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標題: | 腺苷與厚朴酚刺激大鼠腎上腺皮質細胞分泌皮質酮機制之探討 Studies on the Mechanisms of Adenosine- and Magnolol-stimulated Corticosterone Production in Rat Adrenocortical Cells |
作者: | Yung-Chia Chen 陳永佳 |
指導教授: | 王淑美 |
關鍵字: | 腺苷,厚朴酚,固醇類生合成,訊息傳遞, adenosine,magnolool,steroidogenesis,signal transduction, |
出版年 : | 2008 |
學位: | 博士 |
摘要: | 內分泌系統是負責調控動物體內生理功能的重要系統之ㄧ。其中,腎上腺皮質所分泌的荷爾蒙對於動物在遭受外來壓力與疾病之抵抗力非常重要。本實驗室之前研究已發現冬蟲夏草與厚朴酚可促進大鼠腎上腺皮質固醇類生合成,但詳細的訊息機制仍不清楚。在本論文第一部分中我們使用大鼠腎上腺初級培養細胞做為研究模式,探討腺苷促進腎上腺皮質固醇類生合成的機制。我們發現,腺苷可促進腎上腺皮質酮的產量上升。反轉錄-聚合酶酵素連鎖反應的分析偵測到腎上腺皮質細胞有A1,A2A,A2B,及A3共四種腺苷接受器mRNA的表現。藥理學拮抗劑實驗結果顯示,腺苷刺激皮質酮產量上升是透過A2A 及A2B接受器。進一步再利用干擾A2AR核醣核酸 (small interfering ribonucleic acid, SiRNA) 的技術,我們可更確定腺苷刺激皮質酮分泌是透過A2AR。A2AR及A2BR的免疫螢光染色分析可發現A2AR在細胞膜上呈現連續的點狀染色;A2BR的染色只有少量分布在細胞膜上。我們進一步證實腺苷透過活化下列的訊息傳遞路徑達到促進皮質酮產量增加,包括: Janus kinase 2 (JAK2) - mitogen-activated protein kinase kinase 1/2 (MEK1/2)- extracellular signal-regulated kinase 1/2 (ERK1/2),促使細胞核內的轉錄因子cyclic AMP responsive element binding protein (CREB)磷酸化上升,最後可能導致相關的固醇類生合成酵素基因的轉錄、轉譯作用的增加,造成皮質酮產量上升。
由於腺苷刺激皮質酮增加約有1/2是透過JAK2訊息路徑。所以,我們推論還有其他的訊息路徑參與調控腺苷刺激皮質酮分泌的過程。首先,利用專一性的抗體發現腎上腺皮質細胞有protein kinase C (PKC) α,PKCβI, PKCβII, PKCε, PKCδ, PKCζ和PKCμ的表現。藥理學的研究顯示一般的PKC抑制劑calphostin c,PKCα/β/γ/μ的抑制劑Gö6976或PKCε的抑制劑εV1-2皆可以減少腺苷刺激的皮質酮產量,顯示PKCα/β/γ/μ或PKCε參與調控腺苷刺激類固醇增加的作用。在腺苷刺激五分鐘後,磷酸化(活化態)的PKCβII及PKCε表現量增加,證明腺苷也活化了PKCβ及PKCε等訊息傳遞路徑。而PKCβ或PKCε受到腺苷的活化後也會進而活化下游的MEK-ERK1/2與CREB。皮質酮的生成需要受到一系列類固醇水解酵素的作用;我們利用RT-PCR偵測到腺苷會增加CYP11B1 mRNA的表現,所以腺苷也有可能造成CYP11B1蛋白質增多甚至升高其活性來調控皮質酮的生合成,這部分未來須有更多的實驗來闡明。 第三部份研究著重在探討厚朴酚對腎上腺皮質酮分泌的訊息路徑。我們發現MEK 抑制劑PD98059、TK抑制劑genistein以及JAK2抑制劑AG490都可以完全抑制由厚朴酚誘發的皮質酮分泌。此外,厚朴酚可以在短時間內增加MEK,ERK,CREB之磷酸化。同時,決定類固醇生成速率決定步驟的32及30 kDa的固醇類急性調控蛋白;steroidogenic acute regulatory protein (StAR)蛋白之表現也隨著厚朴酚刺激的時間增加而表現增多。為了探討訊息傳遞路徑的上下游關係,我們利用抑制劑阻斷TK及JAK2的活性,發現厚朴酚所引起的MEK、ERK之磷酸化程度會下降。這個結果顯示JAK2是整個訊息路徑最上游的調控分子。而JAK2及MEK的活化很明顯的調控了CREB的磷酸化與StAR 蛋白質的表現。我們的研究結果證明厚朴酚透過一個新的訊息調控機制來影響固醇類生合成。 總結而論,以腺苷或厚朴酚當作刺激源,皆可活化一條新的訊息傳遞路徑而造成腎上腺皮質細胞皮質酮的增加;而皮質酮的上升又有助於抗發炎,所以腺苷與厚朴酚在未來有希望做為在臨床治療免疫相關疾病的輔助用藥。 Endocrine system is one of the important systems which regulate physiologic condition in animals. Adrenal cortex mediates stress response through the production of hormone. We have reported that Cordyceps sinensis and magnolol can stimulate corticosterone production in rat adrenocortical cells; however, their mechanisms are unknown. In the first part of the thesis, we investigated the effects and mechanisms of Adenosine (Ado)-stimulated steroidogenesis. Meanwhile, the adenosine receptors which are involved in Ado-stimulated corticosterone production were also determined. Expression of A1, A2A, A2B, and A3 adenosine receptor (AR) mRNA in rat adrenal cells was shown by reverse transcription- polymerase chain reaction. Ado increased corticosterone production in a time- and dose-dependent manner, and this ado effect was mediated by the A2Rs, since the antagonists specific for the A2A and A2B receptors, and specific silencing the A2AR expression with small interfering RNA significantly blocked the