冬蟲夏草刺激小鼠萊氏細胞性腺素生成之作用機制及基因調控研究(I)

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(1)行政院國家科學委員會專題研究計畫期中精簡報告. 冬蟲夏草刺激小鼠萊氏細胞性腺素生成之作用機制及基因調控研究(1/2). 計畫類別：個別型計畫計畫編號： NSC91-2320-B-006-070執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日執行單位：國立成功大學解剖學科(所). 計畫主持人：黃步敏. 報告類型：精簡報告處理方式：本計畫可公開查詢. 中華. 民國 92 年 5 月 7 日.

(2) Cordyceps sinensis Activates PKA and PKC Signal Pathways to Stimulate Steroidogenesis in MA-10 Mouse Leydig Tumor Cells*. Yung-Chia Chen, Yuan -Li Huang, and Bu-Miin Huang†. From the Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Taina n, Taiwan.. † To whom correspondence may be addressed: Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, 1 Ta -Hsueh Road, Tainan 701, Taiwan. Tel: 886 -6-208-9357; Fax: 886-6-209-3007; E-mail: [email protected].. Running Title: Cordyceps sinensis regulates MA-10 cell function Key Words: CS, Leydig cell, steroidogenesis, regulation, PKA, PKC. * This work was supported by National Science Council Grants NSC 91-2320-B-006-070 to BMH, Taiwan..

(3) SUMMARY Cordyceps sinensis (CS) stimulates steroidogenesis in MA-10 mouse Leydig tumor cells, but the mechanisms remain unclear. In the present study, MA-10 cells were treated with different reagents in the presence or absence of CS. (10 mg/ml) for 3 hr to determine. the mechan isms. Results illustrated that CS might activate the Gs protein subunit, but not Gi, to induce cell steroidogenesis. Inhibitors (SQ 22536 and 2’5’DDA) did not abolish CS-stimulated steroidogenesis; therefore, adenylate cyclase was not involved. Moreover, PKA inhibitors, H89 or PKI, inhibited 37% of CS-stimulated steroidogenesis (p<0.05), which demonstrated that CS might enhance the cAMP-PKA pathway to affect MA-10 cell steroidogenesis. The PKC pathway was also examined due to the incomplete inhibition by PKA inhibitors. PKC inhibitor (calphostin C), phospholipase C inhibitor (neomycin sulfate), and calmodulin antagonist (W7) blocked 35-52% of CS-stimulated steroidogenesis in MA-10 cells, strongly suggesting that CS activated the PKC pathway. Co-treatment of PKA and PKC inhibitors inhibited 61% of CS-stimulated steroid production, indicating that CS simultaneously activated PKA and PKC pathways to stimulate MA-10 cell steroidogenesis. Moreover, CS induced the expression of steroidogenic acute regulatory protein in dose- and time-dependent relationships, and PKA inhibitor, PKC inhibitor, or co-treatment with both inhibitors suppressed it (p<0.05). The role of calcium, which profoundly influences steroidogenesis, was also investigated. Results showed that T-type calcium channels, but not L-type, were required for CS to stimulate MA-10 steroidogenesis. These data support the hypothesis that CS activates both PKA and PKC signal transduction pathways and that calcium ions stimulate MA-10 cell steroidogenesis..

(4) INTRODUCTION Cordyceps sinensis (CS) is a fungal parasite on the larvae of Lepidoptera. In late autumn, the fungus attacks the caterpillars and leisurely devours its host. By early summer of the following year, the fungal infestation has killed the caterpillar and the fruiting body protrudes from its head. Because of its particular life cycle, it is called the "winter-worm, summer-plant". CS has various pharmacological functions, in particular, a remedy for sexual dysfunction (1), and recent studies have illustrated that CS can stimulate Leydig cells to produce sex steroids (2-4). It is well established that steroidogenesis in Leydig cells is regulated by luteinizing hormone/chorionic gonadotropin (LH/CG). LH binds to its receptors to activate G-proteins and, in turn, adenylate cyclase (AC), which can increase cyclic AMP (cAMP) formation (5). cAMP will then stimulate protein kinase A, which will phosphorylate proteins. The phosphorylated proteins will further phosphorylate other proteins or induce new protein synthesis, such as steroidogenic acute regulatory (StAR) protein. The function of StAR protein is to transfer free cholesterol from cytoplasm into the inner membrane of mitochondria, where cytochrome P450 side-chain cleavage enzyme converts cholesterol to pregnenolone (6). Pregnenolone will then be transported to the smooth endoplasmic reticulum for further synthesis to testosterone, an inevitable steroidal hormone for reproduction in males (7). Although PKA-mediated protein phosphorylation is undoubtedly important in regulating steroid synthesis, other signaling systems have also been implicated. It has been shown that activation of the PKC signal pathway can strongly modulate Leydig cell steroidogenesis (8). Likewise, evidence indicates that calcium is also involved in steroidogenesis. It has been shown that the removal of extracellular calcium, or the addition of calmodulin antagonist or calcium channel blocker does blunt Leydig cell.

