Dataset Information

Context-Dependent Release of HMGB1: Cell Death Mode, Cell type and LPS Stress Drive Monomer and Heterocomplex Formation

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Macrophage

SUBMITTER: HARINI PECHIAPPAN

LAB HEAD: Harini Pechiappan

PROVIDER: PXD077444 | Pride | 2026-06-29

REPOSITORIES: Pride

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

Pechiappan Harini H Heinbäck Rebecka R Gravning Sigrid Stråtveit SS Jakobsen Kjerstin K Davies Richard R Aulin Cecilia C Harris Helena Erlandsson HE

Molecular medicine (Cambridge, Mass.) 20260502 1

<h4>Background</h4>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 microen ...[more]

PMID: 42067772

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Project description:Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA expression signature of this response is different from that of a PAMP-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human PBMCs exposed to DAMPcontaining freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6x10-4 and p<3.7x10-3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from HMGB1+/+ mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1-/- MEFs. miR-155 expression in these cultures was negligible, but was significantly higher in PBMCs stimulated with LPS or most other TLR ligands, making it the prototypic PAMPmiR. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKgamma mRNA, a putative target of miR-34c, increased, while protein levels of IKKgamma in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1beta and TNFalpha) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that miR-34c is involved in the inflammasome pathway in response to DAMPs. Our findings suggest that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders. Human PBMC (peripheral blood mononuclear cells) were exposed to 4 conditions for 48 hours. In the first condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1-/- MEF cells (mouse embryonic fibroblast cells). In the second condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1+/+ MEF cells (mouse embryonic fibroblast cells). In the third condition, PBMCs were exposed to LPS (Lipopolysaccharide). In the fourth condition, the PBMCs where left untreated. Four biological repeats were done for each condition for a total of 16 samples.

			Action	DRS
	10_1_Flag-LPS.raw	Raw
	11_2_Flag-LPS.raw	Raw
	12_3_Flag-LPS.raw	Raw
	13_Bead_CTRL.raw	Raw
	1_1_HMG-PBS.raw	Raw