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Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles
Life Sciences 70 (2001) 57–72
Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles Chi-Chuan Yua, Wei-Yi Chenb, P. Shirley Lia,* a
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Rep. of China b Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Rep. of China Received 19 February 2001; accepted 25 June 2001
Abstract The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares’ serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 – 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22Rhydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (1025 M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A–stimulated steroidogenic activity of the preovulatory follicle. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Rat preovulatroy follicles; Cantharidin; Steroidogenic acute regulatory protein; Progesterone
* Corresponding author. Department of Physiology, College of Medicine, National Cheng Kung University, #1, Ta-hsueh Rd, Tainan 70101, Taiwan, Rep. of China. Tel.: 1886-6-235-3535 Ext. 5437; fax: 1886-6-236-2780. E-mail address: [email protected] (P.S. Li) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 6 9 -8
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Introduction Cantharidin takes its name from Cantharis vesicatoria, one of more than 2000 species of beetles of the order Coleoptera, family of Meloidae, which contain a vesicant liquid and are known as “blister beetles”. The beetles are most commonly found in southern Europe, Africa, and Asia. The Mediterranean beetle Cantharis vesicatoria is the most well known as the “Spanish fly”. Cantharidin is the toxic constituent of blister beetles and the active ingradient of the purported aphrodisiac and abortifacient Spanish fly [1]. Cantharidin has been used since ancient times to enhance sexual potency, a practice related to findings of priapisom in men and pelvic organ engorgement in women, which result from high-dose exposure. The aphrodisiac legend stems from cantharidin’s ability to cause vascular congestion and inflammation of the genitourinary tract, a sensation that may be interpreted as enhanced sexuality by some [2]. Recently, cantharidin has been identified as a strong inhibitor of protein phosphatases types 1 (PP1) and 2A (PP2A) [3]. The key role played by protein kinases and protein phosphorylation in the regulation of steroidogenesis is well documented [4, 5]. It is generally accepted that hormone-stimulated steroidogenesis is dependent upon the phosphorylation of protein substrate on serine or threonine [6, 7]. In contrast to the widely studied role of phoshporylation events, the dephosphorylation processes involved in steroidogenesis are not so broadly described. The presence of the serine/threonine phosphatases PP1 and PP2A in rat luteal cells and adrenal cortex has been reported [8, 9]. Regarding their participation in the acute regulation of steroidogenesis, the results are controversial. In isolated luteal cells, the PP inhibitors, okadaic acid and calyculin A, reduced LH-induced progesterone biosynthesis [10], whereas in granulosa cells, inhibition of PP1 and PP2A enhanced FSH-stimulated steroidogenesis [11]. In Leydig cells, cantharidin inhibited testosterone production [12]. The biosynthesis of gonadal steroid hormones — progesterone, estrogens, and androgens — begins from cholesterol. The acute regulated and rate-limiting step for the synthesis of steroid hormones is the delivery of cholesterol from cellular stores to the mitochondrial inner membrane where the cholesterol is then converted to pregnenolone by cytochrome P450 side-chain cleavage (P450scc) enzyme [13, 14]. The transport of cholesterol in steroidogenic cells is thought to be mediated by steroidogenic acute regulatory (StAR) protein [15]. StAR protein has been identified as a mitochondrial phosphoprotein [16], which is rapidly induced by tropic hormones [17, 18]. The decisive demonstration came from an inherited disease that leads to a dramatic deficiency in all steroid hormones, congenital lipoid adrenal hyperplasia (CAH): mutations in the StAR gene have been shown to underlie this disorder [19, 20]. Moreover, mice in which there is a null mutation for StAR have a phenotype similar to lipoid CAH patients [21]. Results from recent studies have demonstrated the pattern of expression and regulation of StAR in ovarian cells during various physiological processes. High levels of StAR protein and mRNA were observed in functional corpus luteum (CL), whereas they were absent in regressed CL [22–24]. Their expression was subject to luteotropic hormones such as eCG, hCG [25], LH [26], and estradiol [27] as well as the luteolytic agent prostaglandin F2a(PGF2a) [23, 25, 26]. StAR is also regulated in a gonadotropin-dependent and stagespecific manner during follicular development [22, 25, 28, 29]. Studies in vitro showed that
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gonadotropins and activators of the PKA pathway increase StAR expression in granulosa cells [22, 28–32], whereas PGF2a and phorbol 12-myristate 13-acetate appeared to be negative regulators of StAR expression in vivo and in vitro [25, 26, 28, 29]. In view of the importance of StAR in basal and hormonally regulated steroidogenesis and the limited knowledge of cantharidin effect on this gene in ovarian follicles, we have used the rat preovulatory follicles as an experimental model to investigate the effects of cantharidin on the expression of StAR protein and progesterone production. Materials and methods Materials Purified porcine pituitary LH (pLH; USDA-pLH-B-1; 1.7 U/mg) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Pregnant mares’ serum gonadotropin (PMSG), BSA (fraction V, fatty-acid free), 8-bromo-cAMP (8-Br-cAMP), cantharidin, and 22R-hydroxycholesterol were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Ham’s F12/Dulbecco’s modified Eagle’s medium (F12/DMEM; 1:1) and other culture supplies were purchased from GIBCO-BRL (Grand Island, NY). Acrylamide, bisacrylamide, and SDS were purchased from Bio-Rad (Richmond, CA). Antiserum directed against amino acids 88–98 of mouse StAR protein was kindly provided by Dr. D.M. Stocco (Department of Cell Biology & Biochemistry, Texas Tech University, Lubbock, TX). Rabbit anti-P450scc antiserum (provided by Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan) was generated by immunization with a human P450scc fusion protein overexpressed in Escherichia coli [33]. Animals Immature (26-day-old) female Wistar rats (National Cheng Kung University Laboratory Animal Center, Tainan, Taiwan) were maintained in 22–24 8C rooms on a 12-h light, 12-h dark schedule (lights on at 0600 h). Food and water were provided ad libitum. The rats received a single subcutaneous injection of 10 IU PMSG in 0.1 ml PBS (pH 7.4) to induce multiple follicle growth. At 48 h later, animals were killed by cervical dislocation, and ovaries were removed, trimmed of adhering tissues and placed in 100-mm glass petri dishes containing F12/DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM) and 0.1% BSA (F12/DMEM-BSA) for follicle dissection. At all times, the animals were treated in accordance with the NIH guide for the Care and Use of Laboratory Animals. Follicle isolation and incubation The largest preovulatory follicles (.400 mm in diameter) were isolated from the ovaries, and follicle culture was performed as previously described [34]. After being cleaned of adhering stromal tissue and/or smaller follicles, eight follicles were incubated in a culture dish (35 3 10 mm) containing 1.5 ml F12/DMEM-BSA in the absence or presence of increasing concentrations of cantharidin, or as indicated, with at least 3 dishes used for each treatment. Cultures were maintained for 24 h at 378C under 5% CO2 and 95% O2. At the end of incubation, follicles were solubilized in TSE buffer (10 mM Tris, 0.25 M sucrose, 0.1 mM EDTA,