(5) steroidogenesis (9-10). We have previously demonstrated that CS enhances steroidogenesis both in mouse Leydig cells and MA-10 Leydig tumor cells (2-4). However, the mechanism through which CS up-modulates Leydig cell steroidogenesis remains elusive. In the present study, we examined the possible signal transduction pathways that could be activated by CS in MA-10 cell steroidogenesis..

(6) EXPERIMENTAL PROCEDURES Chemicals—Fetal bovine serum and lyophilized trypsin-EDTA were purchased from Gibco (Grand Island, NY). Tris/HCl, mercaptoethanol, SDS, sucrose, Tween 20, EDTA, glycerol, bromo-phenol blue, Waymouth MB 752/1 medium, bovine serum albumin (BSA), forskolin, cholera toxin, pertussis toxin, guanosine 5'-[-thio]diphosphate trilithium salt (GDP--S), dextran T-70, dimethyl sulphoxide (DMSO), K+-Na+-tartrate, N-(2-[p-Bromocinnamylamino]ethyl)5-isoquinolinesulfonamide hydrochloride (H-89), sodium azide, dibutyryl-adenosine 3': 5'-cyclic monophosphate (dbcAMP), phorbol-12-miristate-13-acetate (PMA), copper sulfate, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536), nifedipine, and W7 were bought from Sigma Chemical Co. (St. Louis, MO). Tissue-culture-grade sodium bicarbonate, sodium carbonate, sodium chloride, sodium dihydrogen phosphate, potassium chloride, and cadmium chloride-1-hydrate were purchased from Riedel-deHaën® (Seelze, Germany). Sodium hydroxide and hydrochloric acid were purchased from Merck (Darmstadt, Germany). Sodium hydrogen phosphate was purchased from J. T. Baker (Phillipsburg, NJ). Charcoal was purchased from Showa Chemical Inc. (Tokyo, Japan). HEPES was purchased from Mallinckrodt Baker, Inc. (Paris, KY). Calphostin C, PKI (14-22 amide), neomycin sulfate, flunarizine dihydrochloride and 2', 5' dideoxyadenosine were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA). Gentamycin sulfate and folin-ciocalteu's phenol reagent were purchased from Fluka Chemical Co. (Buchs, Switzerland). Renaissance kit and 3H -progesterone used for chemiluminescence and radioimmunoassay, respectively, were purchased from PerkinElmer Life Sciences Inc. (Boston, MA). Donkey anti-rabbit IgG conjugated with horseradish peroxidase was purchased from Amersham International (Arlington Heights, IL). Antibody generated.

