Regulation of adhesion of AML14.3D10 cells by surface clustering of β2-integrin caused by ERK-independent activation of cPLA2
Regulation of adhesion of AML14.3D10 cells by surface clustering of β2-integrin caused by ERK-independent activation of cPLA2
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DOI：
10.1046/j.1365-2567.2002.01486.x
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发表时间：
2002-09-01
期刊：
影响因子：
6.4
通讯作者：
Leff, AR
中科院分区：
文献类型：
--
作者：
Myou, S;Zhu, XD;Leff, AR
我们检测了由蛋白激酶C（PKC）激活的cPLA（2）引起的β（2）-整联蛋白的细胞表面聚集在嗜酸性粒细胞AML 14.3D10（AML）细胞粘附中的作用。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）引起AML细胞与平板牛血清白蛋白（BSA）的时间和浓度依赖性粘附，该粘附被针对β（2）-整合素的抗CD 11b或抗CD 18单克隆抗体（mAb）阻断。用Ro-31-8220或rottlerin抑制PKC以浓度依赖性方式阻断PMA诱导的细胞粘附。用三氟甲基酮或花生四烯酸甲酯抑制胞浆磷脂酶A（2）（cPLA（2））也能阻断PMA诱导的细胞粘附。PMA引起这些细胞中时间依赖性的p42/44丝裂原活化蛋白激酶（MAPK）（ERK）磷酸化。MAPK/细胞外信号调节蛋白激酶激酶（MEK）抑制剂U 0126在阻断PMA诱导的ERK磷酸化的浓度下，对PMA刺激的AML细胞粘附没有影响。PMA不能使p38 MAPK和c-Jun N-末端激酶（JNK）磷酸化。PMA还引起cPLA（2）活性增加，Ro-31-8220可抑制cPLA（2）活性，但U 0126不抑制。共聚焦免疫荧光显微镜显示，PMA引起CD 11b在细胞表面聚集，这被PKC或cPLA（2）抑制剂阻断。PMA刺激也引起AML细胞表面CD 11b的上调。然而，这种上调不受cPLA（2）或PKC抑制的影响。使用单克隆抗体CBRM 1/5，我们还证明了PMA不会诱导CD 11b/CD 18的活性构象。我们的数据表明，PMA通过PKC激活cPLA（2），通过β（2）-整联蛋白引起AML细胞粘附。该途径不依赖于MEK/ERK，并且不需要CD 11b/CD 18改变为其活性构象。我们发现整合素表面聚集引起的亲合力-而不是构象变化或上调CD 11b/CD 18-导致PMA刺激AML细胞的粘附。
We examined the role of cell surface clustering of beta(2) -integrin caused by protein kinase C (PKC)-activated-cPLA(2) in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against beta(2) -integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A(2) (cPLA(2) ) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA(2) activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA(2) inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA(2) - or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through beta(2) -integrin by PKC activation of cPLA(2) . This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering - rather than conformational change or up-regulation of CD11b/CD18 - causes PMA stimulated adhesion of AML cells.