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pH 7.4) for extracting mitochondrial protein for subsequent Western blot analysis, and the media were collected for the determination of progesterone level by RIA. Each set of experiments was performed 2–3 times. Western blot analysis For Western blotting, mitochondria were isolated by mechanical homogenization of the follicles and differential centrifugation, as described previously [34]. Mitochondrial proteins (50 mg/lane) were solubilized in SDS-PAGE sample buffer (58.3 mM Tris-HCl, pH 6.8, 1.7% SDS, 1% 2-mercaptoethanol, 5% glycerol, and 0.002% bromophenol blue), boiled for 5 min, and loaded into a 12.5% mini gel by standard SDS-PAGE procedures, along with prestained molecular weight markers (Bio-Rad). Electrophoresis was performed at 50 mA/min for 45 min at room temperature using a standard SDS-PAGE running buffer (25 mM Tris-base, 192 mM glycine, 0.1% SDS, pH 8.3). The proteins were electrophoretically transblotted to a polyvinylidene difluoride membrane (Bio-Rad) using a semidry electro-transfer apparatus (Hoefer Pharmacia Biotech Inc., San Francisco, CA). Protein transfer was conducted for 40 min at 0.8 mA/cm2 in transfer buffer containing 20 mM Tris-base, 150 mM glycine, 10% methanol, and 0.01% SDS at pH 8.3. Then, the membrane was blotted as previously described [34]. In some experiments, following detection of StAR protein, the membrane was stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol at 508C for 1 h, washed three times for 5 min each in PBS containing 0.5% Tween-20, and then probed with P450scc antiserum (1:2500). The intensity of the bands on Western blots was measured by the Arcus II computer-assisted image system (PDI Inc., Huntington Station, NY). Values obtained were expressed as integrated optical density units, as previously described [34]. RIA for progesterone Quantitation of progesterone directly from aliquots of the medium was performed by RIA as previously described [35]. The antiserum to progesterone-11a-BSA was supplied by Dr. J.E. Hixon (Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL) and used at the dilution of 1:25,000 in 0.1 M Tris buffer (pH 7.4). The sensitivity of the assay was 12.5 pg per assay tube. Statistical analysis All values were expressed as the mean 6 SEM of pooled data from 2–3 experiments. Two means were compared using Student’s t-test. Where there were more than two means, significant differences between means were determined by analysis of variance. The means were then analyzed by Fisher’s PLSD multiple comparison. Results Dose-dependence effect of LH on StAR protein expression and progesterone production To determine the optimal dose of LH, preovulatory follicles were cultured for 24 h with various doses of LH, and StAR protein levels were determined. As shown in Figure 1a, a dose-dependent relationship between StAR protein expression and LH treatment was readily
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Fig. 1. Effect of increasing concentrations of LH on StAR protein levels (a) and progesterone production (b). Preovulatory follicles (.400 mm in diameter) isolated from ovaries of PMSG-primed immature rats were cultured (8 per culture/1.5 ml) in serum-free medium for 24 h in the absence (C, control) or presence of LH (0.01 – 1000 ng/ ml). At the end of culture, mitochondrial protein extracts were dose dependently prepared from 24 follicles of 3 cultures at each dose of LH as described in “Materials and Methods.” For each sample, 50 mg of mitochondrial proteins were analyzed for StAR protein by SDS-PAGE and immunoblotting also as described in “Materials and Methods.” The position of the StAR protein is indicated. StAR protein was detected by the Arcus II computerassisted image system at each treatment. Integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of LH. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 0.01 ng/ml.
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seen in rat preovulatory follicles as determined by Western blot analysis. StAR protein was low in unstimulated follicles, but could be seen with levels of LH as low as 0.01 ng/ml. The level of StAR protein continued to increase, reaching a maximal value at 100 ng/ml. This dose was used in the subsequent experiments. Progesterone production in the culture medium from the same experiment is shown in Figure 1b. LH at concentrations of 0.1, 1, 10, 100, and 1000 ng/ml caused a dose-dependent increase in progesterone production, reaching a maximal effect at a concentration of 100 ng/ml. Effect of treatment with cantharidin on the expression of StAR and P450scc proteins and progesterone production To investigate the effects of cantharidin on the expression of StAR and P450scc proteins, preovulatory follicles were incubated for 24 h with or without increasing concentrations of cantharidin in the presence or absence of LH. As shown in Figure 2a, cantharidin had a significant (P , 0.05) effect in suppressing LH-stimulated StAR protein expression. The inhibitory effect was concentration dependent, and a 24-h exposure of preovulatory follicles to cantharidin concentrations ranging from 100 to 1000 ng/ml decreased StAR protein levels by 24–70%. Basal StAR protein levels were low after 24 h of culture. Treatment with cantharidin had no effect on P450scc protein levels. (Fig. 2a). Results in Figure 2b show that cantharidin at a concentration of 100 ng/ml or higher significantly (P , 0.05) decreased LH-stimulated progesterone production. The inhibited level was about 40–78% of that treated with LH alone (11.861.8 ng/8 follicles/24 h). The basal accumulation of progesterone was 0.4 6 0.1 ng/8 follicles per 24 h which was 4.4 6 2.1% of that in cultures treated with LH alone. Effect of treatment with cantharidin on P450scc activity To test if cantharidin could act by inhibiting the activity of P450scc, follicles were cultured for 24 h with or without cantharidin (500 ng/ml) and 22R-hydroxycholesterol (1026 M) in the presence or absence of 8-Br-cAMP (0.5 mM). The results are presented in Figure 3. Cantharidin had no effect on the conversion of 22R-hydroxycholesterol to progesterone. Effect of treatment with cantharidin on forskolin- and 8-Br-cAMP-stimulated StAR protein expression and progesterone production The influence of cantharidin upon the ability of forskolin (1025 M), an activator of adenylate cyclase, and 8-Br-cAMP (0.5 mM), a cAMP analog, to stimulate StAR protein expression was also investigated. Cantharidin at a concentration higher than 50 ng/ml inhibited (P , 0.05) the stimulatory effect of forskolin on StAR protein to about 97% (Fig. 4a). Progesterone production in control cultures averaged 0.6 6 0.1 ng/8 follicles/24 h and was increased (P , 0.05) by forskolin stimulation (9.361.2 ng/8 follicles/24 h) (Fig. 4b). Cantharidin at a concentration higher than 100 ng/ml reduced the stimulatory effect of forskolin (P , 0.05). Results in Figure 5a show that the stimulatory effect of 8-Br-cAMP on StAR protein expression was inhibited by concomitant treatment with cantharidin (50, 100, 500 or 1000 ng/ ml) in a dose-dependent manner, decreasing from 54.0 6 6.4% to 9.5 6 2.8%. Treatment with 8-Br-cAMP significantly (P , 0.05) increased progesterone production (9.560.9 ng/8 follicles/24 h) as compared to basal levels (0.560.2 ng/8 follicles/24 h) (Fig. 5b), whereas
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Fig. 2. Effect of the dose of cantharidin on LH-stimulated StAR and P450scc protein levels (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) LH (100 ng/ml) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR and P450scc proteins. The positions of the StAR and P450scc proteins are indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from three independent experiments. P , 0.05 between control and concentration above 50 ng/ml.
