Context-dependent release of HMGB1: cell death mode, cell type and LPS stress drive monomer and heterocomplex formation

BACKGROUND: HMGB1 acts as an alarmin when released from stressed or dying cells. In vitro, HMGB1 has previously been demonstrated to readily form complexes with other molecules and through intermolecular disulfide bond formation form homodimers. Recently, dimerized HMGB1 was identified in serum of LPS-challenged mice. In cancer, HMGB1 has been described as having both tumour-promoting and tumour-suppressing features, possibly dependent on the form of HMGB1 released into the tumour microenvironment. Factors determining the form in which HMGB1 is released remain, however, largely unexplored. We therefore investigated the form of HMGB1 released during different cell death modes and in response to LPS stress using various tumour cell lines. METHODS: Supernatants were collected from ten non- and LPS-treated tumour cell lines and necrotic, apoptotic and pyroptotic THP-1 monocytic cells, to assess active and passive secretion of HMGB1. Released proteins were concentrated by TCA-precipitation and analysed by Western blotting under reducing and non-reducing conditions to detect monomeric, dimeric, or HMGB1-protein complexes. Co-immunoprecipitation and LC-MS/MS were used to identify binding partners of extracellular HMGB1. RESULTS: Tumour cells were found to release monomeric HMGB1 and HMGB1 heterocomplexes in the 50-60 kDa range, as indicated by their persistent high molecular weight under reducing conditions. Furthermore, we identified that HMGB1 interacts with ribosomal proteins, histone H2B, and SRP9 following LPS treatment of microglial SIM-A9 cells. CONCLUSIONS: HMGB1 readily formed heterocomplexes, but not homodimers in vitro across multiple cell lines, with differences between LPS-treated and untreated conditions. The form of released HMGB1 was influenced by cell type, cell death mode, and LPS stress.