水解磷脂酸造成卵巢癌SKOV3細胞接合去組裝與其訊息傳遞路徑之探討

Abstract

水解磷脂酸(lysophosphatidic acid, LPA)是一種細胞外調節因子，它參與調控許多生物功能，當水解磷脂酸的調控發生異常時會促進癌細胞的增生，增加癌細胞的轉移。我們實驗室先前的研究發現，水解磷脂酸會活化Src家族蛋白質激酶的成員Fyn，並且經由增加Fyn與p120-catenin的結合來調控黏附接合的穩定性。由於catenin磷酸化狀態的改變會影響黏附接合的完整性，這個過程是經由磷酸激酶與水解磷酸酶所共同調控。本研究的主旨在於了解水解磷脂酸造成黏附接合瓦解時，是如何影響細胞黏附接合蛋白之間的交互作用和在這過程中酪氨酸磷酸酶，包括：Src家族蛋白質激酶、Fer磷酸激酶與酪胺酸水解磷酸酶PTP1B所扮演的角色。本研究以免疫螢光染色觀察水解磷脂酸對卵巢癌SKOV3細胞株細胞形態的影響，和黏附接合蛋白在細胞中分佈位置的改變，以西方墨點分析和免疫沉澱法探討水解磷脂酸如何改變黏附接合蛋白質的交互作用，導致黏附接合的不穩定。水解磷脂酸在短時間內即可導致SKOV3細胞的黏附接合分離，在這過程中細胞黏附接合會以half-junction的形態呈現。免疫沉澱分析顯示水解磷脂酸活化Src家族蛋白質激酶，增加與黏附接合蛋白beta-catenin的結合，並使得beta-catenin與alpha-catenin的結合下降。此外水解磷脂酸也會調節PTP1B與cadherin-catenin複合體的結合，改變黏附接合的穩定度。免疫沉澱分析得知水解磷脂酸會降低p120-catenin與Fer磷酸激酶之間的結合，使得Fer與黏附接合蛋白複合體分開，並減弱水解磷酸酶 PTP1B與beta-catenin和N-cadherin之間的結合。水解磷酸酶PTP1B主要是讓beta-catenin處於低磷酸化狀態，並維持黏附接合蛋白複合體的完整。我們以辨識酪氨酸磷酸化的抗體進行免疫沉澱分析得知，一旦PTP1B從cadherin-catenin複合體離開後，會使beta-catenin的酪氨酸磷酸化上升，而導致黏附接合複合體瓦解。我們也觀察到水解磷脂酸處理SKOV3細胞，會造成p120-catenin的酪氨酸磷酸化上升，卻不會改變N-cadherin與Fer磷酸激酶的酪氨酸磷酸化狀態。而加入Src家族蛋白磷酸激酶的抑制劑PP2後，會回復水解磷脂酸減弱Fer與p120-catenin結合以及beta-catenin分別與PTP1B和alpha-catenin結合的影響。綜合以上的結果我們推測：水解磷脂酸造成黏附接合瓦解的過程中一方面會活化Src家族蛋白質激酶，增加p120-catenin與pSrc的結合，使得p120-catenin的酪氨酸磷酸化上升 ，導致p120-catenin與Fer磷酸酶的結合下降。另一方面也會促進PTP1B從cadherin-catenin複合體分開，造成beta-catenin的酪氨酸磷酸化上升，促使beta-catenin與alpha-catenin的結合下降，細胞間黏附接合瓦解並造成cadherin-catenin複合體內化而進入細胞內。

Lysophosphatidic acid (LPA) is a bioactive lipid with various biological functions, such as cell proliferation, cell survival, and tumor cell invasion. LPA promotes cancer progression and is present at high concentrations in both the plasma and ascites of ovarian cancer patients. In our previous study, we have demonstrated that LPA induces junction dispersal in ovarian cancer SKOV3 cells by activating the Src family kinase Fyn and increasing its association with p120-catenin. However, the mechanism that LPA induces junction dispersal remained unclear. The disassembly of the cadherin-catenin complexes is regulated by the phosphorylation of tyrosine residues on beta-catenin and is correlated to the integrity of adherens junction. Regulation of phosphotyrosine content on beta-catenin is a net result of the balance between the activities of protein tyrosine phosphatase and kinase at the cytoplasmic domain of cadherin. In this study immunoprepcitation, immunoblotting, and immunofluoresence microscopy were used to explore the effect of LPA on junction dispersal in SKOV3 cells and its role in cadherin-bound PTP1B. LPA treatments induced activation of Src family kinase concomitant with a disassociation of PTP1B from cadherin-catenin complexes. Activation of Src family kinase also promoted its association with p120-catenin and induced an increase in tyrosine phosphorylation of p120-catenin. Since Fer kinase is constitutively, indirectly associated with N-cadherin via a binding of p120-catenin, we then explore the effect of LPA on Fer-p120-catenin association. Phosphorylation of p120-catenin on tyrosine residue caused a decrease in its association with Fer and resulted in dissociation of PTP1B from cadherin-catenin complexes. The effects of LPA on the decrease association of Fer and PTP1B with p120-catenin were Src-dependent, since both effects were inhibited by the selective tyrosine kinase inhibitor PP2. These data demonstrate that dissociation of cadherin-bound PTP1B by LPA increases tyrosine phosphorylation of beta-catenin, disrupts beta-catenin-alpha-catenin interaction, and results in intercellular junction dispersal in SKOV3 cells via a Src signaling pathway.