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Fig. 3. Effect of cantharidin on the conversion of 22R-hydroxycholesterol to progesterone. The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (control) cantharidin (500 ng/ml) and 22R-hydroxycholesterol (1026 M) in the absence (control) or presence of 8-Br-cAMP (0.5 mM). The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each treatment. Values are the mean 6 SEM from three independent experiments. Bars with different letters differ significantly (P , 0.05).
concomitant treatment with increasing concentrations of cantharidin reduced progesterone production to 5%. Discussion Using a serum-free culture system of preovulatory follicles isolated from ovaries of immature rats primed with PMSG, we have demonstrated that cantharidin is able to significantly inhibit LH-, forskolin-, or 8-Br-cAMP-induced StAR protein levels. The cantharidin effect is dosedependent and is associated with decreases in progesterone production. These results indicate that inhibiting PP1 and PP2A activities by cantharidin blocks PKA-induced steroidogenesis primarily through down-regulating StAR protein expression. Since cantharidin had no effect on 22R-hydroxycholesterol-supported steroidogenesis, this clearly demonstrates that cantharidin does not interfere with the activity of P450scc. Serine/threonine protein phosphatases can be divided into four groups, the homologous type 1, 2A and 2B, and the distinct 2C. These groups exhibit differences in substrate specificity, subunit structure, sensitivity to inhibitors and cation requirements [36]. Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of PP1 and PP2A [3]. The Ki of cantharidin for PP2A is approximately 0.2 mM and the Ki for PP1 is approximately 1.1 mM [3]. In vitro it inhibits purified catalytic subunit of PP2A (IC50 5 0.16 mM) at a lower concentration than that of PP1 (IC50 5 1.7 mM) and only inhibits the activity of PP2B at high concentrations. In intact cells, cantharidin completely inhibits PP1 and PP2A at a concentration of 100 mM [3]. In this study we observed inhibition of progesterone production at concentrations greater than 0.2 mM (50 ng/ml), suggesting that PP2A, rather than PP1, is involved. Results presented herein are in contrast with a recent study showing that inhibition of PP1 and PP2A by okadaic acid, a marine toxin, enhanced progesterone secretion in FSH-treated
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Fig. 4. Effect of the dose of cantharidin on forskolin-stimulated StAR protein level (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) forskolin (1025 M) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 50 ng/ml for StAR protein and 100 ng/ml for progesterone production.
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Fig. 5. Effect of the dose of cantharidin on 8-Br-cAMP-stimulated StAR protein level (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) 8-Br-cAMP (0.5 mM) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 10 ng/ml for StAR protein and 100 ng/ml for progesterone production.
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preantral primary granulosa cells [11]. The discrepancy in results may be explained by the differences in experimental conditions used, such as the status of differentiation of the follicles. Granulosa cells from diethylstilbestrol-treated rats [11] might be considered nontransformed and undifferentiated or in the process of differentiation, while granulosa cells in preovulatory follicles would be more mature and highly differentiated. It has been reported that in rat luteal cells, cantharidin caused a dose-dependent inhibition of LH-induced progesterone secretion [10]. Thus, it would appear that PP1/2A-induced dephosphorylation reactions may provide a tonic negative mechanism controlling FSH action in granulosa cells in the follicular phase of the ovarian cycle but predominantly enhance LH-stimulated steroidogenesis during the luteal phase. Our results also highlight that regulation of the protein serine/threonine phosphorylation balance is involved in the regulation of follicular steroidogenesis. Moreover, inhibition of PP1 and PP2A has been reported previously to inhibit secretion from a number of different endocrine cell types, including glucose-induced insulin secretion from rat pancreatic islets of Langerhans [37] or mouse b-cells [38], lipoprotein-stimulated corticosterone secretion from rat adrenocortical cells [39], thyrotropin-releasing hormone-induced thyrotropin and prolactin secretion from rat anterior pituitary slices [40] and carbachol-induced secretion of catecholamines from bovine adrenal medullary cells [41]. Whether the inhibitory mechanism of cantharidin is mediated prior or distal to cAMP formation was examined by investigation of the effect of cantharidin on forskolin- and 8-Br-cAMPstimulated progesterone production. Cantharidin treatment suppressed forskolin-stimulated progesterone production. This finding suggests that cantharidin may suppress adenylate cyclase activity, resulting in the reduction of cAMP formation. In addition, to reduce cAMP formation, our observation that cantharidin treatment also attenuated 8-Br-cAMP-stimulated progesterone production indicated that the action of cantharidin is exerted, at least in part, at a point distal to the generation of cAMP. However, the 25% inhibition by cantharidin, at the dose of 500 ng/ml, of 8-Br-cAMP-stimulated progesterone production (data shown in Fig. 3) was not consistent with the 80% inhibition by the same dose of cantharidin on progesterone (data shown in Fig. 5). The inconsistency in the effect of cantharidin on 8-Br-cAMP-stimulated progesterone production may suggest that at least a part of the inhibitory mechanism of cantharidin is mediated prior to cAMP formation. With regard to the direct cellular mechanisms responsible for the effect of cantharidin on hormone-induced steroidogenesis, our present study demonstrates that in rat preovulatory follicles, cantharidin inhibits LH-induced progesterone production, whereas the amount of P450scc is not affected by cantharidin treatment, indicating that the loss of steroidogenic capacity is not a result of inhibition of P450scc. In contrast to the inhibition of 8-Br-cAMPinduced steroidogenesis, the inhibitory effect of cantharidin could be reversed by 22Rhydroxycholesterol. This suggests that the major inhibitory effect of cantharidin is the decrease in the availability of cholesterol substrate in the mitochondria. The results lend support to previous reports, in which inhibitors of PP1/2A did not affect 22R-hydroxycholesterol-supported steroidogenesis [9, 10]. In the present study, we further demonstrated that cantharidin decreased LH-induced, forskolin-induced, or 8-Br-cAMP-induced StAR protein levels. This suggests that in rat preovulatory follicles, inhibiting PP1 and PP2A activities blocks PKA-induced progesterone production primarily through preventing the increase in StAR protein production. Diminished levels of StAR protein would likely result in a reduction in the capacity of
