Hundhausen Christian, Schulte Alexander, Schulz Beate, Andrzejewski Michael G, Schwarz Nicole, von Hundelshausen Philipp, Winter Ulrike, Paliga Krzysztof, Reiss Karina, Saftig Paul, Weber Christian, Ludwig Andreas
Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
J Immunol. 2007 Jun 15;178(12):8064-72. doi: 10.4049/jimmunol.178.12.8064.
CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.
CX3CL1（fractalkine）和CXCL16是趋化因子家族中的独特成员，因为它们不仅以可溶性分子形式存在，还以膜结合分子形式存在。这两种趋化因子作为I型跨膜蛋白表达，其胞外域可从细胞表面被蛋白水解切割，这一过程称为脱落。我们之前的研究表明，解整合素和金属蛋白酶10（ADAM10）介导了大部分组成型CX3CL1和CXCL16的脱落，但不参与佛波酯诱导的可溶性趋化因子的释放（诱导性脱落）。在本研究中，我们引入钙离子载体离子霉素作为CX3CL1和CXCL16脱落的一种新型、非常快速且有效的诱导剂。通过在COS-7细胞和ADAM10缺陷的小鼠胚胎成纤维细胞中进行转染，并结合使用选择性金属蛋白酶抑制剂，我们证明这些趋化因子可溶性形式的诱导性生成依赖于ADAM10的活性。对细胞膜中剩余的C末端切割片段的分析揭示了ADAM10使用的多个切割位点，其中一个在离子霉素刺激后优先被使用。在用表达CX3CL1的ECV-304细胞和细胞因子刺激的内皮细胞进行的黏附研究中，我们证明诱导的CX3CL1脱落导致结合的单核细胞系和外周血单个核细胞从其细胞底物上释放。这些数据为通过ADAM10的诱导性释放机制提供了证据，这一机制可能对白细胞渗出具有重要意义。
