Avraamides C, Bromberg M E, Gaughan J P, Thomas S M, Tsygankov A Y, Panetti T S
Department of Microbiology and Immunology, and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA.
Am J Physiol Heart Circ Physiol. 2007 Jul;293(1):H193-203. doi: 10.1152/ajpheart.00728.2006. Epub 2007 Mar 2.
Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.
内皮细胞迁移对于正常血管发育至关重要。生长因子和基质蛋白发出的信号通过黏着斑蛋白整合，以改变细胞迁移。过氧化氢诱导克隆5（Hic-5）是桩蛋白家族成员，在牛肺动脉内皮（BPAE）细胞的黏着斑中富集，这些细胞在变性胶原蛋白上迁移至溶血磷脂酸（LPA）。在本研究中，我们探究了Hic-5在LPA刺激的内皮细胞迁移中的作用。LPA将Hic-5募集到黏着斑以及铺在胶原蛋白上的BPAE细胞的伪足，这表明Hic-5被募集到黏着斑与内皮细胞迁移有关。敲低内源性Hic-5显著降低向LPA的迁移，证实Hic-5参与迁移。为了阐明Hic-5在内皮细胞迁移中的作用，我们在BPAE细胞中外源表达野生型（WT）Hic-5和绿色荧光蛋白Hic-5 C369A/C372A（LIM3突变体）构建体。WT Hic-5的表达增加BPAE细胞对LPA的趋化性，而表达绿色荧光蛋白Hic-5 C369A/C372A的细胞向LPA的迁移与载体对照细胞相似。此外，在WT Hic-5细胞中，LPA存在时ERK磷酸化增强。MEK活性的药理抑制剂抑制LPA刺激的WT Hic-5细胞迁移和ERK磷酸化，表明Hic-5通过ERK的MEK激活增强迁移。总之，这些研究表明，内皮细胞中的一种黏着斑蛋白Hic-5在LPA存在时被募集到伪足并增强迁移。
