2 activity was blocked in young and old Leydig cells by one of two specific inhibitors, NS398 and DFU (Fig. 7). For these studies, cells were preincubated with inhibitor for 30 min and then were incubated with inhibitor and 0.1 ng/ml LH for an additional 3 h. In cells cultured in the absence of inhibitor, testosterone production was significantly lower by the old than the young cells. With increasing concentrations of each of the two inhibitors, testosterone production was increased significantly in both young and old cells.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Effect of COX2 inhibitors (NS398 and DFU) on testosterone production by Leydig cells from young and aged rats. Leydig cells isolated from 4-month-old (young) or 21-month-old (aged) rats were preincubated with either NS398 (A) or DFU (B) for 30 min. The cells then were treated with LH (0.1 ng/ml) for 3 h with COX2 inhibitor still in the medium. Testosterone in the medium was assayed by RIA. Each bar represents the mean ± SEM of eight experiments. #, Significantly different from young cells without inhibitor (young 0); P < 0.05. *, Significantly different from cells without inhibitor (0) of the same age; P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Incubation of Leydig cells with TGF- or IL-1ß also increased COX2 protein levels significantly above control levels, whereas IGF-I had no effect. Indeed, the stimulatory effect of maximally stimulating LH on COX2 protein was enhanced by TGF- and IL-1ß. Although TGF- and IL-1ß up-regulated COX2 protein, they had little effect on testosterone production.

The results of our in vivo studies were consistent with the in vitro observation that LH stimulated both COX2 and testosterone but not COX1. Suppression of pituitary LH secretion with testosterone-containing SILASTIC capsules resulted in decreases in both testosterone production and COX2 protein. This was seen at 3 d postimplantation, the earliest time examined. Surprisingly, basal COX2 levels did not remain suppressed in the absence of LH stimulation, but rather were increased above control levels in Leydig cells isolated from the 14-d implanted rats, whereas testosterone production remained suppressed. The explanation for this unexpected finding is not known. Clearly, the fact that increased COX2 occurred despite LH suppression indicates that Leydig cell COX2 is regulated by factors in addition to LH in vivo. Based on the observations that TGF- and IL-1ß are capable of up-regulating Leydig cell COX2 protein in vitro, it is possible that testicular growth factors and/or cytokines from macrophages are involved in COX2 regulation. It should be noted, however, that macrophages do not have LH receptors and therefore any effect of LH withdrawal on macrophage function presumably must be mediated through Leydig cells (30). It also is possible that COX2 is regulated by factors from the seminiferous tubules. For example, on LH withdrawal, spermatogenesis is affected in approximately 2 wk (31), about the time frame in which COX2 increases were seen in the implanted rats. However, there are no known suppressors of COX2 in the testis and therefore it is unknown how decreases in spermatogenesis and/or changes in Sertoli cell function might up-regulate COX2 in Leydig cells. Interestingly, although the Leydig cells from the 2-wk implanted rats responded to LH stimulation with increased testosterone production, the cells did not produce increased COX2. Again, the explanation is not known. Finally, in rats in which implants were left in place for 14 d and then removed, COX2 protein levels, both basal and LH stimulated, were at control levels 2 wk thereafter, indicating that the effects of LH withdrawal on steroidogenesis and COX2 protein are reversible.

Previous studies have suggested that COX2 might play a regulatory role in the reduction in testosterone production that characterizes aged Leydig cells (19). Evidence for such a role is that COX2 mRNA and protein levels have been shown to increase in aged compared with young cells, whereas testosterone production and StAR decrease (18, 19, 20, 32); the incubation of aged rat Leydig cells with a COX2 inhibitor resulted in significantly increased testosterone biosynthesis by these cells (19); and the administration of a COX2 inhibitor to aged rats through the diet resulted in increased serum testosterone levels (19). In the present study, we also noted that COX2 expression is greater in old than in young cells and confirmed that old Leydig cells respond to inhibition of COX2 in vitro with increased testosterone production. However, we also found that this effect of COX2 inhibition was not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. These results suggest that the proposed relationship between COX2 and testosterone production is not unique to aged Leydig cells.

The very issue of the proposed role of COX2 in the suppression of Leydig cell steroidogenesis is called into question by the observations relating LH stimulation to COX2 and testosterone production. In particular, we found that COX2 protein in both young and old Leydig cells increased when the cells were incubated with LH as did testosterone production and StAR expression by these cells. Clearly, the observation that COX2, StAR, and testosterone all increased significantly in response to LH is inconsistent with the contention that increased COX2 causes reduced steroidogenesis. Understanding the seemingly opposing observations that, on the one hand, the inhibition of COX2 can result in increased steroidogenesis in both young and aged cells, and on the other that LH stimulation can result in the up-regulation of COX2, StAR, and steroidogenesis, requires further study.

The mechanism by which increases in COX2 protein occur as Leydig cells age is uncertain. One possibility is that cytokine-induced COX2 production may occur with aging. This is based on previous studies showing that aging typically is associated with increase in cytokine production (33) and, by our observation, that specific cytokines (IL-1, for example) can significantly increase COX2 production by adult Leydig cells. A second possibility is based on the seemingly conflicting observations that although acute (hours-long) exposure to LH stimulates adult Leydig cell COX2 protein, longer-term (weeks-long) in vivo suppression of LH actually increases basal levels of Leydig cell COX2 with the cells losing their ability to produce COX2 in response to acute LH stimulation. Aged Leydig cells also are characterized by relatively high basal levels of COX2 and reduced COX2 production in response to LH. Although serum LH does not decrease significantly in old Brown Norway rats (20, 34), Leydig cells isolated from these animals have a reduced ability to produce cAMP under LH stimulation (35). The defect in the LH signal pathway of old cells thus may contribute to the age-related increase in COX2 protein production in Leydig cells much as LH suppression leads to increased COX2 protein in adult Leydig cells. The mechanism, however, is far from clear.

	Footnotes

 
This work was supported by National Institutes of Health Grants AG026721 (to H.C.) and AG21092 (to B.R.Z.) from the National Institute on Aging.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: COX, Cyclooxygenase; dbcAMP, dibutyryl cAMP; DFU, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl) phenyl-2((5 )H)-furanone; PKA, protein kinase A; SDS, sodium dodecyl sulfate; StAR, steroidogenic acute regulatory protein.

Received July 11, 2006.

Accepted for publication October 16, 2006.

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