Molecular perspectives on HMGB1 as a multifunctional alarmin

High mobility group box 1 (HMGB1) is an alarmin activating and regulating inflammation upon release to the extracellular space. During homeostasis, HMGB1 resides in the nuclei regulating DNA transcription, repair and recombination. Following cell stress, HMGB1 is hyperacetylated leading to cytosolic translocation and subsequent release to the extracellular space in a calcium-dependent manner. In the extracellular space, HMGB1 binds and activates multiple receptors, such as Toll-like receptor 4, Receptor for advanced glycation end-products and C-X-C motif chemokine receptor 4, activating cytokine release, cell differentiation and cell migration. The molecular role of HMGB1 is determined by its redox status, subcellular localization and surrounding microenvironment. HMGB1 is involved in the pathogenesis of multiple diseases, such as cancer, endotoxemia and arthritis and is regarded as a promising therapeutic target. As HMGB1 can mediate both protective and detrimental effect in chronic inflammatory diseases and cancers, a deeper mechanistic understanding of how HMGB1 operates in different cellular compartments and interacts with immune and tumor cells is necessary. This thesis aimed to elucidate molecular properties and mechanisms of HMGB1 in different subcellular compartments. More specifically, we aimed to characterize the intracellular HMGB1 interactome and to identify previously undescribed intracellular roles. In addition, we aimed to identify factors determining the form of HMGB1 being released from different tumor- and myeloid-derived cells and to elucidate the difference between HMGB1-induced macrophage polarization and classical M1 or lipopolysaccharide stimulations. To identify the intracellular HMGB1 interactome and alterations induced by LPS stress, we used BioID2-mediated proximity labelling. We identified >100 proteins being part of the interactome, whereof eight being significantly different between resting and LPS-stressed monocytic THP-1 cells. Following enrichment analysis, we identified both known and unknown HMGB1-related pathways. We further confirmed the interaction between HMGB1 and selected proteins of the interactome using in silico protein-protein docking and proximity ligation assay. Since multiple proteins within the interactome are involved in actin polymerization, we investigated whether HMGB1 regulates actin polymerization and cell migration. Hmgb1 knockout mouse embryonic fibroblasts displayed a dysregulation of actin polymerization and an increased rate of cell migration compared to wild-type cells. In addition, shRNA-mediated knockdown of HMGB1 in THP-1 cells resulted in enhanced chemotaxis. Since HMGB1 has previously been associated to filopodia and that the actin-regulating proteins in the interactome are cytosolic, we propose a novel role of cytosolic HMGB1 in actin polymerization. Based on the HMGB1 interactome, we hypothesized that HMGB1 is involved in Wnt signaling. Following RNA sequencing of Hmgb1 knockout and wild-type mouse embryonic fibroblasts, genes belonging to the Wnt signaling pathway were shown to be differentially expressed. We further confirmed that HMGB1 interacts with Dishevelled 2 in mouse cells using in silico protein-protein docking and proximity ligation assay. Hmgb1 knockout mouse embryonic fibroblasts had significantly increased levels of active β-catenin in both resting and Wnt-3a-activated cells. However, the levels of phosphorylated Low-density lipoprotein receptor-related protein 6 were unaffected, suggesting a role downstream of the receptor. Since the proliferative rate was similar between Hmgb1knockout and wild-type cells, we suggest an alternative downstream response. Based on previous publications, we hypothesize that over-activation of Wnt/β-catenin signaling results in differentiation into myofibroblast-like cells characterized by actin reorganization and enhanced migratory capacity. During stress, HMGB1 is released to the extracellular environment and its structural form determines receptor interaction. We identified several factors impacting which form of HMGB1 is being released, including cell type, cell death mode and cell stress induced by LPS. During cell death, monomeric HMGB1 is released whereas it is found in complexes with other molecules when secreted from various tumor- and myeloid-derived cells. The observed molecular mass (on immunoblots) of HMGB1 released from different cells varies, suggesting that different HMGB1-protein complexes form depending on cell type. For example, in SIM-A9 cells, HMGB1 was shown to bind ribosomal proteins, histones and signal recognition particle 9 and future studies should determine their joint role for receptor binding and cell activation. Once present in the extracellular environment, HMGB1 signals through several receptors leading to activation and differentiation of target cells. To gain in-depth understanding of this process, bone marrow-derived macrophages were stimulated with frHMGB1, dsHMGB1, LPS or LPS/IFNγ, and thereafter analyzed by RNA sequencing. Bioinformatic analysis revealed that dsHMGB1 induced a transcriptomic shift similar to LPS, whereas frHMGB1 only had minor effects. Even though dsHMGB1 showed a large overlap with LPS, there were distinct differences, including foam cell differentiation, arginine metabolism and TLR7/9 signaling. To further investigate these differences, we induced foam cell differentiation following two hours of stimulation with frHMGB1, dsHMGB1, LPS or LPS/IFNγ. LPS- and LPS/IFNγ treatment enhanced the uptake of oxLDL and the formation of foam cells whereas HMGB1 did not, suggesting a difference between PAMPs and DAMPs. Taken together, even though dsHMGB1-treated macrophages are transcriptomically similar to LPS- and LPS/IFNγ-treated cells, we have observed distinct functional differences. By sequencing the transcriptome of dsHMGB1-polarized macrophages, we have in-detail defined their position on the macrophage polarization spectrum. In conclusion, this thesis expands current knowledge on the molecular role of the multifunctional alarmin HMGB1 by identifying previously undescribed roles. Due to its multi-functionality, it is of high importance to understand all molecular aspects in order to successfully develop targeting therapeutics with minimal adverse effects. Taken together, this thesis constitutes a foundation to advance future molecular studies of HMGB1 and its role in cell biology and disease pathogenesis. List of scientific papers I. Heinbäck R, van der Zijde M, Tsang CH, Tršelič T, Espinosa A, Aulin C, Erlandsson Harris H. Mapping the intracellular HMGB1 interactome and alterations induced by Toll-like receptor 4 activation. J Biol Chem. 2025 Oct 27;301(12):110866. https://doi.org/10.1016/j.jbc.2025.110866 II. Heinbäck R, Pechiappan H, Tsang CH, Schulte G, Espinosa A, Aulin C, Erlandsson Harris H. HMGB1 interacts with DVL2 and regulates ß- catenin-mediated signaling. [Manuscript] III. Pechiappan H, Heinbäck R, Stråtveit Gravning S, Jakobsen K, Davies R, Aulin C, Erlandsson Harris H. Cell type, cell death mode and LPS stimulation modulate HMGB1 release. [Manuscript] IV. Qu H, Heinbäck R, Salo H, Ewing E, Espinosa A, Aulin C, Erlandsson Harris H. Transcriptomic Profiling Reveals That HMGB1 Induces Macrophage Polarization Different from Classical M1. Biomolecules. 2022 Jun 2;12(6):779. https://doi.org/10.3390/biom12060779