雌性素和雌性素接受體基因轉殖對心肌細胞保護之分子機轉探討

Investigation of the Protective Mechanisms of 17beta-Estradiol and/or Estrogen Receptor-alpha in Cardiomyocyts

劉忠榮(Chung-Jung Liu)

指導教授 ： 劉哲育

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2008年)

https://doi.org/10.6834/CSMU.2008.00128

摘要

Part-I：
女性罹患心血管疾病的機率要比同年齡的男性來得低，然而停經
後則呈緩慢增加的現象，此現象與更年期卵巢功能急速消退、女性雌激素分泌缺乏有關。目前雖然已知雌性素具保護心血管功能，但對心肌細胞的保護機轉仍不清楚。心肌細胞凋亡是心臟衰竭的重要原因之一，已知β-adrenergic pathway 過度活化會造成心肌細胞凋亡，然而雌性素(E2) 以及雌性素接受體(ER) 是否能有效地抑制β-adrenergicpathway 所誘發之心肌細胞凋亡，來保護心肌細胞，有待釐清。為了探討此機制，將胚胎大白鼠心肌轉型細胞株H9c2 建立double-stableTet-On/ERα expression system 以及使用ERα-transfected primarycardiomyocytes 進行分析。實驗結果發現：隨著isoproterenol (Iso)濃度的增加，過度活化β-adrenergic pathway 時，心肌細胞存活率隨著減少，同時Iso 造成心肌細胞明顯的DNA 斷裂以及凋亡小體產生之現象。使用MTT assay、DNA fragmentation assay、TUNEL assay 及DAPI nucleus staining 分析發現雌性素(17β-estradiol)及雌性素接受體α (ERα)可有效地抑制β-adrenergic pathway 所誘發之心肌細胞凋亡。實驗發現E2、ERα和BSA-E2 均可快速誘發Akt 和ERK1/2 活化、促進Bad 磷酸化並且抑制Iso 所誘發凋亡蛋白Bad 大量上升以及cytochrome c 從粒線體大量釋出之作用，進而抑制active caspases 3 &9 的活化，有效地抑制Iso 所誘導的心肌細胞凋亡。然而PI3K siRNA、LY294002 (PI3K inhibitor)、MEK1 siRNA 和U0126 (MEK inhibitor)卻有效地抑制此心肌保護之作用。實驗中進一步探討E2、ERα和BSA-E2 藉由何種mediators 調控Akt 與ERK1/2 的活化，於是使用Gαi protein inhibitor、 IGFIR inhibitor (AG1024)、Src inhibitor (PP2)、MMP2/9 inhibitor 和EGFR inhibitor (AG1478)處理Tet-On/ERα H9c2 cells，從實驗結果得知：Src-IGFIR pathway 調控E2 與BSA-E2 所誘發Akt 的活化：Src-MMP2/9-EGFR pathway 調控E2 與BSA-E2 所誘發ERK1/2 的活化。Src 調控ERα overexpression 所誘發Akt 的活化：Src-MMP2/9-EGFR pathway 調控ERα overexpression 所誘發ERK1/2活化。Src 在E2 與ERα的訊息途徑調控中的確扮演主要決定的角色，參與E2-ERα/ERα心肌保護的機制。

Part-II：除了上述β-adrenergic pathway 過度活化會造成心肌細胞凋亡之外，心臟衰竭是敗血病患中經常併發的病症。內毒素lipopolysaccharide(LPS)會造成病患的左右心室收縮舒張功能急速減低。心室功能減低的敗血症病人經常伴隨著血液中細胞激素上升。然而LPS 直接刺激心(臟)肌細胞，是否誘發細胞病理性肥大、細胞凋亡與心肌纖維化的影響與詳細的機轉，目前尚待釐清。於是在實驗中同時進行這方面的探討。使用U0126 (ERK1/2 innhibitor) 、SB203580 (p38 MAPKinhibitor)、SP600125 (JNK1/2 inhibitor)、CsA (calcineurin inhibitor)、FK506 (calcineurin inhibitor)或QNZ (NFκB inhibitor)進行實驗分析
後，實驗結果發現：(A) LPS 主要透calcineurin/NFAT-3 pathway 誘發ANP 和BNP 基因與蛋白的表現、病理性心肌細胞肥大。(B) LPS 透過JNK1/2 signaling pathway 誘發促凋亡蛋白TNFα, active caspase-8,t-Bid, Bax, Bak, active caspase-9 和active caspase-3 上升與DNA 斷裂以及心肌細胞凋亡。(C) 在心肌細胞與心臟纖維母細胞中，LPS 主要透過ERK1/2 signaling pathway 誘發cardiac fibrosis-related factors uPA,MMP-2 與MMP-9 蛋白表現以及MMP-2 和MMP-9 的活性上升。

