Role of mitofusin-2 in high mobility group box-1 protein-mediated apoptosis of T cells in vitro.

Background: High mobility group box-1 protein 9HMGB1), a ubiquitous nuclear protein, which is recognized as a danger-associated molecular pattern 9DAMP) triggering activation of the innate immune system. Previous studies have shown that HMGB1 also plays a role in T cell-mediated immunity, but the effect of HMGB1 on apoptosis of T cells and its precise mechanism remain to be determined. Methods: Two kinds of apoptosis assay techniques were used, i. e., Annexin V-FITC conjunction with PI to identify early apoptotic cells, Hoechst 33342 staining for double-stranded DNA to observe nuclear fragmentation or apoptotic body. The activation status of caspase-3, caspase-8, as well as caspase-9 was examined by colorimetric assay. The dynamic changes in intracellular calcium concentration ([Ca2+] i) was monitored by flow cytometry. Overexpression of Mfn2 was preformed by lentiviral vector transfection. The mRNA and protein levels of Mfn2 were determined by RT-PCR and Western-blotting. Results: Treatment of Jurkat T cells with recombinant human HMGB1 9rhHMGB1) causes a significant dose-dependent increase in percentage of apoptotic cells. When T cells are incubated with HMGB1 they express decreased mitochondria fusion-related protein mitofusin-2 9Mfn2) and activate mitochondrial apoptotic pathway via elevation of [Ca2+] i, Bax insertion, and activation of caspase. Furthermore, overexpression of Mfn2 ameliorates the apoptosis of T cells induced by HMGB1. This occurs at least partly through Mfn2 keeps Ca2+ homeostasis in T cells evidenced by monitoring [Ca2+] i dynamics. Conclusion: HMGB1 can trigger apoptosis of T lymphocytes through mitochondrial death pathway associated with [Ca2+] i elevation. Mfn2 plays a pivotal role in this process, and it might be a novel therapeutic target in T cell apoptosis related disorders. Copyright (c) 2014 S. Karger AG, Basel