(7) against residues 89-99 of human StAR was a gift from Dr. Jerome Struass, III (University of Pennsylvania, Philadelphia, PA, USA). Antiserum to progesterone was obtained from Dr. Paulus Shyi-Gang Wang (National Yang -Ming University, Taipei, Taiwan). Cell Culture and Experiments—The MA-10 cell line was a gift from Dr. Mario Ascoli (The University of Iowa, Iowa City, IA) and was maintained by standard techniques. This mouse Leydig tumor cell line produces progesterone as the major steroid in response to trophic hormones (LH and hCG) and cAMP analogues (Ascoli, 1981). Cells (5  104) were placed in 96-well plates and grown for 24 h in Waymouth medium containing 15% fetal bovine serum. Medium was then removed and the cells were washed twice with PBS+. Cells in medium without serum were then treated with CS alone or CS plus different reagents for 3 h, and the media were collected and stored at -20 C until assayed for progesterone by radioimmunoassay. The cells were maintained in a humidified atmosphere containing 95% air/5% CO2 at 37 C. Immunoblot analysis—The total proteins were solubilized in the sample buffer (25 mM Tris, pH 6.8, 1% SDS, 5% -mercaptoethanol, 1 mM EDTA, 4% glycerol, and 0.01% bromophenol blue) and loaded onto a 12.5% SDS-PAGE mini-gel (Mini-Protean® II; Bio-Rad Laboratories (Life Science), Hercules, CA). SDS-PAGE was performed at 200 V for 45 min using standard running buffer (24 mM Tris, 0.19 M glycine, and 0.5% SDS, pH 8.3). The proteins were then transferred to a polyvinyldiene difluoride membrane (PVDF) (Bio-Rad) at 80 mA for 1 h at room temperature with transfer buffer (20 mM Tris, 150 mM glycine, 10% methanol, and 0.01% SDS). The PVDF membrane with transferred protein was incubated in blocking buffer (PBS buffer containing 5% Carnation® non-fat dry milk, and 0.5% Tween 20) at room temperature for 1 h and then incubated in fresh blocking buffer.

(8) containing the primary antibody for 16-18 h at 4 C. The membrane was then washed three times (10 min each) with PBS including 0.5% Tween 20. It was then incubated for 30 min at room temperature with fresh blocking buffer containing the secondary antibody donkey anti-rabbit IgG, which was conjugated with horseradish peroxidase (Amersham). The membrane was washed and the specific signal was determined by using the Renaissance chemiluminescence reagent as described by the manufacturer (NEN; DuPont, Boston, MA). Proteins of interest were quantitated by a computer-assisted image analysis system (Quantity One, Huntington Station, NY) (11). The amount of -actin in each lane was also detected as a control to correct the expression of StAR protein. Radioimmunoassay (RIA)—Media from cultures with different treatments were collected and diluted with plain medium to fall in the standard curves for the respective assays. Twenty-five l of diluted sample was withdrawn to a glass tube and 100 l of progesterone antiserum and 100 l of 3H-progesterone were added. Equilibrium reaction occurred at room temperature for 2 h and was stopped by putting the tubes in ice. Charcoal was added and incubated for 15 min at 4 C and then centrifuged at 12000  g for 10 min to precipitate the charcoal bound with free 3. H-steroids. The supernatant was poured into 3 ml of scintillation fluid and samples. were counted in beta-counter for 2 min. The progesterone level in each treatment was then normalized by total protein concentration measured by the Lowry method (12). Statistical Analysis—Results are expressed as the mean  S.E. in percentage of progesterone production in three experiments with triplicates of each treatment. Statistically significant differences between treatments and the control were determined by one-way ANOVA and the Fisher -PLSD multiple comparison procedure. Statistical significance was set at (p<0.05)..

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(10) RESULTS The effects of CS on G protein related to steroidogenesis in MA-10 cells—Studies have suggested that cAMP-PKA is the major signal transduction pathway involved in the regulation of Leydig cell steroidogenesis by LH/CG (13). Accordingly, we examined whether CS could affect any locus along the cAMP-PKA pathway to activate MA-10 cell steroidogenesis. The effect of CS on G protein was first investigated. It is well known that G proteins are heterotrimers composed of  and  subunits, and at least four  subunits (Gs, Gi, Gq and G12) that can regulate the activity of adenylate cyclase have been identified (14). In the present study, general G protein inhibitor, GDP--S (100 M), reduced 18  2% of CS-stimulated steroidogenesis in MA-10 cells (p<0.05)(Fig. 1a), which suggested that CS might mediate G protein activity. To further determine whether CS affects the Gs o r Gi subunit, we added different concentrations of Gs protein activator, cholera toxin (CTX) (1-100 ng/ml), or Gi protein inhibitor, pertussis toxin (PTX) (10-1000 ng/ml), without or with CS (10 mg/ml), to cells for 3 h of incubation. CTX (1-100 ng/ml) alone caused a dosage-dependent increase in progesterone production in MA-10 cells (Fig. 1b). CTX at 100 ng/ml significantly stimulated progesterone production compared to the control (p<0.05). Moreover, CS induced more progesterone with CTX (10 and 100 ng/ml) present than without (p<0.05), which indicated that CS might affect Gs protein to stimulate MA-10 cell steroidogenesis. However, CS did not induce more progesterone with PTX present (p>0.05) (Fig. 1c), and PTX alone at different dosages did not affect MA-10 cell steroidogenesis (p>0.05), which strongly suggested that Gs protein might not be involved in CS-stimulated steroidogenesis. The effects of CS on adenylate cyclase related to steroidogenesis in MA-10.

