Aziz Monowar, Yang Weng-Lang, Corbo Lana M, Chaung Wayne W, Matsuo Shingo, Wang Ping
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Int J Mol Med. 2015 Jul;36(1):18-28. doi: 10.3892/ijmm.2015.2196. Epub 2015 Apr 23.
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.
我们之前已经证明，乳脂肪球表皮生长因子8（MFG-E8）在急性肺损伤（ALI）小鼠模型中可减少中性粒细胞浸润。在本研究中，我们旨在阐明MFG-E8减轻中性粒细胞迁移的机制。重组人MFG-E8（rhMFG-E8）在我们的实验室中表达并纯化。使用重组白细胞介素8（IL-8）作为趋化因子，将人分化的中性粒细胞系dHL-60用rhMFG-E8处理，并在Boyden小室中进行细胞迁移试验。通过流式细胞术或蛋白质印迹分析评估表面CXCR2和细胞内G蛋白偶联受体激酶2（GRK2）的水平。通过蛋白质印迹分析测定丝裂原活化蛋白（MAP）激酶的水平。用rhMFG-E8处理导致dHL-60细胞迁移以剂量依赖性方式受到显著抑制。在经rhMFG-E8处理的dHL-60细胞中，CXCR2表达下降46%，这与GRK2表达增加32%相关。在dHL-60细胞中，用rhMFG-E8处理在10-30分钟内促进了p38和细胞外信号调节激酶（ERK）的磷酸化。使用p38抑制剂SB203580和ERK抑制剂PD98059可使dHL-60细胞迁移恢复，而rhMFG-E8处理可显著抑制这种迁移。此外，用抗αv整合素中和抗体（Ab）阻断MFG-E8受体αvβ3/αvβ5整合素可抑制p38和ERK的激活，并逆转rhMFG-E8诱导的dHL-60细胞迁移抑制。最后，用SB203580和PD98059处理dHL-60细胞可中和rhMFG-E8诱导的CXCR2表达下调和GRK2表达上调，以及对细胞迁移的抑制作用。我们的研究结果揭示了MFG-E8的一种新作用机制，即它通过αvβ3整合素依赖性MAP激酶激活来抑制中性粒细胞迁移。