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preovulatory follicles for progesterone production, in response to gonadotropins, by limiting the availability of cholesterol to the P450scc complex, located in the inner mitochondrial membrane. Previous studies of the StAR protein have indicated that it has an indispensable role in steroid hormone biosynthesis [19], and it has been further postulated that this role is in regulating cholesterol transfer to the inner mitochondrial membrane [15, 16]. It appears that the cantharidin-induced depression in progesterone production in preovulatory follicles is to be due to the inhibition of StAR protein synthesis. This observation is highly consistent with previous studies in which inhibition of steroid hormone biosynthesis, such as cycloheximide [17], lipopolysaccharide [42], diethylumbelliferyl phosphate [43], PGF2a [25], and estrogen withdrawal [27], have all been demonstrated to decrease StAR protein content. Studies in Y1 mouse adrenocortical cells also showed that both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A [44]. Moreover, a StAR-dependent reduction in steroidogenesis could be manifested through a reduction in the expression and/or activity of the protein. It is possible that the inhibitory effect of cantharidin on StAR protein occurs as a result of a reduction in cAMP-mediated transcription of the StAR gene. A more recent study of Y1 cells with the PP1/2A inhibitor calyculin A demonstrated that calyculin A-induced reductions in StAR protein levels were a consequence of a marked reduction in the levels of StAR mRNA expression [45], suggesting that a dephosphorylation event is involved in cAMP-induced increase in StAR mRNA levels. Since StAR is transcriptionally regulated by the orphan nuclear receptor, steroidogenic factor 1 (SF-1) [46], the possible target for the action of PP1/2A in the regulation of StAR gene may be SF-1, whose activity is regulated by phosphorylation. DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1), which also belongs to the orphan nuclear receptor family and has been found to inhibit SF-1 transactivation [47], is another potential target of PP1/2A action. The possibility that cycles of phosphorylation/dephosphorylation may be involved in the regulation of other activating factors or co-regulators that have been implicated in the regulation of StAR gene expression, such as the CCAAT/enhancer binding protein b [48, 49], GATA-4 [50], and the sterol regulatory element binding protein [51], also remains. Cantharidin is the active constituent of mylabris, the dried body of the Chinese blister beetle, which can be traced back more than 2000 years as a traditional medicine in China and is still used as a folk medicine in Asia today [1]. Cantharidin also has a history of medical usage in Europe. Although occasionally utilized as a topical vesicant for the removal of warts [52], cantharidin was considered as too toxic for use as an internal medicine by the early 1900’s. Nevertheless, folklore surrounding the purported properties of “Spanish fly” as an aphrodisiac and abortifacient continues to result in human poisonings today, with 26–50 mg estimated as a lethal dose for humans [53]. Most poisonings are unwitting ingestions resulting from a prospective mate’s attempt to arouse sexual interest. Livestock toxicosis due to the consumption of feed containing blister beetles also continues to present a problem for ranchers [54, 55]. The toxic properties of mylabris/cantharidin are well characterized. Blisters with a distinctive vesiculobullous eruption are caused by dermal contact. Severe irritation and ulceration of the gastrointestinal and urinary tract epithelial linings resulted from oral ingestion [53]. In clinics, cantharidin and its analogs, e.g., norcantharidin, have been used for cancer therapies in hepatoma patients in China [1]. The antitumor action might be explained by their cytotoxic
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effects. In an in vitro study, the dose of 0.1 mg/ml cantharidin caused significant disruption to HeLa cells, but a dose of 1–2 mg/ml had a lesser effect on murine erythroleukemia cells [56]. This would suggest that a cell-specificity exists in the cytotoxic effect. The damage of cantharidin to rat Leydig cells was also found to be insignificant at the dose of 1 mg/ml, which caused 80% inhibition of LH-induced testosterone production in vitro [12]. Thus, the inhibitory action of cantharidin on progesterone production from preovulatory follicles results mainly from the interference of steroidogenesis in the cells. Our results, however, provide evidence for the ability of cantharidin to modulate the gonadotropin-stimulated regulation of follicular function. Our results also suggest that ingestion of cantharidin can decrease ovarian steroidogenesis. A decline in serum steroid hormone levels may disrupt reproductive function. Moreover, the effects of other cantharidin’s analogs, including the herbicide endothall needs to be further examined. In conclusion, the results obtained in this study indicate that in rat preovulatory follicles, cantharidin acts to repress the LH-induced expression of StAR protein and progesterone production. These observations may indicate that cAMP-induced increases in StAR protein levels are dependent upon phosphoprotein phosphatase activities. Identification of substrates phosphorylated after cantharidin treatment would increase our understanding of the intermediate targets implicated in the LH-stimulated steroidogenic activity of the preovulatory follicle. Acknowledgments We thank Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center for his supply of antiserum for the StAR protein and Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica for her supply of P450scc antibody. Special thanks are given to Li-Ming Huang, Tsurng-Juhn Huang, and JyhChen Lin for their skilled technical assistance. This study was partly supported by a grant (NSC87-2314-B006-091) from the National Science Council, Republic of China. References 1. Wang GS. Medical uses of mylabris in ancient China and recent studies. Journal of Ethnopharmacology 1989; 26:147–162. 2. Till JS, Majmudar BN. Cantharidin poisoning. Southern Medical Journal 1981; 74:444–447. 3. Honkanen RE. Cantharidin: another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A. FEBS Letters 1993; 330:283–286. 4. Cooke BA, Lindh ML, Janszen FHA. Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone. Biochemical Journal 1976; 160:439–446. 5. Podesta E, Milani A, Steffen H, Neher R. Steroidogenesis in isolated adrenocortical cells. Biochemical Journal 1979; 180:355–363. 6. Green EG, Orme-Johnson NR. Inhibition of steroidogenesis in rat adrenal-cortex cells by a threonine analogue. Journal of Steroid Biochemistry and Molecular Biology 1991; 40:421–429. 7. Stocco DM, Clark BJ. The requirement of phosphorylation or a threonine residue in the acute regulation of steroidogenesis in MA-10 Leydig tumor cells. Journal of Steroid Biochemistry and Molecular Biology 1991; 46:337–347. 8. Ford SL, Abayasekara DRE, Persaud SJ, Jones PM. Role of phosphoprotein phosphatases in the corpus luteum: I. Identification and characterization of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells. Journal of Endocrinology 1996; 150:205–211.