(11) cells—Adenylate cyclases are a family of membrane-bound enzymes that catalyze the formation of cAMP from ATP. They are regulated by numerous neurotransmitters and hormones via G protein-linked cell surface receptors (15). To investigate whether CS might act on adenylate cyclase to affect MA-10 cell steroidogenesis, cells were treated with the adenylate cyclase activator forskolin and the inhibitors SQ 22536 (SQ) or 2',5' dideoxyadenosine (2',5' DDA), without or with CS (10 mg/ml) for 3 h of incubation. Figure 2a illustrates that forskolin (1-100 M) caused a dosage-dependent increase in progesterone production and that, in the presence of forskolin (50 M), CS-stimulated progesterone production was significantly greater (p<0.05). Neither inhibitor, however, suppressed CS-stimulated steroidogenesis in MA-10 cells (p>0.05) (Fig. 2, b and c), respectively, and neither SQ nor 2',5' DDA alone at different dosages had any effect on MA-10 cells (p>0.05). These results suggest that CS might not affect adenylate cyclase to stimulate steroidogenesis in MA-10 cells. The effects of CS on PKA related to steroidogenesis in MA-10 cells—PKA is a major pathway for stimulation of Leydig cell steroid synthesis (6). To see whether CS acts on PKA to regulate MA-10 cell steroidogenesis, the PKA activator dbcAMP and the PKA inhibitors, H89 and PKI, without or with CS (10 mg/ml), were added to cells for 3 h of incubation. dbcAMP (0.05-1.0 mM) caused a dosage-dependent increase in progesterone production, and CS induced significantly more progesterone in the presence of dbcAMP (p<0.05) (Fig. 3a). Moreover, H89 (50 M) and PKI (4 M) significantly inhibited CS-stimulated progesterone production by 37  9% and 40  6%, respectively (p<0.05) (Fig. 3, b and c). These results strongly indicate that CS activated PKA to stimulate MA-10 cell steroidogenesis. The effect of CS on PKC related to steroidogenesis in MA-10 cells—Because PKA inhibitors reduced only about 40% of the steroidogenesis in MA-10 cells, it is.

(12) possible that CS might activate other signal pathways. In steroidogenic cells, the PKC signal transduction pathway and calcium ions play important roles in steroidogenesis regulation (10, 16). We therefore examined the PKC pathway. The PKC activator PMA phorbol ester, the PKC inhibitor calphostin C, and the phospholipase C (PLC) inhibitor neomycin sulfate, without or with CS (10 mg/ml), were added to cells for 3 h of incubation. PMA (2.5 ug/ml) significantly enhanced progesterone production in the presence of CS (p<0.05) (Fig. 4a). Moreover, calphostin C (5-500 nM) and neomycin sulfate (5-500 uM) reduced CS-stimulated steroidogenesis (ca. 52% and 40%, respectively; p<0.05) (Fig. 4, b and c). These results strongly suggested that activation of PKC and PLC was responsible for the CS-stimulated steroidogenesis in MA-10 cells. To see whether PKA and PKC p athways were stimulated at same time by CS in MA-10 cells, PKI (4 M), calphostin C (500 nM), and CS (10 mg/ml) were added to cells for 3 h of incubation. Co-treatment of PKI with calphostin C inhibited CS-stimulated progesterone production by 61  8% (p<0.05), which suggested that the stimulatory effect of CS might travel the PKA and PKC pathways simultaneously. The effect of CS on the expression of StAR protein in MA-10 cells—The induction of StAR protein expression is concomitant with the increase of steroid production in steroidogenic cells (17). The present study illustrated that the expression of StAR protein significantly increased as the CS dosages (0-10 mg/ml) and the treatment interval (0-360 min) increased (Fig. 5, a and b) (p<0.05). Moreover, treatment with PKA inhibitor or PKC inhibitor, or co-treatment with both inhibitors, significantly reduced the expression of StAR protein (Fig. 5, c, d and e) (p<0.05). These data confirmed the possibility that CS activates StAR protein expression through PKA and PKC signal transduction pathways to induce MA-10 cell steroidogenesis..