ado-induced steroidogenesis. Using pharmacological approaches, we further demonstrated that Janus kinase 2 (JAK2) was the downstream molecule next to the A2ARand A2BR. Inhibition of JAK2 prevented the ado-induced steroidogenesis and phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal- regulated kinase 1/2 (ERK1/2), demonstrating that JAK2 was the upstream effector of the MEK pathway. Pretreatment with A2R, JAK2, or MEK inhibitors significantly decreased the adenosine-induced phosphorylation of 3’, 5’-cyclic adenosine monophosphate responsive element binding protein (CREB). In conclusion, these data show that ado-stimulated steroidogenesis is mediated via the A2AR and A2BR, activation of which triggers the JAK2-MEK-ERK signaling pathway and CREB phosphorylation. In the second part of the thesis, we identified the signaling pathways besides A2R -JAK2-MEK-ERK signaling pathway, which were responsible for Ado-stimulated steroidogenesis. Using pharmacological approaches, we found that the non-specific protein kinase C (PKC) inhibitor, calphostin c, inhibited the Ado-stimulated steroidogenesis. Moreover, PKCα/β/γ/μ inhibitor, Gö6976 and PKCε inhibitor εV1-2 blocked the Ado-stimulated corticosterone production, indicating that PKCα/β/γ/μ and PKCε are participated in this event. Antagonization of A2R inhibited the Ado-stimulated PKCα/βII or PKCε phosphorylation, indicating that PKC activation was downstream of A2R. Inhibition of PKC or PKCε prevented the Ado-induced phosphorylation of MEK-ERK, implying that PKCε is the upstream molecules of the MEK-ERK pathway. Ado increased the translocation of PKCα or PKCεto the membrane fraction, concomitantly with the enhanced PKCα/βII and PKCε phosphorylation. Inhibition of PKCα/β and PKCε reduced the Ado-induced of CREB phosphorylation, supporting that CREB is a downstream effector for PKCβ and PKCε. In conclusion, these data indicate that Ado-stimulated steroidogenesis might be mediated through PKCβ/ε-MEK-ERK-CREB cascade. In the third part of the thesis, we focused on identifying the signalling mediating the effect of magnolol on corticosterone production. Magnolol-induced corticosterone production was completely inhibited by MEK-inhibitor PD98059, tyrosine kinase (TK)-inhibitor genistein or JAK2-inhibitor AG490, suggesting that ERK and JAK2 are both involved in this signaling cascade. Further, magnolol induced the transient phosphorylation of MEK, ERK, CREB and the expression of 32 and 30 kDa steroidogenic acute regulatory protein (StAR) in a time-dependent manner. Inhibition of TK or JAK2 activities blocked magnolol-induced phosphorylation of MEK and ERK, again supporting the upstream role of JAK2. The activation of JAK2 or MEK apparently mediated the magnolol-induced phosphorylation of CREB and the upregulation of StAR. These findings demonstrate a novel pathway for magnolol to induce the expression of StAR, which regulates the rate-limiting step in sterodiogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37654 |
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