Dental pulp cell-derived powerful inducer of TNF-alpha comprises PKR containing stress granule rich microvesicles (GFvsDP-1)

It is well known that dental pulp tissue can evoke some of the most severe acute inflammation observed in the human body. We found that dental pulp cells secrete a factor that induces tumor necrosis factor-α production from macrophages, and designated this factor, dental pulp cell-derived tumor necrosis factor-α-inducing factor (DPTIF). DPTIF was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPTIF activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPTIF-containing supernatants from pulp cells resulted in activation of both nuclear factor-κB and MAP kinases like JNK and p38. Proteomics analyses revealed that many stress granule-related proteins were present in supernatants from dental pulp cells as well as microvesicle marker proteins like GAPDH, β-actin, HSPA8, HSPB1, HSPE1, and HSPD1. Furthermore, giant molecule AHNAK and PKR were detected in microvesicles derived from dental pulp cells, and gene silencing of AHNAK in pulp cells led to reduced DPTIF activity. Thus, it appeared that the core protein of DPTIF was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPTIF for TNF-α induction was far superior to that of gram-negative bacterial endotoxin. Therefore, we, report for the first time, that active PKR is transported via microvesicles as stress granule aggregates and induces powerful inflammatory signals in macrophages. To determine the differences in gene expression between cells with and without dental pulp cell-derived tumor necrosis factor-α-inducing factor (DPTIF) activity, we compared gene expression profiles between immortalized dental pulp calls (DP-1) with DPTIF activity and gingival tissue-derived fibroblasts without DPTIF activity

众所周知，牙髓组织（dental pulp tissue）可引发人体中最为剧烈的急性炎症反应之一。本研究发现牙髓细胞（dental pulp cell）可分泌一种能诱导巨噬细胞（macrophage）产生肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）的因子，并将该因子命名为牙髓细胞源性肿瘤坏死因子-α诱导因子（dental pulp cell-derived tumor necrosis factor-α-inducing factor, DPTIF）。DPTIF可在牙髓细胞中被诱导产生，并通过微囊泡（microvesicles）转运至受体细胞。用蛋白激酶R（protein kinase R, PKR）抑制剂处理牙髓细胞后，DPTIF的活性显著受到抑制，且细胞内本就存在弱激活的干扰素信号通路。在受体巨噬细胞中，用含有DPTIF的牙髓细胞上清液进行刺激，可同时激活核因子-κB（nuclear factor-κB, NF-κB）以及c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）、p38等丝裂原活化蛋白激酶（MAPK）。蛋白质组学（proteomics）分析显示，牙髓细胞上清液中存在多种应激颗粒（stress granule）相关蛋白，同时也含有GAPDH、β-肌动蛋白（β-actin）、HSPA8、HSPB1、HSPE1及HSPD1等微囊泡标记蛋白。此外，在牙髓细胞来源的微囊泡中还检测到了大分子蛋白AHNAK及PKR；对牙髓细胞中的AHNAK进行基因沉默（gene silencing）后，DPTIF的活性出现降低。由此可见，DPTIF的核心蛋白为PKR，PKR可与AHNAK共同存在于应激颗粒聚集体中并保持激活状态，随后通过微囊泡进行转运。DPTIF诱导TNF-α产生的活性远优于革兰氏阴性细菌内毒素（gram-negative bacterial endotoxin）。因此，本研究首次报道：激活状态的PKR可作为应激颗粒聚集体通过微囊泡进行转运，并在巨噬细胞中诱导强效的炎症信号通路。为明确具有DPTIF活性与不具有DPTIF活性的细胞之间的基因表达差异，本研究对比了具有DPTIF活性的永生化牙髓细胞（immortalized dental pulp cell, DP-1）与不具有DPTIF活性的牙龈组织成纤维细胞（gingival tissue-derived fibroblast）的基因表达谱。