Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor

Abstract

Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.

Keywords: Hsp70; RNA interference (RNAi); TNFR1; Tag7; Tag7-Hsp70 complex; apoptosis; cell death; cytotoxicity; heat shock protein (HSP); necroptosis; tumor necrosis factor (TNF).

FIGURE 1.

Tag7-Hsp70 has a cytotoxic effect on the L-929 cell clones. The clones were isolated by a limiting dilution method and were incubated with Tag7-Hsp70 (0.1 nm). The proportion of dead cells (identified by trypan blue staining) was determined after 3 and 20 h. All data are presented as the means ± S.E. from at least five independent assays. p values of less than 0.05 were considered significant (*, p < 0.05; **, p < 0.005).

FIGURE 2.

Tag7-Hsp70 interacts with L-929 cells to induce apoptosis after 3 h and necroptosis after 20 h. A, Tag7-Hsp70 (0.1 nm) induces an increase in caspase-3 activity (see “Experimental Procedures”). Neither Tag7 alone nor Hsp70 alone induces caspase-3 activation. B, effects of a 1-h preincubation with the caspase-3 (5 mm DEVD-CHO), caspase-8 (5 mm IEDT-CHO), caspase-9 (5 mm LEHD-FMK), and RIP1 kinase (5 mm necrostatin 1) inhibitors on the cytotoxicity of Tag7-Hsp70 (0.1 nm) after 3 and 20 h of exposure to the L-929 heterogeneous cultures. C, effects of the 1-h preincubation with the caspase-3 (5 mm DEVD-CHO), caspase-8 (5 mm IEDT-CHO), and RIP1 kinase (5 mm necrostatin 1) inhibitors on the cytotoxicity of Tag7-Hsp70 (0.1 nm) after 3 and 20 h of incubation with the clones that were sensitive to the 3-h death and the 20-h death. D, all data are presented as the means ± S.E. from at least five independent assays. p values of less than 0.05 were considered significant (*, p < 0.05; **, p < 0.005).

FIGURE 3.

Tag7-Hsp70 interacts with TNFR1. A, sTNFR1 was applied to the anti-Tag7-conjugated Sepharose 4B, with pre-adsorbed Tag7-Hsp70 complex. The retained proteins were eluted with triethanolamine, fractioned by SDS-PAGE (12%), and analyzed by Western blotting with rabbit antibodies to TNFR1 (1:10,000), Tag7 (1:10,000), or Hsp70 (1:10,000). B, the Tag7-Hsp70 complex was applied to anti-TNFR1-conjugated Sepharose 4B, with pre-adsorbed sTNFR1. The retained proteins were eluted with triethanolamine, fractionated by SDS-PAGE (12%), and analyzed by Western blotting with rabbit antibodies to TNFR1 (1:10,000), Tag7 (1:10,000), or Hsp70 (1:10,000). C, the Tag7-Hsp70 complex containing biotinylated Tag7 (10 nm) was added to L-929 cells and incubated for 2 h at 4 °C, and then 0.2 mm BS³ was added. After 30 min of incubation, the cells were washed and the membrane fraction was solubilized and applied to the anti-TNFR1 column. Lane 1, the retained proteins were analyzed by Western blotting with streptavidin-peroxidase. Lane 2, the same experiment was performed with the Tag7-Hsp70 complex containing biotinylated Hsp70. Lanes 3–5, the membrane fraction of the L-929 cells (lane 3), the Tag7-Hsp70 complex with biotinylated Tag7 (lane 4), and the Tag7-Hsp70 complex with biotinylated Hsp70 (lane 5) were analyzed by Western blotting with streptavidin-peroxidase and are shown as the controls.

FIGURE 4.

The interaction with TNFR1 is necessary for TNF-α- and Tag7-Hsp70-induced cell death. A, the cytotoxic effects of TNF-α and Tag7-Hsp70 (1 nm each) on both apoptotic (3 h) and necroptotic (20 h) cell death were inhibited after the L-929 cells were preincubated with anti-TNFR1 antibodies (100 nm). B, the same experiment was performed on the 3-h-sensitive clones for the apoptotic (3 h) cell death and on the 20-h-sensitive clones for the necroptotic (20 h) cell death. C, preincubation of TNF-α (0.05 nm) and Tag7-Hsp70 (0.1 nm) with increasing concentrations of sTNFR1 inhibits their cytotoxic activity in L-929 cells. D, TNFR1 knockdown blocks the cytotoxic effect of TNF-α and Tag7-Hsp70 (see “Experimental Procedures”). All data are presented as the means ± S.E. from at least five independent assays. p values of less than 0.05 were considered significant (*, p < 0.05).

FIGURE 5.

The Tag7 protein inhibits the cytotoxic effects of both the Tag7-Hsp70 complex and TNF-α. A, the 3-h apoptotic effects of TNF-α (0.05 nm) and Tag7-Hsp70 (0.1 nm) are inhibited after preincubating the L-929 and HEK293 cells with Tag7 (1 nm). B, the 20-h necroptotic effects of TNF-α (0.05 nm) and Tag7-Hsp70 (0.1 nm) are inhibited after preincubating the L-929 and HEK293 cells with Tag7 (1 nm). C, the 20-h necroptotic effects of TNF-α (0.05 nm) and Tag7-Hsp70 (0.1 nm) are inhibited after preincubating the L-929 cells with increasing concentrations of Tag7. D, the cytotoxic effects of TNF-α (0.05 nm) and Tag7-Hsp70 (0.1 nm) are inhibited after preincubating the L-929 clones with Tag7 (1 nm). The apoptotic death of the 3-h-sensitive clones and necroptotic death of the 20-h-sensitive clones are shown. All data are presented as the means ± S.E. from at least five independent assays. p values of less than 0.05 were considered significant (*, p < 0.05; **, p < 0.005).