Adhikari Pratikshya, Ayo Tolulope E, Vines John C, Sugita Shuzo, Xu Hao
Center for Molecular and Cellular Biosciences, School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi, Hattiesburg, MS, 39406, USA.
Division of Fundamental Neurobiology, University Health Network, Toronto, ON, M5T 2S8, Canada.
Inflamm Res. 2023 Mar;72(3):639-649. doi: 10.1007/s00011-023-01698-z. Epub 2023 Feb 1.
Mast cells utilize SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins to secrete/exocytose a variety of proinflammatory mediators. However, whether a common SNARE-SM machinery is responsible remains unclear.
Four vesicle/granule-anchored SNAREs (VAMP2, VAMP3, VAMP7, and VAMP8) and two Munc18 homologs (Munc18a and Munc18b) were systematically knocked down or knocked out in RBL-2H3 mast cells and antigen-induced release of β-hexosaminidase, histamine, serotonin, and TNF was examined. Phenotypes were validated by rescue experiments. Immunofluorescence studies were performed to determine the subcellular distribution of key players.
The reduction of VAMP8 expression inhibited the exocytosis of β-hexosaminidase, histamine, and serotonin but not TNF. Unexpectedly, however, confocal microscopy revealed substantial co-localization between VAMP8 and TNF, and between TNF and serotonin. Meanwhile, the depletion of other VAMPs, including knockout of VAMP3, had no impact on the release of any of the mediators examined. On the other hand, TNF exocytosis was diminished specifically in stable Munc18b cells, in a fashion that was rescued by exogenous, RNAi-resistant Munc18b. In line with this, TNF was co-localized with Munc18b (47%) to a much greater extent than with Munc18a (13%).
Distinct exocytic pathways exist in mast cells for the release of different mediators.
肥大细胞利用可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）和Sec1/Munc18（SM）蛋白来分泌/胞吐多种促炎介质。然而，是否存在共同的SNARE - SM机制尚不清楚。
在RBL - 2H3肥大细胞中，系统性地敲低或敲除四种囊泡/颗粒锚定的SNAREs（VAMP2、VAMP3、VAMP7和VAMP8）以及两种Munc18同源物（Munc18a和Munc18b），并检测抗原诱导的β - 己糖胺酶、组胺、血清素和肿瘤坏死因子（TNF）的释放。通过挽救实验验证表型。进行免疫荧光研究以确定关键蛋白的亚细胞分布。
VAMP8表达的减少抑制了β - 己糖胺酶、组胺和血清素的胞吐作用，但对TNF无影响。然而，出乎意料的是，共聚焦显微镜显示VAMP8与TNF之间以及TNF与血清素之间存在大量共定位。同时，其他VAMPs的缺失，包括VAMP3的敲除，对所检测的任何介质的释放均无影响。另一方面，TNF胞吐作用在稳定的Munc18b细胞中特异性降低，外源性、RNAi抗性的Munc18b可使其恢复。与此一致的是，TNF与Munc18b的共定位程度（47%）远高于与Munc18a的共定位程度（13%）。
肥大细胞中存在不同的胞吐途径用于释放不同的介质。
