Study of Signal Transduction Pathways Activated by Cordycepin on Steroidogenesis in Mouse Leydig Cells

Autor: Hsiang-Yin Pao, 包翔引
Rok vydání: 2008
Druh dokumentu: 學位論文 ; thesis
Popis: 96
Cordycepin (3'-deoxyadenosine) is an adenosine analog isolated from Cordyceps sinensis, which is a widespread component used in traditional Chinese medicine to reduce fatigue, and is prescribed for various diseases, such as pulmonary disease, chronic inflammation or even cancer therapy. Previously, we have demonstrated that cordycepin might act through the adenosine receptors (A1, A2a, and A3 subtypes) to activate StAR protein expression and regulate steroidogenesis in normal mouse Leydig cells. Correspondingly, the stimulatory effect could also be observed in MA-10 mouse Leydig tumor cells. However, the intracellular signal transduction pathways mediated by cordycepin in MA-10 cells remain unclear. Here, we hypothesized that cordycepin may act through adenosine receptors to induce cAMP/PKA signal transduction pathway and regulate steroidogenesis in Leydig cells. To investigate whether cordycepin can regulate Leydig cell steroidogenesis, radioimmunoassay and immunoblot assay were used in this study to determine steroidogenic hormones production and specific protein expression. Results demonstrated that, compared to control, 100 μM cordycepin significantly induced progesterone production in MA-10 cells (p < 0.01). This stimulatory effect reached 3-fold after 12 and 24 hr treatment. GF109203X (PKC inhibitor; 1 μM), U73122 (PLC inhibitor; 20 μM), and sodium orthovanadate (IP3-Ca2+ inhibitor; 40 μM) significantly inhibited cordycepin-activated progesterone production (p < 0.05), whereas H89 (PKA inhibitor; 5 ~ 100 μM), wortmannin (PI3K inhibitor; 10 ~ 200 nM), U0126 (MEK1/2 inhibitor; 1 ~ 20 μM), PD98059 (ERK1/2 inhibitor; 5 ~ 50 μM ), SB202190 (p38 MAPK inhibitor; 1 ~ 25 μM), and SP600125 (JNK inhibitor; 20 ~ 500 nM) did not. Interestingly, U0126 (1 μM) and SP600125 (500 nM) enhance cordycepin-mediated progesterone production for 2.5 and 1.8 folds (p < 0.05), respectively. The increasing trend of steroidogenic acute regulatory (StAR) protein expression in MA-10 cells was not observed under cordycepin treatment, but the increasing effect could be significantly observed after PD98059 (ERK1/2 inhibitor; 50 μM) and U0126 (MEK1/2 inhibitor; 20 μM) alone treatment or co-treatment of cordycepin compared to control (p < 0.05). In contrast, the phosphorylation of ERK 1/2 and JNK could be detected after 15 min cordycepin treatment (p < 0.05). Moreover, the inhibition of PKC significantly enhance the expression of cordycepin-activated ERK1/2 protein phosphorylation (p < 0.05), suggesting that ERK1/2 might have inhibitory role on cordycepin-activated steroidogenesis. Furthermore, the cordycepin (40 mg/kg) was administrated by intraperitoneal (IP) injection into 6-week old male B6 mouse to investigate its effect in vivo. Data illustrated that 7-day cordycepin treatment would increase the testes weight and plasma testosterone concentration (p < 0.05). In addition, with cell morphological observation and MTT cell viability assay, we also demonstrated that cordycepin caused MA-10 cell death, which was possible through the activation of PKC pathway. This death effect was not caused by the accumulation of progesterone, which was produced by MA-10 cell induced by cordycepin. Together, our study demonstrated that cordycepin significantly induced mouse Leydig cell steroidogenesis both in vitro and in vivo, and this effect might go through PKC and MAPK signal transduction pathways. Moreover, cordycepin might activate PKC signal transduction pathway to induce MA-10 mouse Leydig tumor cell death.
Databáze: Networked Digital Library of Theses & Dissertations
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