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Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles Chi-Chuan Yua, Wei-Yi Chenb, P. Shirley Lia,* a
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Rep. of China b Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Rep. of China Received 19 February 2001; accepted 25 June 2001
Abstract The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares’ serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 – 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22Rhydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (1025 M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A–stimulated steroidogenic activity of the preovulatory follicle. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Rat preovulatroy follicles; Cantharidin; Steroidogenic acute regulatory protein; Progesterone
* Corresponding author. Department of Physiology, College of Medicine, National Cheng Kung University, #1, Ta-hsueh Rd, Tainan 70101, Taiwan, Rep. of China. Tel.: 1886-6-235-3535 Ext. 5437; fax: 1886-6-236-2780. E-mail address: [email protected] (P.S. Li) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 6 9 -8
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Introduction Cantharidin takes its name from Cantharis vesicatoria, one of more than 2000 species of beetles of the order Coleoptera, family of Meloidae, which contain a vesicant liquid and are known as “blister beetles”. The beetles are most commonly found in southern Europe, Africa, and Asia. The Mediterranean beetle Cantharis vesicatoria is the most well known as the “Spanish fly”. Cantharidin is the toxic constituent of blister beetles and the active ingradient of the purported aphrodisiac and abortifacient Spanish fly [1]. Cantharidin has been used since ancient times to enhance sexual potency, a practice related to findings of priapisom in men and pelvic organ engorgement in women, which result from high-dose exposure. The aphrodisiac legend stems from cantharidin’s ability to cause vascular congestion and inflammation of the genitourinary tract, a sensation that may be interpreted as enhanced sexuality by some [2]. Recently, cantharidin has been identified as a strong inhibitor of protein phosphatases types 1 (PP1) and 2A (PP2A) [3]. The key role played by protein kinases and protein phosphorylation in the regulation of steroidogenesis is well documented [4, 5]. It is generally accepted that hormone-stimulated steroidogenesis is dependent upon the phosphorylation of protein substrate on serine or threonine [6, 7]. In contrast to the widely studied role of phoshporylation events, the dephosphorylation processes involved in steroidogenesis are not so broadly described. The presence of the serine/threonine phosphatases PP1 and PP2A in rat luteal cells and adrenal cortex has been reported [8, 9]. Regarding their participation in the acute regulation of steroidogenesis, the results are controversial. In isolated luteal cells, the PP inhibitors, okadaic acid and calyculin A, reduced LH-induced progesterone biosynthesis [10], whereas in granulosa cells, inhibition of PP1 and PP2A enhanced FSH-stimulated steroidogenesis [11]. In Leydig cells, cantharidin inhibited testosterone production [12]. The biosynthesis of gonadal steroid hormones — progesterone, estrogens, and androgens — begins from cholesterol. The acute regulated and rate-limiting step for the synthesis of steroid hormones is the delivery of cholesterol from cellular stores to the mitochondrial inner membrane where the cholesterol is then converted to pregnenolone by cytochrome P450 side-chain cleavage (P450scc) enzyme [13, 14]. The transport of cholesterol in steroidogenic cells is thought to be mediated by steroidogenic acute regulatory (StAR) protein [15]. StAR protein has been identified as a mitochondrial phosphoprotein [16], which is rapidly induced by tropic hormones [17, 18]. The decisive demonstration came from an inherited disease that leads to a dramatic deficiency in all steroid hormones, congenital lipoid adrenal hyperplasia (CAH): mutations in the StAR gene have been shown to underlie this disorder [19, 20]. Moreover, mice in which there is a null mutation for StAR have a phenotype similar to lipoid CAH patients [21]. Results from recent studies have demonstrated the pattern of expression and regulation of StAR in ovarian cells during various physiological processes. High levels of StAR protein and mRNA were observed in functional corpus luteum (CL), whereas they were absent in regressed CL [22–24]. Their expression was subject to luteotropic hormones such as eCG, hCG [25], LH [26], and estradiol [27] as well as the luteolytic agent prostaglandin F2a(PGF2a) [23, 25, 26]. StAR is also regulated in a gonadotropin-dependent and stagespecific manner during follicular development [22, 25, 28, 29]. Studies in vitro showed that
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gonadotropins and activators of the PKA pathway increase StAR expression in granulosa cells [22, 28–32], whereas PGF2a and phorbol 12-myristate 13-acetate appeared to be negative regulators of StAR expression in vivo and in vitro [25, 26, 28, 29]. In view of the importance of StAR in basal and hormonally regulated steroidogenesis and the limited knowledge of cantharidin effect on this gene in ovarian follicles, we have used the rat preovulatory follicles as an experimental model to investigate the effects of cantharidin on the expression of StAR protein and progesterone production. Materials and methods Materials Purified porcine pituitary LH (pLH; USDA-pLH-B-1; 1.7 U/mg) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Pregnant mares’ serum gonadotropin (PMSG), BSA (fraction V, fatty-acid free), 8-bromo-cAMP (8-Br-cAMP), cantharidin, and 22R-hydroxycholesterol were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Ham’s F12/Dulbecco’s modified Eagle’s medium (F12/DMEM; 1:1) and other culture supplies were purchased from GIBCO-BRL (Grand Island, NY). Acrylamide, bisacrylamide, and SDS were purchased from Bio-Rad (Richmond, CA). Antiserum directed against amino acids 88–98 of mouse StAR protein was kindly provided by Dr. D.M. Stocco (Department of Cell Biology & Biochemistry, Texas Tech University, Lubbock, TX). Rabbit anti-P450scc antiserum (provided by Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan) was generated by immunization with a human P450scc fusion protein overexpressed in Escherichia coli [33]. Animals Immature (26-day-old) female Wistar rats (National Cheng Kung University Laboratory Animal Center, Tainan, Taiwan) were maintained in 22–24 8C rooms on a 12-h light, 12-h dark schedule (lights on at 0600 h). Food and water were provided ad libitum. The rats received a single subcutaneous injection of 10 IU PMSG in 0.1 ml PBS (pH 7.4) to induce multiple follicle growth. At 48 h later, animals were killed by cervical dislocation, and ovaries were removed, trimmed of adhering tissues and placed in 100-mm glass petri dishes containing F12/DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mM) and 0.1% BSA (F12/DMEM-BSA) for follicle dissection. At all times, the animals were treated in accordance with the NIH guide for the Care and Use of Laboratory Animals. Follicle isolation and incubation The largest preovulatory follicles (.400 mm in diameter) were isolated from the ovaries, and follicle culture was performed as previously described [34]. After being cleaned of adhering stromal tissue and/or smaller follicles, eight follicles were incubated in a culture dish (35 3 10 mm) containing 1.5 ml F12/DMEM-BSA in the absence or presence of increasing concentrations of cantharidin, or as indicated, with at least 3 dishes used for each treatment. Cultures were maintained for 24 h at 378C under 5% CO2 and 95% O2. At the end of incubation, follicles were solubilized in TSE buffer (10 mM Tris, 0.25 M sucrose, 0.1 mM EDTA,