(13) The effects of CS on calcium related to steroidogenesis in MA-10 cells—Deprivation of extracellular calcium will result in a decrease in LH-stimulated testosterone production in adult rat Leydig cells (18). To evaluate whether calcium is involved in the CS stimulatory effect on MA-10 cell steroidogenesis, calcium ionophore (A23187; 0.5-50 M), non-specific calcium channel blocker (cadmium; 0.01-1 mM), T-type calcium channel blocker (flunarizine dihydrochloride; 0.2-20 M), L-type calcium channel blocker (nifedipine; 5-500 M), and calmodulin antagonist (W7; 10 M), with or without CS (10 mg/ml) were added to cells for 3 h of incubation. The results demonstrated that the calcium ionophore promoted (Fig. 6a) but that the cadmium (Fig. 6b) inhibited CS-stimulated progesterone production. Moreover, flunarizine dihydrochloride (Fig. 6c), but not nifedipine (Fig. 6d), suppressed CS-stimulated steroidogenesis in MA-10 cells. In addition, W7 inhibited CS-stimulated steroidogenesis by 45  3% (p<0.05) (Fig. 6e). These data strongly suggested that calcium and the T-type calcium channel played important roles in CS-stimulated steroidogenesis in MA-10 cells..

(14) DISCUSSION The present study demonstrates that CS activates both PKA and PKC signal transduction pathways, and that calcium is involved in the stimulation of MA-10 cell steroidogenesis. The characteristics of this stimulation indicate that along both signal transduction pathways, CS has effects on different loci, such as G protein, PKA protein, PKC protein and phospholipase C, and that the presence of intracellular calcium is essential. Several lines of evidence indicate that LH receptor-mediated effects on Leydig cell steroidogenesis are mostly through the activation of the Gs/adenylate cyclase/cAMP/PKA pathway (13). Many receptors can activate more than one type of G protein, and the G protein subunits can interact with many different types of receptor (19). The general G protein inhibitor GDP--S significantly inhibits CS-stimulated progesterone production (Fig. 1a), which indicates that CS may affect at least one kind of G protein in MA-10 cells. To validate this conclusion, it was necessary to determine which G s ubfamily protein CS would activate. CS enhanced cholera toxin-treated progesterone production in a dosage-dependent manner (Fig. 1b). The Gi protein inhibitor pertussis toxin did not abolish CS-stimulated steroidogenesis (Fig. 1c). Thus, our study at least shows that CS affects Gs, but not Gi, to regulate MA-10 cell steroidogenesis. Although forskolin-induced progesterone production was significantly increased by CS (Fig. 2a), suggesting that CS might enhance adenylate cyclase (AC) to effect MA-10 cell steroidogenesis, our results show that neither of the AC inhibitors, SQ 22536 and 2',5' DDA, eliminated the stimulatory effect of CS, which suggested that CS might not affect AC activity in MA-10 cell steroidogenesis (Fig. 2, b and c). At least nine isoforms of adenylate cyclase (AC1 through AC9), each with different.

(15) functions, are regulated by different G subunit proteins (15). The lack of steroidogenesis inhibition by both SQ 22536 and 2',5' DDA might not mean that CS had no effect on AC. An investigatio n of the effects of other available inhibitors for different AC isoforms will help further pinpoint which isoforms of AC are affected by CS in MA-10 cells. In the present study, the PKA inhibitors H89 and PKI significantly diminished CS-stimulated progesterone production, which unquestionably illustrated that CS did activate PKA activity to stimulate steroidogenesis in MA-10 cells. However, both inhibitors decreased only 30-40% of steroid production, which strongly suggested that other signal pathways might also be involved. Phorbol ester (PMA) can activate protein kinase C and increase testosterone production (20). Moreover, one study demonstrated that the PKC inhibitor calphostin C blocks the stimulatory effect of the water-soluble extract of CS on corticosterone production in cultured adrenocortical cells (21). These studies strongly suggest that the PKC pathway is involved in CS-stimulated steroidogenesis. Our study shows that PMA enhanced CS-stimulated progesterone production and that calphostin C abolished the stimulatory effect of CS on MA-10 cell steroidogenesis, both consistent with the observations by Wang et al. (21). We also found that CS-stimulated steroidogenesis could be blocked by neomycin sulfate, a PLC inhibitor. Consequently, CS did activate PKC signal transduction pathway in MA-10 cells. In fact, MA-10 cells do express an isoform of PLC that can be activated by Gq (13). Previous studies illustrate that PLC activation requires high concentrations of LH/hCG (22). Present results suggest that CS (10 mg/ml) might up-regulate LH receptors or function as an LH analog to activate the inositol phosphate/diacylglycerol pathway to stimulate steroidogenesis in MA-10 cells, which.

