Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo 113‑8421, Japan.
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo 113‑8421, Japan.
Mol Med Rep. 2018 Dec;18(6):4951-4959. doi: 10.3892/mmr.2018.9532. Epub 2018 Oct 2.
LL‑37 is the sole antimicrobial peptide of human cathelicidin comprising 37 amino acids, which is expressed mainly in epithelial cells and neutrophils, and activates mast cells. In the present study, in order to elucidate the mechanism of mast cell activation by LL‑37, the associations between the internalization of LL‑37 and Mas‑related gene X2 (MrgX2)‑mediated mast cell activation (degranulation) was investigated using the human mast cell line, LAD2. LL‑37 was rapidly internalized into the cells, and induced degranulation, as assessed by the extracellular release of β‑hexosaminidase. Pertussis toxin, a G‑protein inhibitor, significantly suppressed the internalization of LL‑37 and the degranulation of LAD2 cells. Furthermore, small interfering (si)‑RNA‑mediated knockdown of MrgX2, a putative G protein‑coupled receptor for LL‑37, inhibited the internalization of LL‑37 and degranulation of LAD2 cells. Notably, LL‑37 internalization was enhanced by the stable expression of MrgX2 in HMC‑1 and 293 cells. In addition, the internalized LL‑37 mainly colocalized with MrgX2 in the perinuclear region of LAD2 cells. Furthermore, neuraminidase treatment, which removes negatively charged sialic acid from the cell surface, markedly reduced the internalization of LL‑37 and degranulation of LAD2 cells, and clathrin‑mediated endocytosis inhibitors (dynasore and chlorpromazine) inhibited the internalization and degranulation of LAD2 cells. Taken together, these observations indicated that LL‑37 may bind the negatively charged cell surface molecules, rapidly internalize into the cells via clathrin‑mediated endocytosis and interact with MrgX2 to activate mast cells (LAD2 cells).
LL-37 是人类防御素唯一的抗菌肽，由 37 个氨基酸组成，主要在表皮细胞和嗜中性粒细胞中表达，并激活肥大细胞。在本研究中，为了阐明 LL-37 激活肥大细胞的机制，使用人肥大细胞系 LAD2 研究了 LL-37 内化与 Mas 相关基因 X2（MrgX2）介导的肥大细胞激活（脱颗粒）之间的关联。LL-37 迅速被细胞内化，并通过β-己糖胺酶的细胞外释放来评估诱导脱颗粒。百日咳毒素，一种 G 蛋白抑制剂，显著抑制 LL-37 的内化和 LAD2 细胞的脱颗粒。此外，MrgX2 的小干扰 (si)-RNA 介导的敲低，一种 LL-37 的假定 G 蛋白偶联受体，抑制了 LL-37 的内化和 LAD2 细胞的脱颗粒。值得注意的是，MrgX2 在 HMC-1 和 293 细胞中的稳定表达增强了 LL-37 的内化。此外，内化的 LL-37 主要与 LAD2 细胞的核周区域中的 MrgX2 共定位。此外，神经氨酸酶处理，从细胞表面去除带负电荷的唾液酸，显著降低了 LL-37 的内化和 LAD2 细胞的脱颗粒，网格蛋白介导的内吞抑制剂（dynasore 和氯丙嗪）抑制了 LAD2 细胞的内化和脱颗粒。综上所述，这些观察结果表明，LL-37 可能与带负电荷的细胞表面分子结合，通过网格蛋白介导的内吞作用迅速内化到细胞内，并与 MrgX2 相互作用激活肥大细胞（LAD2 细胞）。