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pH 7.4) for extracting mitochondrial protein for subsequent Western blot analysis, and the media were collected for the determination of progesterone level by RIA. Each set of experiments was performed 2–3 times. Western blot analysis For Western blotting, mitochondria were isolated by mechanical homogenization of the follicles and differential centrifugation, as described previously [34]. Mitochondrial proteins (50 mg/lane) were solubilized in SDS-PAGE sample buffer (58.3 mM Tris-HCl, pH 6.8, 1.7% SDS, 1% 2-mercaptoethanol, 5% glycerol, and 0.002% bromophenol blue), boiled for 5 min, and loaded into a 12.5% mini gel by standard SDS-PAGE procedures, along with prestained molecular weight markers (Bio-Rad). Electrophoresis was performed at 50 mA/min for 45 min at room temperature using a standard SDS-PAGE running buffer (25 mM Tris-base, 192 mM glycine, 0.1% SDS, pH 8.3). The proteins were electrophoretically transblotted to a polyvinylidene difluoride membrane (Bio-Rad) using a semidry electro-transfer apparatus (Hoefer Pharmacia Biotech Inc., San Francisco, CA). Protein transfer was conducted for 40 min at 0.8 mA/cm2 in transfer buffer containing 20 mM Tris-base, 150 mM glycine, 10% methanol, and 0.01% SDS at pH 8.3. Then, the membrane was blotted as previously described [34]. In some experiments, following detection of StAR protein, the membrane was stripped in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol at 508C for 1 h, washed three times for 5 min each in PBS containing 0.5% Tween-20, and then probed with P450scc antiserum (1:2500). The intensity of the bands on Western blots was measured by the Arcus II computer-assisted image system (PDI Inc., Huntington Station, NY). Values obtained were expressed as integrated optical density units, as previously described [34]. RIA for progesterone Quantitation of progesterone directly from aliquots of the medium was performed by RIA as previously described [35]. The antiserum to progesterone-11a-BSA was supplied by Dr. J.E. Hixon (Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL) and used at the dilution of 1:25,000 in 0.1 M Tris buffer (pH 7.4). The sensitivity of the assay was 12.5 pg per assay tube. Statistical analysis All values were expressed as the mean 6 SEM of pooled data from 2–3 experiments. Two means were compared using Student’s t-test. Where there were more than two means, significant differences between means were determined by analysis of variance. The means were then analyzed by Fisher’s PLSD multiple comparison. Results Dose-dependence effect of LH on StAR protein expression and progesterone production To determine the optimal dose of LH, preovulatory follicles were cultured for 24 h with various doses of LH, and StAR protein levels were determined. As shown in Figure 1a, a dose-dependent relationship between StAR protein expression and LH treatment was readily
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Fig. 1. Effect of increasing concentrations of LH on StAR protein levels (a) and progesterone production (b). Preovulatory follicles (.400 mm in diameter) isolated from ovaries of PMSG-primed immature rats were cultured (8 per culture/1.5 ml) in serum-free medium for 24 h in the absence (C, control) or presence of LH (0.01 – 1000 ng/ ml). At the end of culture, mitochondrial protein extracts were dose dependently prepared from 24 follicles of 3 cultures at each dose of LH as described in “Materials and Methods.” For each sample, 50 mg of mitochondrial proteins were analyzed for StAR protein by SDS-PAGE and immunoblotting also as described in “Materials and Methods.” The position of the StAR protein is indicated. StAR protein was detected by the Arcus II computerassisted image system at each treatment. Integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of LH. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 0.01 ng/ml.
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seen in rat preovulatory follicles as determined by Western blot analysis. StAR protein was low in unstimulated follicles, but could be seen with levels of LH as low as 0.01 ng/ml. The level of StAR protein continued to increase, reaching a maximal value at 100 ng/ml. This dose was used in the subsequent experiments. Progesterone production in the culture medium from the same experiment is shown in Figure 1b. LH at concentrations of 0.1, 1, 10, 100, and 1000 ng/ml caused a dose-dependent increase in progesterone production, reaching a maximal effect at a concentration of 100 ng/ml. Effect of treatment with cantharidin on the expression of StAR and P450scc proteins and progesterone production To investigate the effects of cantharidin on the expression of StAR and P450scc proteins, preovulatory follicles were incubated for 24 h with or without increasing concentrations of cantharidin in the presence or absence of LH. As shown in Figure 2a, cantharidin had a significant (P , 0.05) effect in suppressing LH-stimulated StAR protein expression. The inhibitory effect was concentration dependent, and a 24-h exposure of preovulatory follicles to cantharidin concentrations ranging from 100 to 1000 ng/ml decreased StAR protein levels by 24–70%. Basal StAR protein levels were low after 24 h of culture. Treatment with cantharidin had no effect on P450scc protein levels. (Fig. 2a). Results in Figure 2b show that cantharidin at a concentration of 100 ng/ml or higher significantly (P , 0.05) decreased LH-stimulated progesterone production. The inhibited level was about 40–78% of that treated with LH alone (11.861.8 ng/8 follicles/24 h). The basal accumulation of progesterone was 0.4 6 0.1 ng/8 follicles per 24 h which was 4.4 6 2.1% of that in cultures treated with LH alone. Effect of treatment with cantharidin on P450scc activity To test if cantharidin could act by inhibiting the activity of P450scc, follicles were cultured for 24 h with or without cantharidin (500 ng/ml) and 22R-hydroxycholesterol (1026 M) in the presence or absence of 8-Br-cAMP (0.5 mM). The results are presented in Figure 3. Cantharidin had no effect on the conversion of 22R-hydroxycholesterol to progesterone. Effect of treatment with cantharidin on forskolin- and 8-Br-cAMP-stimulated StAR protein expression and progesterone production The influence of cantharidin upon the ability of forskolin (1025 M), an activator of adenylate cyclase, and 8-Br-cAMP (0.5 mM), a cAMP analog, to stimulate StAR protein expression was also investigated. Cantharidin at a concentration higher than 50 ng/ml inhibited (P , 0.05) the stimulatory effect of forskolin on StAR protein to about 97% (Fig. 4a). Progesterone production in control cultures averaged 0.6 6 0.1 ng/8 follicles/24 h and was increased (P , 0.05) by forskolin stimulation (9.361.2 ng/8 follicles/24 h) (Fig. 4b). Cantharidin at a concentration higher than 100 ng/ml reduced the stimulatory effect of forskolin (P , 0.05). Results in Figure 5a show that the stimulatory effect of 8-Br-cAMP on StAR protein expression was inhibited by concomitant treatment with cantharidin (50, 100, 500 or 1000 ng/ ml) in a dose-dependent manner, decreasing from 54.0 6 6.4% to 9.5 6 2.8%. Treatment with 8-Br-cAMP significantly (P , 0.05) increased progesterone production (9.560.9 ng/8 follicles/24 h) as compared to basal levels (0.560.2 ng/8 follicles/24 h) (Fig. 5b), whereas
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Fig. 2. Effect of the dose of cantharidin on LH-stimulated StAR and P450scc protein levels (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) LH (100 ng/ml) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR and P450scc proteins. The positions of the StAR and P450scc proteins are indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from three independent experiments. P , 0.05 between control and concentration above 50 ng/ml.
