Pacurari M, Cox I, Bible A N, Davern S
Department of Biology College of Science Engineering and Technology Jackson State University, Jackson, MS 39217, USA.
RCMI Center for Environmental Health College of Science Engineering and Technology Jackson State University, 1400 Lynch Street, 18750, Jackson, MS 39217, USA.
Biochem Res Int. 2024 Aug 2;2024:5527895. doi: 10.1155/2024/5527895. eCollection 2024.
CC-chemokine ligand 18 also known as MIP-4 is a chemokine with roles in inflammation and immune responses. It has been shown that MIP-4 is involved in the development of several diseases including lung fibrosis and cancer. How exactly MIP-4 is regulated and exerts its role in lung fibrosis remains unclear. Therefore, in the present study, we examined how MIP-4 is regulated and whether it acts via its potential receptor Nir-1.
A549 cells were grown and maintained in DMEM : F12 (1 : 1) and supplemented with 10% FBS and 1000 U of penicillin/streptomycin and maintained as recommended by the manufacturer (ATCC). Cell migration and invasion, immunohistochemistry (IHC), Western blot, qPCR, and siRNA Nir-1 were used to determine MIP-4 regulation and its role in cell migration.
Cell migration was increased following stimulation of cells with recombinant (r) MIP-4 and bleomycin (BLM), whereas quenching rMIP-4 with its antibody (Ab) or addition of the Ab to BLM or HO diminished rMIP-4-induced cell migration. Along with cell migration, rMIP-4, BLM, and HO induced the formation of actin filaments dynamic structures whereas costimulation with MIP-4 Ab limited BLM- and HO-induced effects. MIP-4 mRNA and protein were increased by BLM and HO, and the addition of its Ab significantly reduced treatments effect. Experiments with siRNA investigating whether Nir-1 is a potential MIR-4 receptor indicated that the inhibition of Nir-1 decreased cell migration/invasion but did not totally inhibit rMIP-4-induced cell migration.
Therefore, our data indicate that MIP-4 is regulated by BLM and HO and costimulation with its Ab limits the effects on MIP-4 and that the Nir-1 receptor partially mediates MIP-4's effects on increased cell migration. These data also evidenced that MIP-4 is regulated by fibrotic and oxidative stimuli and that quenching MIP-4 with its Ab or therapeutically targeting the Nir-1 receptor may partially limit MIP-4 effects under fibrotic or oxidative stimulation.
CC趋化因子配体18也被称为巨噬细胞炎症蛋白-4（MIP-4），是一种在炎症和免疫反应中发挥作用的趋化因子。研究表明，MIP-4参与了包括肺纤维化和癌症在内的多种疾病的发展过程。然而，MIP-4在肺纤维化中究竟是如何被调控以及发挥作用的，目前仍不清楚。因此，在本研究中，我们探究了MIP-4是如何被调控的，以及它是否通过其潜在受体Nir-1发挥作用。
A549细胞在DMEM∶F12（1∶1）培养基中培养，并添加10%胎牛血清和1000 U青霉素/链霉素，按照美国典型培养物保藏中心（ATCC）的建议进行培养。使用细胞迁移和侵袭实验、免疫组织化学（IHC）、蛋白质免疫印迹法、定量聚合酶链反应（qPCR）以及Nir-1小干扰RNA（siRNA）来确定MIP-4的调控及其在细胞迁移中的作用。
用重组（r）MIP-4和博来霉素（BLM）刺激细胞后，细胞迁移增加，而用其抗体（Ab）淬灭rMIP-4或向BLM或过氧化氢（HO）中添加该抗体可减少rMIP-4诱导的细胞迁移。与细胞迁移情况一致，rMIP-4、BLM和HO诱导了肌动蛋白丝动态结构的形成，而与MIP-4抗体共刺激则限制了BLM和HO诱导的效应。BLM和HO使MIP-4的信使核糖核酸（mRNA）和蛋白质水平升高，添加其抗体可显著降低处理效果。用siRNA研究Nir-1是否为潜在的MIR-4受体的实验表明，抑制Nir-1可降低细胞迁移/侵袭，但并未完全抑制rMIP-4诱导的细胞迁移。
因此，我们的数据表明，MIP-4受BLM和HO调控，与其抗体共刺激可限制对MIP-4的影响，且Nir-1受体部分介导了MIP-4对细胞迁移增加的影响。这些数据还证明，MIP-4受纤维化和氧化刺激的调控，用其抗体淬灭MIP-4或对Nir-1受体进行治疗性靶向干预可能会在纤维化或氧化刺激下部分限制MIP-4的作用。