(16) is not unprecedented. It is well established that StAR protein is essential for steroidogenesis, and that the activation of PKA will induce StAR protein expression and then steroidogenesis (6, 17). Our data are consistent with the principle that CS activated the PKA signaling pathway and StAR expression to induce steroidogenesis in MA-10 cells. However, the activation of PKC generally inhibits Leydig cell steroidogenesis. Interestingly, our data showed that the CS-induced activation of PKC stimulated StAR protein expression and then steroidogenesis in MA-10 cells. In fact, it has been shown that the activation of the PKC signaling pathway may inhibit StAR protein expression in granulosa cells (23) or stimulate it in adrenal cells (24). The effect of PKC on StAR protein, however, has never been shown in Leydig cells. Accordingly, t he stimulatory effect of CS on StAR protein related to PKC is worthy of further investigation. Whether CS will simultaneously activate the PKA and PKC signal transduction pathways to induce MA-10 cell steroidogenesis is also of interest. Our data demonstrated that co-treatment of cells with PKI and calphostin C did abolish the stimulatory effect of CS. Another study (25) showed that stimulation of calcium uptake by distal convoluted tubule cells requires the activation of both PKA and PKC. Hence, our observation was not unexpected. The co-treatment of cells with PKI and calphostin C, however, reduced steroid production by only 61%, an amount almost equal to the total reduction by PKA inhibitor (30-40%) and PKC inhibitor (30-40%) together. This strongly suggests that systems other than the PKA and PKC pathways may also be implicated in steroidogenesis in MA-10 cells. Because calcium has been shown to play a pivotal role in steroidogenesis (10, 26), calcium was the next best candidate. Our data demonstrated that the increase of cytosolic calcium promoted CS-stimulated steroidogenesis in MA-10 cells, and that.

(17) the blockage of extracellular calcium influx, especially through the T-type calcium channel, significantly inhibited progesterone production. It is possible that, in the present study, CS directly promoted a calcium influx into cells or that CS activated the PKC pathway and then induced the release of calcium into cytoplasm from the endoplasmic reticulum. Regardless of the mechanism, calcium stimulated by CS will bind to calmodulin and then induce MA-10 cell steroidogenesis, because our data illustrated that the calmodulin antagonist W7 significantly inhibited (45  3%) CS-stimulated steroidogenesis in MA-10 cells. In conclusion, the mechanisms underlying CS-stimulated steroidogenesis in MA-10 mouse Leydig tumor cells possibly go through the PKA and PKC pathways and involve calcium ions..

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(20) FIGURE LEGENDS Figure 1. The effects of CS on G protein related to steroidogenesis in MA-10 cells. Cells were treated with CS (10 mg/ml) without or with GDP--S (100 M) (a); without or with cholera toxin (CTX) (1-100 ng/ml) (b); without or with pertussis toxin (PTX) (10-1000 ng/ml) (c) for 3 h of incubation. Different numbers above the columns indicates that the means are significantly (p<0.05) different between each group. Figure 2. The effects of CS on adenylate cyclase related to steroidogenesis in MA-10 cells. Cells were treated with CS (10 mg/ml) without or with forskolin (1-100 M) (a); without or with SQ 22536 (SQ) (1-100 ) (b); without or with 2',5' dideoxyadenosine (2',5' DDA) (0.3-30 ) (c) for 3 h of incubation. Different numbers above the columns indicates that the means are significantly (p<0.05) different between each group.. Figure 3. The effects of CS on PKA related to stero idogenesis in MA-10 cells. Cells were treated with CS (10 mg/ml) without or with dbcAMP (0.05-1.0 mM) for 3 h of incubation (a); cells were pretreated with H89 (50 ) for 30 min without CS, and then without or with H89 (50 M) plus CS for 3 hr incubation (b); without or with PKI (4 ) (c) for 3 h of incubation. Different numbers above the columns indicates that the means are significantly (p<0.05) different between each group.. Figure 4. The effect of CS on PKC related to steroidogenesis in MA-10 cells. Cells were treated with CS (10 mg/ml) without or with PMA (2.5 g/ml) (a); without or with calphostin C (Cal) (500 nM) (b); without or with neomycin sulfate (Neo) (5-500 M) (c); without or with the co-treatment of PKI (4 M) and calphostin C (500 nM) (d) for 3 h of incubation. Different numbers above the columns indicates that the.