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Fig. 3. Effect of cantharidin on the conversion of 22R-hydroxycholesterol to progesterone. The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (control) cantharidin (500 ng/ml) and 22R-hydroxycholesterol (1026 M) in the absence (control) or presence of 8-Br-cAMP (0.5 mM). The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each treatment. Values are the mean 6 SEM from three independent experiments. Bars with different letters differ significantly (P , 0.05).
concomitant treatment with increasing concentrations of cantharidin reduced progesterone production to 5%. Discussion Using a serum-free culture system of preovulatory follicles isolated from ovaries of immature rats primed with PMSG, we have demonstrated that cantharidin is able to significantly inhibit LH-, forskolin-, or 8-Br-cAMP-induced StAR protein levels. The cantharidin effect is dosedependent and is associated with decreases in progesterone production. These results indicate that inhibiting PP1 and PP2A activities by cantharidin blocks PKA-induced steroidogenesis primarily through down-regulating StAR protein expression. Since cantharidin had no effect on 22R-hydroxycholesterol-supported steroidogenesis, this clearly demonstrates that cantharidin does not interfere with the activity of P450scc. Serine/threonine protein phosphatases can be divided into four groups, the homologous type 1, 2A and 2B, and the distinct 2C. These groups exhibit differences in substrate specificity, subunit structure, sensitivity to inhibitors and cation requirements [36]. Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of PP1 and PP2A [3]. The Ki of cantharidin for PP2A is approximately 0.2 mM and the Ki for PP1 is approximately 1.1 mM [3]. In vitro it inhibits purified catalytic subunit of PP2A (IC50 5 0.16 mM) at a lower concentration than that of PP1 (IC50 5 1.7 mM) and only inhibits the activity of PP2B at high concentrations. In intact cells, cantharidin completely inhibits PP1 and PP2A at a concentration of 100 mM [3]. In this study we observed inhibition of progesterone production at concentrations greater than 0.2 mM (50 ng/ml), suggesting that PP2A, rather than PP1, is involved. Results presented herein are in contrast with a recent study showing that inhibition of PP1 and PP2A by okadaic acid, a marine toxin, enhanced progesterone secretion in FSH-treated
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Fig. 4. Effect of the dose of cantharidin on forskolin-stimulated StAR protein level (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) forskolin (1025 M) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 50 ng/ml for StAR protein and 100 ng/ml for progesterone production.
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Fig. 5. Effect of the dose of cantharidin on 8-Br-cAMP-stimulated StAR protein level (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) 8-Br-cAMP (0.5 mM) in the presence or absence (B; C, control) of increasing concentrations of cantharidin (10 – 1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of cantharidin. Values are the mean 6 SEM from two independent experiments. P , 0.05 between control and concentration above 10 ng/ml for StAR protein and 100 ng/ml for progesterone production.
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preantral primary granulosa cells [11]. The discrepancy in results may be explained by the differences in experimental conditions used, such as the status of differentiation of the follicles. Granulosa cells from diethylstilbestrol-treated rats [11] might be considered nontransformed and undifferentiated or in the process of differentiation, while granulosa cells in preovulatory follicles would be more mature and highly differentiated. It has been reported that in rat luteal cells, cantharidin caused a dose-dependent inhibition of LH-induced progesterone secretion [10]. Thus, it would appear that PP1/2A-induced dephosphorylation reactions may provide a tonic negative mechanism controlling FSH action in granulosa cells in the follicular phase of the ovarian cycle but predominantly enhance LH-stimulated steroidogenesis during the luteal phase. Our results also highlight that regulation of the protein serine/threonine phosphorylation balance is involved in the regulation of follicular steroidogenesis. Moreover, inhibition of PP1 and PP2A has been reported previously to inhibit secretion from a number of different endocrine cell types, including glucose-induced insulin secretion from rat pancreatic islets of Langerhans [37] or mouse b-cells [38], lipoprotein-stimulated corticosterone secretion from rat adrenocortical cells [39], thyrotropin-releasing hormone-induced thyrotropin and prolactin secretion from rat anterior pituitary slices [40] and carbachol-induced secretion of catecholamines from bovine adrenal medullary cells [41]. Whether the inhibitory mechanism of cantharidin is mediated prior or distal to cAMP formation was examined by investigation of the effect of cantharidin on forskolin- and 8-Br-cAMPstimulated progesterone production. Cantharidin treatment suppressed forskolin-stimulated progesterone production. This finding suggests that cantharidin may suppress adenylate cyclase activity, resulting in the reduction of cAMP formation. In addition, to reduce cAMP formation, our observation that cantharidin treatment also attenuated 8-Br-cAMP-stimulated progesterone production indicated that the action of cantharidin is exerted, at least in part, at a point distal to the generation of cAMP. However, the 25% inhibition by cantharidin, at the dose of 500 ng/ml, of 8-Br-cAMP-stimulated progesterone production (data shown in Fig. 3) was not consistent with the 80% inhibition by the same dose of cantharidin on progesterone (data shown in Fig. 5). The inconsistency in the effect of cantharidin on 8-Br-cAMP-stimulated progesterone production may suggest that at least a part of the inhibitory mechanism of cantharidin is mediated prior to cAMP formation. With regard to the direct cellular mechanisms responsible for the effect of cantharidin on hormone-induced steroidogenesis, our present study demonstrates that in rat preovulatory follicles, cantharidin inhibits LH-induced progesterone production, whereas the amount of P450scc is not affected by cantharidin treatment, indicating that the loss of steroidogenic capacity is not a result of inhibition of P450scc. In contrast to the inhibition of 8-Br-cAMPinduced steroidogenesis, the inhibitory effect of cantharidin could be reversed by 22Rhydroxycholesterol. This suggests that the major inhibitory effect of cantharidin is the decrease in the availability of cholesterol substrate in the mitochondria. The results lend support to previous reports, in which inhibitors of PP1/2A did not affect 22R-hydroxycholesterol-supported steroidogenesis [9, 10]. In the present study, we further demonstrated that cantharidin decreased LH-induced, forskolin-induced, or 8-Br-cAMP-induced StAR protein levels. This suggests that in rat preovulatory follicles, inhibiting PP1 and PP2A activities blocks PKA-induced progesterone production primarily through preventing the increase in StAR protein production. Diminished levels of StAR protein would likely result in a reduction in the capacity of
