P2X7R-NF-κB-PDI signal pathway regulates LPS-induced impaired GSH synthesis by modulating ATF4-mediated xCT upregulation, S-nitrosylation of ASCT2 and GSHS expressions in the mouse hippocampus

Protein disulfide isomerase (PDI) augments lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation by integrating Toll-like receptor 4 (TLR4) and P2X7 receptor (P2X7R) signaling pathways in a positive feedback manner. However, it has been largely unknown whether PDI is involved in altered glutathione (GSH) biosynthesis, which is mediated by P2X7R, in response to LPS. In the present study, LPS-induced NF-κB activation increased PDI expression, but decreased solute carrier 1 A5 (ASCT2) level in the P2X7^+/+ mouse hippocampus. PDI knockdown attenuated ASCT2 downregulation and S-nitrosylated (SNO-) ASCT2 level in response to LPS. This LPS-induced NF-κB-PDI activation also increased activating transcription factor 4 (ATF4) expression in astrocytes, which elicited cystine:glutamate transporter (xCT) upregulation, but decreased ASCT2 and GSH synthetase (GSHS) expression. Furthermore, S-nitrosylation of PDI modulated ATF4-mediated xCT upregulation in response to LPS. SN50 (a NF-κB inhibitor), PDI knockdown and ATF4 siRNA mitigated the decreased GSH content induced by LPS. Under physiological condition, P2X7R deletion did not affect basal PDI, ATF4, xCT and SNO-ASCT2 levels. However, it increased ASCT2 expression and decreased SNO-PDI level. P2X7R ablation ameliorated (1) PDI, ATF4 and xCT2 upregulations, (2) S-nitrosylation of ASCT2 and PDI and (3) ASCT2 downregulation in response to LPS. These findings indicate that P2X7R-NF-κB-PDI signal pathway may inhibit GSH biosynthesis in response to LPS by modulating expression/S-nitrosylation of ASCT2 and ATF4-mediated xCT regulation in response to LPS.