(21) means are significantly (p<0.05) different between each group.. Figure 5. The effect of CS on the expression of StAR protein in MA-10 cells. Cells were treated with CS (0-10 mg/ml) for 3 h of incubation (a); with CS (10 mg/ml) for 0 to 6 h of (b); with CS (10 mg/ml) without or with H89 (50 M) (c); calphostin C (500 nM) (d), or the co-treatment of H89 and calphostin C for 3 h of incubation (e). Different numbers above the columns indicates that the means are significantly (p<0.05) different between each group.. Figure 6. The effects of CS on calcium related to steroidogenesis in MA-10 cells. Cells were treated with CS (10 mg/ml) without or with calcium ionophore A23187 (0.5-50 M) (a); without or with cadmium (0.01-1 mM) (b); without or with flunarizine dihydrochloride (flu) (0.2-20 M) (c); without or with nifedipine (5-500 M) (d); without or with W7 (10 M) (e) for 3 h of incubation. Different numbers above the columns indicates that the means are significantly (p<0.05) different between each group..

(22) PT X1 0n g/m PT l X1 00 ng/ ml PT X1 μ g/m l CS 10 mg PT X1 /m l 0n g/m PT l +C X1 S 00 ng/ ml +C PT S X1 μ g/m l+C S. Ba sal. progesterone level (% of CS). Ba sa CS 10m l g/m l CT X1 ng/ m CT X1 l 0n g/m CT l X1 0 CT 0 ng/m X1 l ng CT /ml+ C X1 0n S g/m CT l+C X1 S 00 ng/ ml +C S. progesterone level (% of CS). (a). 100. 200 180 160 140 120 100 80 60 40 20 0. 1. 700. (b). 200 1. 1. 1. 1 1. 1. GD P-β -S+ CS. 20. CS 10m g/m l. Ba sal GD P-β -S 100 μ M. progesterone level (% of CS). 120 2. 100 3. 80. 60. 40. 1. 0. 2. 600 2. 500. 400 2. 300. 1. 1. 0. (c). 2. 2. 2. 1. 2.

(23) 160 140 120 100 80 60 40 20 0. 1. forskolin forskolin+CS. 2. 2. 1 1. 1 1. (c). 2. 1. Figure 2 100 μM. 1. 50μ M. 2. 10μ M. 150. 1μ M. CS. 300. CS 10m g/m l SQ 1μ M+ CS SQ 10 μM +C S SQ 100 μ M+ CS. 200 180 160 140 120 100 80 60 40 20 0. SQ 10 μ M SQ 100 μ M. Ba sal. progesterone level (% of CS). 50. SQ 1μ M. Ba sal. progesterone level (% of CS). (a). CS 10 2',5 mg 'D /ml DA 0.3 μM +C 2',5 S 'D DA 3μ M+ 2',5 CS 'D DA 30 μM +C S. 2',5 Ba sal 'D DA 0.3 μM 2',5 'D DA 3μ M 2',5 'D DA 30 μM. progesterone level (% of CS). 350 3. 3. 250 3. 200. 1,2 1,2. 100. 1 1,2. 0. (b) 2. 2 2. 1. 2. 2. 2. 1.

(24) progesterone level (% of CS). 900 800. 3. 2,3. dbcAMP. (a). 3. dbcAMP+CS. 3. 3. 700 2,3. 600 1,2. 500. 1,3. 400 300 1. 200 100. 1. 1. 1. 0 CS. M 5m 0.0. mM 0.1. 2. (b). 100 3. 80 60 40 1. 1. 20 0. M 1m. mM 0.5. M 5m 0.2. progesterone level (% of CS). 120. 120. 2. (c). 100 3. 80 60 40 20. 1. 1. Figure 3. PKI +CS. CS 10m g/m l. PKI 4μ M. Basa l. H89 +CS. CS 10m g/m l. H89 50μ M. 0 Bas al. progesterone level (% of CS). al Bas.

