CD98 활성화에 의한 사람유방암세포 MCF-7의 침윤성 증가
CD98 Activation Increases the Invasion of Human Breast Carcinoma MCF-7 Cells
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초록
CD98 단백질은 125-kDa 크기의 제2형 막횡단 당단백질로서, β1 인테그린의 기능을 조절한다. 이는 이 분 자가 암세포의 침윤과정에 관여함을 시사한다. 본 연구의 목적은 암세포의 부착과 침윤에 대한 CD98신호의 영향 을 조사하는 데에 있다. MCF-7 세포 표면에서의 CD98의 발현 여부는 면역조직화학법으로 확인하였다. CD98 활성화가 MCF-7 세포의 부착과 침윤에 미치는 영향은 세포-세포밖기질 부착도 및 침윤도측정법을 통하여 조사하였다. Dominant-negative 형태의 FAK 유전자의 일시적 발현을 유도하기 위하여 리포솜 제제를 이용하여 pcDNA3-FAK related non-kinase (FRNK)와 pcDNA3-FAK Y397F plasmid DNA를 각각 세포안으로 도입하였다. CD98 분자가 단클론항체에 의하여 활성화될 경우, 사람유방암세포주인 MCF-7 세포의 파이브로넥틴, 라미닌과 제4형 콜라겐에 대한 부착력과 침윤력이 증가되었다. EDTA나 β1 인테그린 억제 항체를 CD98 항체와 함께 처리 할 경우, 침윤력에 대한 CD98 효과가 억제되었다. FAK 인산화 억제제인 PP2를 처리하면 CD98에 의해 증가된 MCF-7 세포의 침윤이 감소하였다. 이 결과는 dominant-negative 형태의 FAK 유전자의 과발현을 통하여서도 확인 할 수 있었다. 또한, 세포골격단백질의 재구성을 억제하는 약제인 cytochalasin D나 phalloidin도 CD98 신호를 저해 하였다. PP2, cytochalasin D나 phalloidin의 CD98 신호 억제효과는 β1 인테그린의 형태변화를 유도하는 Mn++을 처리할 경우 감소하였다. 본 연구는 CD98분자가 활성화되면 MCF-7 사람유방암세포의 침윤도가 증가되며, 이 과정에 β1 인테그린의 세 포밖기질에의 부착도의 증가, FAK의 인산화와 그에 따른 세포골격의 재구성에 관여함을 보여준다.
Abstract
CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates the func-tions of β1 integrin, suggesting that it may play a role in tumor cell invasion. In this study, the effects of CD98 signaling on the adhesion and invasion of tumor cells were investigated. The expression of CD98 on MCF-7 human breast carcinoma cells was confirmed by immunohistochemistry. The effects of CD98 activation on the adhesion to extracellular matrix (ECM) and invasion of MCF-7 cells were determined by adhesion assay and cell invasion assay. Dominant negative forms of focal adhesion kinase (FAK) were transiently transfected into MCF-7 cells using liposome reagents. CD98 stimulation increased the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV. Activation of CD98 augmented the invasion rate of MCF-7 cells through ECM. EDTA or a function-blocking anti-β1 integrin mAb suppressed the effect of CD98 on invasiveness. Inhibition of phosphorylation of FAK by PP2, an inhibitor of Src family kinase, reduced CD98-induced invasion of MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, cytochalasin D or phalloidin inhibited CD98-mediated induction of tumor cell invasion. Inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated invasion of MCF-7 cells were diminished by pretreatment of cells with Mn++, which is shown to induce conformational change of β1 intgerin. These results provide the first evidence that CD98 activation increases tumor cell invasion by activating β1 integrin affinity, and that FAK phosphorylation and subsequent cytoskeletal reorganization may be essential for CD98-mediated regulation of cell motility.
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