Dental pulp cell-derived powerful inducer of TNF-\u03b1 comprises PKR containing stress granule rich microvesicles

Shigeki Suzuki,Tomomi Sano,Kouji Ohta,Jun Nakanishi,Satoru Yamada,Kazuma Yoshida,Takao Fukuda,Shintaro Nagayasu,Fusanori Nishimura,Shizu Hirata-Tsuchiya,Hideki Shiba,Akiko Yamashita,Yuki Nakao

doi:10.1038/s41598-019-40046-2

Shigeki Suzuki, Tomomi Sano + Show 11 more

Open Access

https://doi.org/10.1038/s41598-019-40046-2

Copy DOI

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.

Highlights

Dental pulp cells are continuously exposed to various environmental stresses such as hot and cold temperatures, mechanical stress, and bacterial irritation[1]
We found that dental pulp cells, both immortalized cells[2] and primary cells, secrete a factor that strongly stimulates differentiated THP-1 cells to induce tumor necrosis factor (TNF)-α at both the gene and protein levels (Fig. 1a: left, 1b: left), and designated this factor dental pulp cell-derived powerful inducer of TNF-α (DPIT)
DPIT activity was seen in both immortalized dental pulp cells (DP-1) and primary dental pulp cells (PriDPC) and the activity was superior to that of a gram-negative bacterial endotoxin, lipopolysaccharide (LPS) when differentiated THP-1 (dTHP-1) cells were incubated for 2 hrs (Fig. 1a: right, 1b: right)

Summary

Methods

Differentiated THP-1 cells were washed with serum-free medium and stimulated with supernatants from DP-1cells or priDPC cells, matrix vesicles from DP-1, priDPC, HEK293, and MCF-7 cells, Pam3CSK4, and LPS. The siRNAs were reverse-transfected into DP-1 cells and forward-transfected into differentiated THP-1 cells at final concentrations of 10 and 50 nM, respectively, using Lipofectamine RNAiMAX reagent (Life Technologies) and incubated for 48 h. 80 ng of PKH67-labeled matrix vesicles was incubated with 2 × 105 primed THP-1 cells for 100 min. DP-1 and priDPC cells were seeded into 8-well non-coated glass chamber slides (SCS-N08; Matsunami, Osaka, Japan), fixed with 3.7% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in DPBS for 5 min, blocked with 1% BSA in DPBS for 1 h at room temperature, and incubated with anti-AHNAK antibody (1:200 dilution) overnight at 4°C. The solubilized formazan product was spectrophotometrically quantified by measuring the absorbances at 575 nm and 650 nm on a microtiter plate reader (Multiskan)

Author Contributions

Findings

Additional Information