Part-III：
從文獻得知：臨床上心肌梗塞(myocardial infarction, MI)、燒燙傷(burns)、創傷(trauma)與敗血症(sepsis)病患中，女性較不易有過度發炎反應、器官衰竭與致死的現象。Schroder 發現女性敗血症病患有較低的TNFα含量與較低的死亡率。燒燙、創傷傷害(burn trauma injury)病患中也發現女性有較低細胞介素生成(cytokines production)、較低的心肌(臟)發炎反應和有較好的心肌(臟)收縮功能。於是我們也進一步探討雌性素(E2)以及雌性素接受體(ER)是否有效抑制LPS 所誘發的心肌細胞凋亡並且釐清透過何種機轉達到心肌的保護作用。實驗結果發現：(1)E2, BSA-E2 和ERα overexpression 會抑制LPS 所誘導的IκB磷酸化、IκB 降解、NFκB 磷酸化/活化、NFκB 進核與NFκB-DNAactivity，進而抑制LPS 所誘發的促凋亡蛋白TNFα, active caspases-8,t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3 和心肌細胞凋亡。(2)JNK1/2 扮演一個重要的角色，JNK1/2 參與/調控LPS 所誘發IκB 磷酸化、IκB 降解、TNFα表現以及心肌細胞凋亡。(3)E2, BSA-E2 和ERα overexpression 主要透過PI3K-Akt signaling pathway 來抑制LPS 所活化的JNK1/2，進而抑制IκB 降解與NFκB
進核、阻止LPS 誘發TNFα和active caspase-3 的表現與心肌細胞凋
亡。此實驗或許對於臨床上女性為何比較不容易有心肌(臟)發炎反
應、心肌(臟)衰竭和有較好的心肌(臟)收縮功能提供一個合理的解釋與機轉。

關鍵字

雌性素 ； 雌性素接受體 ； 心肌細胞 ； 基因轉殖

並列摘要

Part-I：
It is well established today that premenopausal women display a lower prevalence of cardiovascular diseases compared with age-matched men, contributing to a low incidence of heart disease after menopause following receiving estrogen replacement. The sympathetic nervous system is one of the most important mechanisms to affect the cardiac function by accelerating contraction and relaxation through β-adrenergic stimulation. However, increased plasma norepinephrine (NE) has been observed in patients with heart failure, and it has been found that stimulation of β-AR by NE and isoproterenol (Iso) can promote the apoptosis of ventricular myocytes and dilated cardiomyopathy in vivo and in vitro. To identify the cardioprotective effects and mechanisms of estrogen (E2) and estrogen receptor (ER), in the present study using
doxycycline (Dox)-inducible Tet-On/ERα H9c2 myocardiac cells and ERα-transfected primary cardiomyocytes overexpressing ERα, we further investigate the effects of 17β-estradiol (E2) and estrogen receptor α (ERα) on β-adrenergic pathway-induced apoptosis by analyzing the
activation of survival and death signaling pathways. We found that treatments involving E2, membrane-impermeable BSA-E2 and overexpression of ERα significantly phosphorylated Akt and ERK1/2 and inhibited isoproterenol (Iso)-induced apoptosis analyzed by MTT analysis, TUNEL assay, DNA fragmentation, DAPI nucleus staining and Western blot for Bcl-2 family proteins (including Bcl-2, phospho-Bad, Bid and tBid), released cytochrome c, active caspase-9 and active caspase-3. The abilities of E2, ERα overexpression and BSA-E2 to inhibit the Iso-induced
apoptosis and promote cell survival through the phosphorylation of Akt, ERK1/2 and downstream Bad were attenuated by applying LY294002 (PI3K inhibitor), U0126 (MEK1/2 inhibitor), PI3K siRNA and MEK1 siRNA. Furthermore, the treatments using ICI 182,780 (ICI), ERs antagonist, and melatonin (Mel), an ERα-specific inhibitor, completely blocked the cardioprotective properties of E2, overexpression of ERα and BSA-E2, accompanied by the deactivation of Akt and ERK1/2. After
administrations of Gαi protein inhibitor, IGFIR inhibitor (AG1024), Src inhibitor (PP2), MMP2/9 inhibitor and EGFR inhibitor (AG1478), we found that Src plays key role mediating BSAE2-ERα, E2-ERα-activated Src-IGFIR-PI3K-Akt pathway and Src-EGFR-MEK1/2-ERK1/2 pathway, and that Src mediates ERα overexpression-activated Src-PI3K-Akt pathway and Src-EGFR- MEK1/2-ERK1/2 pathway. Taken together,these data suggest that Src mediates the inhibitory effects of E2-ERα and/or ERα overexpression on β-adrenergic pathway-induced cardiomyocyte apoptosis through activation of PI3K-Akt and MEK1/2-ERK1/2 pathways.

