Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both β-arrestin-1 and -2
Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both β-arrestin-1 and -2
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DOI：
10.1074/jbc.m410312200
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发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
DeFea, K
中科院分区：
文献类型：
--
作者：
Ge, L;Shenoy, SK;DeFea, K
蛋白酶激活受体-2（PAR-2）由胰蛋白酶样丝氨酸蛋白酶激活，可通过ERK 1/2依赖性途径促进细胞迁移，包括在细胞前缘形成支架复合物。以前的研究也表明，β-arrestin-1的显性负片段的表达减少PAR-2刺激的内化，ERK 1/2激活和细胞迁移;然而，这种试剂可能会阻止许多蛋白质的关联，包括β-arrestin-2与网格蛋白包被的小凹。在这里，我们调查PAR-2的转移性乳腺癌细胞系，MDA MB-231的组成性迁移的作用，并使用小干扰RNA，以确定每个β-arrestin的贡献，这一过程。我们证明了MDA MB-231细胞分泌的胰蛋白酶样蛋白酶可以通过自分泌激活PAR-2促进细胞迁移，这与PAR-2，β-arrestin-2和激活的ERK 1/2的伪足组成性定位相关。添加MEK-1抑制剂、胰蛋白酶抑制剂、乱序PAR-2肽和用小干扰RNA沉默β-抑制蛋白也减少MDA MB-231细胞的基线迁移。相比之下，转移性较低的PAR-2表达乳腺癌细胞系不表现出组成性迁移，伪足形成，或胰蛋白酶分泌，在这些细胞中，PAR-2更均匀地分布在细胞周围。这些数据表明PAR-2介导的运动需要两种β-抑制蛋白，并表明分泌的蛋白酶对PAR-2的自分泌激活可能通过β-抑制蛋白依赖性ERK 1/2激活促进转移性肿瘤细胞的迁移。
Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.