雌激素受體β拑制ZAK所誘發之心肌細胞肥大訊息機轉探討

CMUR > College of Medicine > Graduate Institute of Basic Medical Science > Theses & dissertations > Item 310903500/41340

Please use this identifier to cite or link to this item: http://ir.cmu.edu.tw/ir/handle/310903500/41340

Title:	雌激素受體β拑制ZAK所誘發之心肌細胞肥大訊息機轉探討 Mechanisms of Estrogen Receptor β Suppressive Effects on ZAK-Induced Hypertrophy in Myocardiac Cells
Authors:	蘇家琪
Contributors:	基礎醫學研究所碩士班
Keywords:	小泛素修飾分子;泛素蛋白;雌激素接受體β SUMO-1;Ubiquitin;p-JNK;p-p38;c-Jun;GATA-4;Erβ
Date:	2011-08-15
Issue Date:	2011-10-17 16:52:08 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	Part I 心肌肥大為心臟在面對各種刺激下的適應性反應。長期壓力刺激之下，使得心肌由生理性肥大轉向病理性肥大，終將導致心肌細胞凋亡、心肌纖維化與心肌衰竭。在本實驗室先前研究中已發現ZAK在心肌梗塞病人檢體中會高度表現，且野生型的ZAK會誘發心肌細胞肥大及肥大指標蛋白-鈉利尿素蛋白 (ANP) 的表現。我們在本實驗中，以Tet-on ZAK wild-type H9c2處理四環黴素(Doxycycline)，發現隨Dox濃度的增加，心肌肥大指標腦利尿素蛋白(BNP)及細胞骨架大小均隨著劑量增加而上升。ZAK蛋白經Dox的誘發表現，亦促進p38、p-JNK訊息及肥大轉錄因子p-GATA4及p-c-Jun的表現活化，但p-ERK蛋白及NFATc3轉錄因子均未能改變上述tet-on ZAK wild-type H9c2原本呈現的現象，tet-on DN均未在H9c2細胞中發現。核質分離及免疫螢光的實驗更進一步確認Dox促進tet-on ZAK wild-type細胞中的p-GATA4及p-c-Jun核轉置現象。之後我們在上述實驗中分別加入了U0126 (ERK1/2 抑制劑), SP600125(JNK1/2 抑制劑), SB203580(p38 MAPK 抑制劑)和CsA(Calcineurin 抑制劑)來進一步探討ZAK誘發BNP及肥大之機轉，發現JNK1/2抑制劑和p38抑制劑確實可以抑制ZAK所誘導的心肌細胞肥大指標，BNP的蛋白表現量，並同時阻斷轉錄因子p-GATA-4、p-c-Jun之活化現象。最後再觀察JNK 及p38 siRNA轉殖至初生小鼠之初代培養後的效應，亦發現其顯著阻斷ZAK誘發之心肌細胞肥大之現象。綜合我們的研究證實，ZAK主要經由p38和JNK的訊息途徑，刺激活化轉錄因子GATA-4和c-Jun，同時導致BNP的大量表現進而誘發心肌細胞肥大。期盼未來ZAK基因表現之阻斷或下游訊息路徑之抑制可進一步應用於治療因ZAK大量表現所造成之心肌病理性肥大患者。 Part II 臨床的研究發現，停經前的女性罹患心臟肥大相關疾病的危險性明顯低於同年齡男性，但一旦進入更年期，停經後婦女罹患心臟肥大相關疾病的情形卻大幅增加，在給予適當的荷爾蒙補充後症狀得以獲得改善，然而雌激素與雌激素接受體究竟是透過何種機轉達到保護心臟仍然不甚清楚。在本實驗室先前研究中已發現，經由卵巢切除的母鼠在給予雌激素的補充後，可降低其心肌肥大與心臟疾病的不良影響；另外在LPS所誘導的心肌細胞凋亡實驗中，雌激素補充及雌激素接受體α過度表現確實可達到抑制的效果。除此之外，我們也發現到ZAK在人體病態組織中有高度的表現，並導致病理性的肥大和纖維化。故本實驗主旨在探討雌激素與雌激素接受體β對於心肌保護影響的機轉究竟為何。實驗中我們首先使用了雌激素接受體β活化劑 (DPN) 以及短暫轉殖雌激素接受體β基因到Tet-on ZAK 野生型 H9c2細胞中，結果顯示雌激素接受體β確實會去抑制ZAK過度表現而誘發之心肌細胞肥大的現象，及肥大指標BNP蛋白的表現，同時ZAK下游之p-JNK, p-p38 和轉錄因子c-Jun, GATA-4蛋白活化亦皆受到抑制，但在加入雌激素接受體抑制劑 (ICI) 後，雌激素接受體β所造成的抑制效果則完全消失。同時本實驗更顯示，在正常情況下，雌激素接受體β就與ZAK產生鍵結，而ZAK的過度活化則降低其結合情形，當短暫轉殖雌激素接受體β基因則更促進兩者間的結合，並進一步抑制了ZAK蛋白表現。而我們的實驗結果也證明其並非經由促ZAK蛋白質降解而減少蛋白量，我們推測雌激素接受體β可經由直接抑制ZAK mRNA基因的轉錄而造成此影響。此外我們也發現，雌激素接受體β可經抑制ZAK專一性E3 ligase-CBL的蛋白表現量，使得CBL與PI3K的結合能力下降而活化了細胞存活的訊息途徑並達到保護心肌的功能。同時由實驗得知，雌激素接受體β過度表現亦會抑制SUMO-1的修飾作用，而阻斷了ZAK蛋白進合的能力，使其大量堆積在細胞質中，而抑制了ZAK的下游訊息途徑。我們的結果更證明雌激素接受體β的過度表現在無雌激素的補充下，仍可達到抑制ZAK所造成的心肌肥大現象，期盼未來對停經後的女性可採用雌激素接受體β過度表現基因療法來達到治療因ZAK誘發肥大所導致之心肌傷害，同時可避免因雌激素過度給予所造成的乳癌及子宮頸癌之副作用。 Part I Cardiomyocyte hypertrophy is an adaptive response of heart under varied types of stress. During the period of accumulation of stress, the transition from physiological hypertrophy to pathological hypertrophy resulted in promotion of heart failure. Our previous studies found ZAK highly expressed in heart specimen of myocardial infarction patients and demonstrated that overexpression of ZAK by Tet-on system induced cardiac hypertrophy and elevated the pathiological hypertrophy marker, Atrial natriuretic peptide （ANP） expression. In this study, we firstly applied tet-on ZAK WT H9c2 and treated them with Doxycycline (Dox), finding the increases in both cell size and hypertrophic marker BNP in a dose-dependent manner. ZAK expression was induced by Dox triggering the p38 and JNK pathway and activating the p-GATA4 and p-c-Jun transcription factors, without the involvement of p-ERK or NFATc3. However, tet-on ZAK DN showed no effect on the above signaling. Moreover, by the nuclear immunostaining and nuclear-cytoplasm isolation assay, we further confirmed that activated tet-on ZAK WT H9c2 cell may have highly enhanced the p-GATA4 and p-c-Jun nuclear translocation. To identify which signal pathways were involved in the mechanism behind ZAK-induced cardiomyocyte hypertrophy, we further applied the following inhibitors including U0126 (ERK1/2 inhibitor), SP600125 (JNK1/2 inhibitor), SB203580 (p38 MAPK inhibitor) and