Part-II：
Evidences suggest that lipopolysaccharide (LPS) participates in the inflammatory response in the cardiovascular system and causes the cardiovascular collapse and even death observed in patients with bacterial
sepsis; however, the cellular mechanisms involved in LPS-induced cardiac hypertrophy, cardiac apoptosis and expression of cardiac fibrosis-related factors still remains unclear. (A) Here we show that after administration of inhibitors including U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor), FK506 (calcineurin inhibitor) and QNZ (NFκB inhibitor), LPS-induced hypertrophic characteristic features such as
increases in cell size, actin fibers and levels of ANP and BNP, and the nuclear localization of NFAT-3 are markedly inhibited only by calcineurin inhibitors, CsA and FK506. These results suggest that LPS leads to myocardiac hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 cells. (B) The LPS-increased TUNEL-positive
myocardiac cells, DNA fragmentation, and up-regulations in TNFα, active caspases-8, -9, -3, tBid, Bax, Bak and released cytochrome c proteins in myocardiac cells treated with LPS were completely inhibited by SP600125 (JNK1/2 inhibitor). The results demonstrate that LPS-induced myocardiac cell apoptosis is mainly mediated through JNK1/2 signaling pathway and these findings give JNK1/2 a novel role in LPS-induced heart failure, particularly in patients with sepsis. (C) The LPS-induced expressions/activities of uPA, MMP-2 and MMP-9 were
significantly blocked by ERK1/2 pathway inhibitor, U0126 in H9c2 cells and primary cardiac fibroblasts. These results suggest that ERK1/2 signaling pathway mediates LPS-upregulated uPA, MMP-2 and MMP-9 expression and activation.

Part-III：
Evidences show that females have lower TNFα level, heart
dysfunction and motility in sepsis. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen (E2) and estrogen receptor (ER), we used doxycycline (Dox)-inducible Tet-On
H9c2 myocardiac cells and ERα-transfected primary cardiomyocytes overexpressing ERα to investigate the effects of 17β-estradiol (E2) and estrogen receptor α (ERα) on LPS-induced apoptosis by analyzing the activation of survival and death signaling pathways. We found that LPS challenge activated JNK1/2, and then induced IκB degradation, NFκB activation, TNFα upregulation and subsequent myocardiac apoptotic responses. In addition, treatments involving E2, membrane-impermeable BSA-E2 and/or ERα overexpression significantly inhibited LPS-induced apoptosis through suppressions of LPS-upregulated JNK1/2 activity, IκB degradation, NFκB activation and pro-apoptotic proteins (e.g. TNFα, active caspases-8, t-Bid, Bax, released cytochrome c, active caspase-9, active caspase-3) in myocardiac cells. However, the cardioprotective properties of E2, BSA-E2 and ERα overexpression to inhibit LPS-induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that the activation of PI3K−Akt pathway is required for E2, BSA-E2 and ERα overexpression to inhibit LPS-induced IκB degradation and NFκB activation through suppressing JNK1/2 activity, and then further contributes to the cardioprotective effects of E2, BSA-E2 and ERα by
inhibiting LPS-increased TNFα, active caspase-3 and apoptosis in myocardiac cells.

並列關鍵字

Estrogen ； Estrogen Receptor ； Apoptosis ； Cardiomyocyte ； Gene Transfection

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