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preovulatory follicles for progesterone production, in response to gonadotropins, by limiting the availability of cholesterol to the P450scc complex, located in the inner mitochondrial membrane. Previous studies of the StAR protein have indicated that it has an indispensable role in steroid hormone biosynthesis [19], and it has been further postulated that this role is in regulating cholesterol transfer to the inner mitochondrial membrane [15, 16]. It appears that the cantharidin-induced depression in progesterone production in preovulatory follicles is to be due to the inhibition of StAR protein synthesis. This observation is highly consistent with previous studies in which inhibition of steroid hormone biosynthesis, such as cycloheximide [17], lipopolysaccharide [42], diethylumbelliferyl phosphate [43], PGF2a [25], and estrogen withdrawal [27], have all been demonstrated to decrease StAR protein content. Studies in Y1 mouse adrenocortical cells also showed that both steroidogenesis and StAR protein expression were inhibited by two structurally dissimilar inhibitors of PP1 and PP2A activities, okadaic acid and calyculin A [44]. Moreover, a StAR-dependent reduction in steroidogenesis could be manifested through a reduction in the expression and/or activity of the protein. It is possible that the inhibitory effect of cantharidin on StAR protein occurs as a result of a reduction in cAMP-mediated transcription of the StAR gene. A more recent study of Y1 cells with the PP1/2A inhibitor calyculin A demonstrated that calyculin A-induced reductions in StAR protein levels were a consequence of a marked reduction in the levels of StAR mRNA expression [45], suggesting that a dephosphorylation event is involved in cAMP-induced increase in StAR mRNA levels. Since StAR is transcriptionally regulated by the orphan nuclear receptor, steroidogenic factor 1 (SF-1) [46], the possible target for the action of PP1/2A in the regulation of StAR gene may be SF-1, whose activity is regulated by phosphorylation. DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1), which also belongs to the orphan nuclear receptor family and has been found to inhibit SF-1 transactivation [47], is another potential target of PP1/2A action. The possibility that cycles of phosphorylation/dephosphorylation may be involved in the regulation of other activating factors or co-regulators that have been implicated in the regulation of StAR gene expression, such as the CCAAT/enhancer binding protein b [48, 49], GATA-4 [50], and the sterol regulatory element binding protein [51], also remains. Cantharidin is the active constituent of mylabris, the dried body of the Chinese blister beetle, which can be traced back more than 2000 years as a traditional medicine in China and is still used as a folk medicine in Asia today [1]. Cantharidin also has a history of medical usage in Europe. Although occasionally utilized as a topical vesicant for the removal of warts [52], cantharidin was considered as too toxic for use as an internal medicine by the early 1900’s. Nevertheless, folklore surrounding the purported properties of “Spanish fly” as an aphrodisiac and abortifacient continues to result in human poisonings today, with 26–50 mg estimated as a lethal dose for humans [53]. Most poisonings are unwitting ingestions resulting from a prospective mate’s attempt to arouse sexual interest. Livestock toxicosis due to the consumption of feed containing blister beetles also continues to present a problem for ranchers [54, 55]. The toxic properties of mylabris/cantharidin are well characterized. Blisters with a distinctive vesiculobullous eruption are caused by dermal contact. Severe irritation and ulceration of the gastrointestinal and urinary tract epithelial linings resulted from oral ingestion [53]. In clinics, cantharidin and its analogs, e.g., norcantharidin, have been used for cancer therapies in hepatoma patients in China [1]. The antitumor action might be explained by their cytotoxic
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effects. In an in vitro study, the dose of 0.1 mg/ml cantharidin caused significant disruption to HeLa cells, but a dose of 1–2 mg/ml had a lesser effect on murine erythroleukemia cells [56]. This would suggest that a cell-specificity exists in the cytotoxic effect. The damage of cantharidin to rat Leydig cells was also found to be insignificant at the dose of 1 mg/ml, which caused 80% inhibition of LH-induced testosterone production in vitro [12]. Thus, the inhibitory action of cantharidin on progesterone production from preovulatory follicles results mainly from the interference of steroidogenesis in the cells. Our results, however, provide evidence for the ability of cantharidin to modulate the gonadotropin-stimulated regulation of follicular function. Our results also suggest that ingestion of cantharidin can decrease ovarian steroidogenesis. A decline in serum steroid hormone levels may disrupt reproductive function. Moreover, the effects of other cantharidin’s analogs, including the herbicide endothall needs to be further examined. In conclusion, the results obtained in this study indicate that in rat preovulatory follicles, cantharidin acts to repress the LH-induced expression of StAR protein and progesterone production. These observations may indicate that cAMP-induced increases in StAR protein levels are dependent upon phosphoprotein phosphatase activities. Identification of substrates phosphorylated after cantharidin treatment would increase our understanding of the intermediate targets implicated in the LH-stimulated steroidogenic activity of the preovulatory follicle. Acknowledgments We thank Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center for his supply of antiserum for the StAR protein and Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica for her supply of P450scc antibody. Special thanks are given to Li-Ming Huang, Tsurng-Juhn Huang, and JyhChen Lin for their skilled technical assistance. This study was partly supported by a grant (NSC87-2314-B006-091) from the National Science Council, Republic of China. References 1. Wang GS. Medical uses of mylabris in ancient China and recent studies. Journal of Ethnopharmacology 1989; 26:147–162. 2. Till JS, Majmudar BN. Cantharidin poisoning. Southern Medical Journal 1981; 74:444–447. 3. Honkanen RE. Cantharidin: another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A. FEBS Letters 1993; 330:283–286. 4. Cooke BA, Lindh ML, Janszen FHA. Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone. Biochemical Journal 1976; 160:439–446. 5. Podesta E, Milani A, Steffen H, Neher R. Steroidogenesis in isolated adrenocortical cells. Biochemical Journal 1979; 180:355–363. 6. Green EG, Orme-Johnson NR. Inhibition of steroidogenesis in rat adrenal-cortex cells by a threonine analogue. Journal of Steroid Biochemistry and Molecular Biology 1991; 40:421–429. 7. Stocco DM, Clark BJ. The requirement of phosphorylation or a threonine residue in the acute regulation of steroidogenesis in MA-10 Leydig tumor cells. Journal of Steroid Biochemistry and Molecular Biology 1991; 46:337–347. 8. Ford SL, Abayasekara DRE, Persaud SJ, Jones PM. Role of phosphoprotein phosphatases in the corpus luteum: I. Identification and characterization of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells. Journal of Endocrinology 1996; 150:205–211.
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