(25) A PM. 100. 20. l g/m μ 2.5. 1 l g/m m 10 CS. 120. 80. 60. 40. 1. 0. (b) 2. 3. 0. Figure 4. 20. 20. 1. 120. 100. 1. 1. 80. 1 1. CS 10 mg /m l PK I+C al+ CS. 1 120. PK I+C al. 1. μ M. 3. Ca l 50 0n M. PK I4. CS A+ PM Ba sal. 2. Ba sal Ne o5 μM Ne o5 0μ M Ne o5 00μ M CS 10m g/m Ne l o5 μM +C Ne S o5 0μ M +C Ne S o5 00μ M+ CS. 300 250. progesterone level (% of CS). (a). progesterone level (% of CS). 200 150. Ca l+C S. sal Ba. CS 10 mg /ml. progesterone level (% of CS) 100 50. Ca l 50 0n M. Ba sal. progesterone levle (% of CS). 400 350. (c) 2. 100 80. 60 3. 40 1 1. 0. (d) 2 3 3. 60. 40. 1. 0 3.

(26) 30 kDa. 43 kDa. 43 kDa 3. 2.5. 2,3 1,2,3. 2 1,2,3. 1.5 1 1,2. 0.5. 1. 0 Basal CS 2. CS 4 CS 6. 2 1.5 3. 1 0.5. 1. 1. 0 al Bas. S +C Cal. l g/m 0m 1 CS. nM 500 Cal. 30 kDa. 43 kDa. 43 kDa. 3. 1.5 2. 1 1. 0.5. 1. 2. 1.4. (b). 1. optic density (StAR/b-actin). 2. (e). 1.2 1 0.8 0.6 0.4. 1. 0.2. 3. 1. 120'. 180'. 360'. time elapse 30 kDa 43 kDa 2. 2. (c). 1.5 1 0.5. 3. 1. 1. al Bas. M 0u 95 8 H. 0 l /m mg 0 1 CS. CS 9+ H8. Figure 5. H8 9+C al+ CS. 60'. Ba sal. 0'. CS 10 mg /ml. 0. 0. optimal density (StAR/ -actin). 2. 2.5. CS 8 CS 10. 30 kDa. optic density (StAR/ -actin). (d). H8 9+ Ca l. optic density (StAR/ -actin). 3. optic density (StAR/ -actin). 30 kDa.

(27) Ba sal flu 0.2 μ M flu 2μ M flu 20 μM CS 10 mg flu /ml 0.2 μM +C S flu 2μ M+ CS flu 20 μ M+ CS. progesterone level (% of CS). 20. 120. (b). 40. 140. 120. 1 1. 1 1. (c) 2. 1 1. 1. 2 2. 80. 60. 3. 1. 2 3. 0. 2. 100 3. 80. 60. 40. 1. 0. Figure 6. 1 1. 120. 20 1. CS 10 Nif mg edi /m pin l e5 μ Nif M+ edi CS pin e5 0μ Nif M+ edi CS pin e5 00 μ M+ CS. Nif Ba sal edi pin e5 μ Nif M edi pin e5 0μ Nif M edi pin e5 00 μM. 2. progesterone level (% of CS). 2. progesterone level (% of CS). Ba sal CS 10m g/m A2 l 318 70 .5 μ M A2 318 75 μM A2 318 75 A2 0μ 318 M 70 .5 μ M+ A2 CS 318 75 μM A2 +C 318 S 75 0μ M+ CS. progesterone level (% of CS). 2. 1. W7 +C S. 1 3. CS 10 mg /ml. 20. (a). 500 450 400 350 300 250 200 150 100 50 0. W7 10μ M. 100 1 1. Ba sal. cad miu Ba sal m0 .01 mM cad miu m0 .1m M cad miu m 1m M CS cad 10 miu mg m0 /ml .01 mM cad +C S miu m0 .1 m M+ cad CS mi um 1m M+ CS. progesterone level (% of CS). 400 350 300 250 200 150 100 50 0 2. (d) 2. 2. 2. 1. (e) 2. 100. 80 3. 60. 40. 1. 0.

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