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標題: | 細胞外酸性pH對HepG2細胞黏附接合之影響 Effects of acidic extracellular pH on Adherens Junctions of HepG2 cells |
作者: | Po-Yuan Tung 董伯元 |
指導教授: | 王淑美 |
關鍵字: | 黏附接合, adherens junction, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 細胞外酸性pH(pHe)是腫瘤細胞生長環境的特徵之一,並且它會影響腫瘤的侵犯能力。過去多著重於研究分解細胞外基質(extracellular matrix)的能力,鮮少探討adherens junction(AJ)參與的角色。我們假設pHe可能藉由破壞AJ而增加細胞移行能力。結果顯示,細胞培養在酸性的培養液(pH 6.6)中,會引發細胞形態的改變,從原本緊密接合的群聚狀態,變為比較扁平的分散細胞,且伴隨著增強的細胞移行能力。pH 6.6處理之後,活化的c-Src磷酸酶短時間內即有顯著的增加。和pH 7.4的組別相比,pH 6.6處理組的細胞於AJ處的E-cadherin和β-catenin染色減少,且細胞質內含有E-cadherin的vesicle增加。同時,細胞膜上E-cadherin和β-catenin的蛋白質的量也顯著減少。使用Src的抑制劑PP2處理,可以阻止因為pHe所造成上述兩種蛋白質在細胞膜上減少的狀況,同時也會抑制因酸性pHe而增加的細胞移行能力。pH 6.6可以造成β-catenin 酪氨酸(tyrosine)磷酸化的程度顯著上升,並且伴隨著連接在β-catenin上的E-cadherin相對減少。這些效應可能是因為活化的c-Src可以藉由磷酸化β-catenin的Y654,促使cadherin/catenin complex瓦解的緣故。除此之外,利用免疫沉澱法,可觀察到pH 6.6處理後E-cadherin酪氨酸磷酸化的程度增加,而且與E-cadherin結合的Hakai和ubiquitinized E-cadherin的蛋白質量也分別增加。故推測c-Src也可以使E-cadherin酪氨酸磷酸化,使得Hakai (cbl-like ubiquitin ligase)連接到E-cadherin上,進一步促進ubiquitinated E-cadherin的形成,而增加E-cadherin endocytosis。酸性pHe所引發之E-cadherin在細胞膜上分布的減少,也受到其他機制的調控。在pH 7.4對照組的細胞,actin bundles會嵌入AJ處。pH 6.6處理使AJ處E-cadherin 減少及cortical actin bundles去聚合,伴隨著活化的PKC An acidic extracellular pH (pHe) is a feature of the tumor microenvironment and increases tumor invasion. However, little attention has been paid to the role of adherens junctions (AJs) in acidic pHe-induced cell invasion. We hypothesized that an acidic pHe may increase cell migration by disruption of the AJ via modulation of cell-cell adhesion molecules. Our results showed that incubation of HepG2 cells in acidic medium (pH 6.6) induced a phenotypic change from tight cell clusters to dispersed cells with a flattened morphology and this change was accompanied by an increase of cell migration. Rapid phosphorylation of c-Src kinase was detected after incubation of the cells at pH 6.6. In comparison to cells incubated at pH 7.4, the decrease of E-cadherin and β-catenin staining at AJ and an increase in E-cadherin-containing cytoplasmic vesicles were observed in pH 6.6-treated cells. The lower protein levels in the membrane fractions of E-cadherin and β-catenin were also noticed. Pretreatment with a Src kinase inhibitor, PP2, prevented the acidic pHe-induced decrease in protein levels and immunoreactivity of membrane-bound E-cadherin and β-catenin. The acidic pHe-induced c-Src activation increased tyrosine phosphorylation of E-cadherin and β-catenin, and resulted in the decreased amount of E-cadherin associated with β-catenin. These effects may be caused by disruption of cadherin/ catenin complex through tyrosine phosphorylation β-catenin at Y654 in response to c-Src kinase activation. Furthermore, the protein amounts of Hakai (cbl-like ubiquitin ligase) bound to E-cadherin and the levels of ubiquitinated E-cadherin were both increased by pH 6.6 treatments by immunoprecipitation and immunoblotting analysis. The results inferred that the activation of c-Src kinase led to the binding of Hakai to tyrosine phosphorylated E-cadherin at AJ, which promoted increased ubiquitination of E-cadherin and accelerated E-cadherin endocytosis. In addition, other mechanisms may contribute to the acidic pHe-induced downregulation of membranous E-cadherin. In pH 7.4-treated cells, cortical actin bundles inserted at AJ. pH 6.6 treatment caused the disappearance of E-cadherin staining coincided with the dissolution of junctional actin bundle, and induced a rapid activation of PKCε. Our results showed that the inhibition of PKCε by eV1-2 prevented the depolymerization of junctional actin bundles and downregulation of junctional E-cadherin induced by acidic pHe. These data confirmed the hypothesis that normal distribution of E-cadherin at AJ is intimately associated with cortical actin bundles. Either inhibition of PKCε by eV1-2 or of c-Src by PP2 prevented acidic pHe induecd cell migration. Acidic pHe enhanced the association of active PKC |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28203 |
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顯示於系所單位: | 解剖學暨細胞生物學科所 |
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