CsA (Calcineurin inhibitor) to individually block the specific pathway. The results showed both inhibitors of JNK1/2 and p38 significantly suppressed ZAK-induced BNP expression. In addition, transfected the siRNA of p38 and JNK both blocked the hypertrophy-related c-Jun and GATA-4 transcription factor activation. Moreover, the application of p38 and JNK siRNA obviously reduced the cell size of neonatal cardiomyocytes induced by ZAK overecpression. All our findings strongly suggest ZAK activated through p38 and JNK signaling pathways, and further promote GATA-4 and c-Jun activation induced hypertrophic marker BNP overexpression, and caused cardiomyocyte hypertrophy. We proposed that the gene silencing of ZAK and/or blocking ZAK downstream p38 and JNK pathway could be applied to ameliorate the heart pathiological hypertrophy symptom of ZAK-overexpressed patients. Part II Previously reports in our lab indicated estrogen supplement reduced pathiological cardiac hypertrophy and heart failure in ovariectomized female rats, and E2 and/or ERβ inhibited LPS-induced myocardial cell apoptosis. The estrogen level and abundant ERα expression might contribute to a low incidence of heart disease in pre- and post-menopause following receiving estrogen replacement women, but the effect of ERβ needed more investigation. Besides, we found aberrantly expression analysis of ZAK protein in human myocardial abnormal tissues, and found ZAK lead to pathiological hypertrophy and fibrosis. Therefore, we aim to further identify the cardioprotective effects and mechanisms of estrogen (E2) and estrogen receptor β (ERβ) against ZAK protein overexpression. We applied DPN (ERβ agonist) and transient transfection to overexpress ERβ into the ZAK tet-on H9c2 cells. Our data showed the significantly increase of cell size and pathiological hypertrophy BNP protein induced by Dox of ZAK WT overexpressed tet-on H9c2 cells. However, treatment of ERβ and E2 plus ERβ could totally suppress the ZAK overexpression effect. Results also showed the induction of p-JNK, p-p38 and their downstream c-Jun, GATA-4 transcription factors in ZAK overexpressed cells, but significantly inhibited by ERβ and E2 plus ERβ as well. However, the suppressive effects of E2 and/or ERβ in ZAK WT H9c2 cells were totally reversed by ER antagonist, ICI. Moreover, our results even indicate that ERβ indeed bind with ZAK in normal situation, after treatment of Dox to overexpress ZAK along reduced ZAK-ERβ association significantly. However, the overexpressed ERβ with or without E2 both enhanced the binding strength of ERβ and ZAK, and ERβ overexpression reduced ZAK protein level, but not mediated through the ubiquitination effect to induce ZAK protein degradation, which suggest the ERβ might directly inhibit the ZAK gene mRNA transcription. Furthermore, the ZAK protein E3 ligase-CBL was significantly suppressed by ERβ overexpression, which might attenuate CBL-PI3K protein association to prevent PI3K protein degradation and maintain the cardiomyocyte survival capabilities. Moreover, we observed the overexpressed ERβ and E2 plus ERβ overexpression both significantly block ZAK nuclear translocation via the inhibition of SUMO-1 modification. Taken together, our results further suggest that even without estrogen supplement, and only ERβ overexpression still strongly suppressed ZAK-induced cardiomyocyte hypertrophy, which implied that ERβ gene overexpression therapy might be a potential treatment for myocardial damage and would avoid the side effects of breast and vaginal cancer occur under estrogen over supplement of post-menopause women.
Appears in Collections:	[Graduate Institute of Basic Medical Science] Theses & dissertations

Files in This Item:

File	Description	Size	Format
cmu-100-9882006-1.pdf		2544Kb	Adobe PDF	182	View/Open
index.html		0Kb	HTML	10	View/Open

Loading...