Dental pulp cell-derived powerful inducer of TNF-α comprises PKR containing stress granule rich microvesicles

Abstract

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 powerful inducer of TNF-α (DPIT). DPIT was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPIT activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPIT-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 dental pulp cells led to reduced DPIT activity. Thus, it appeared that the core protein of DPIT was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPIT 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.

The S100B Protein and Partners in Adipocyte Response to Cold Stress and Adaptive Thermogenesis: Facts, Hypotheses, and Perspectives

“…AHNAK1 is highly sensitive to metal-dependent proteases [ 91 ] and calcium-dependent proteolysis leads to the cleavage of AHNAK1 at specific sites on N- and C- terminal domains [ 92 ]. AHNAK1 contained in purified EVs migrates as a set of shorter peptides compared to the full length AHNAK1, supporting the hypothesis that AHNAK1 proteolysis accompanies vesicle formation (this evidence is supported by comparing Figure 10 panel c and panel f in [ 93 ] and in Figure 7a of [ 94 ]). Such a scenario is further supported by the finding that targeting the C-terminal domain of AHNAK1 (residues 5645–5673) to the membrane bound annexin2/S100A10 complex, in place of the full length AHNAK1, was sufficient to cause membrane blebbing that evolves into vesicular structures ( Figure 4 ).…”

“…Dynamic S100B release into the blood and into the cerebrospinal fluid during acute brain injury may serve as a repair mechanism [ 98 ] but also contribute to neural disorders [ 37 ]. EVs are relevant carriers of both S100 proteins [ 99 ] and AHNAK [ 90 , 93 , 94 , 100 , 101 , 102 ]. Hence, an alternative pathway for S100B release into circulating fluids using EVs is plausible.…”

Exosomes from TNF-α-treated human gingiva-derived MSCs enhance M2 macrophage polarization and inhibit periodontal bone loss

“…Amounts of protein in exosome were quantified using the BCA Protein Assay Kit (Takara Bio, Otsu, Japan). Transmission electron microscopy (TEM) analyses were performed by the Hanaichi Ultrastructure Research Institute (Okazaki, Japan) as previously reported [ 34 ]. The Nanoparticle Characterization System (NanoSight, Malvern Instruments, UK) was used to measure size distribution and concentration of exosomes.…”

Extracellular vesicles derived from GMSCs stimulated with TNF-α and IFN-α promote M2 macrophage polarization via enhanced CD73 and CD5L expression

“…IFN-α enhances TLR3-mediated antiviral activity by inducing protein kinase R (PKR) 25 . We previously reported that PKR in the dental pulp cell-derived microvesicles is critical as a powerful inducer of inflammation 26 . However, to our knowledge, no study has been published regarding the effects of IFN-α on MSC priming, and the effect of IFN-α in combination priming with TNF-α on the negative feedback loop of GMSCs remains unclear.…”