title: Tanshinone IIA sodium sulfonate facilitates endocytic HMGB1 uptake.
abstract: Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.
authors.affiliation: The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.
authors.forename: Yusong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Shu
authors.initials: S
authors.lastname: Zhu
authors.forename: Arvin
authors.initials: A
authors.lastname: Jundoria
authors.forename: Jianhua
authors.initials: J
authors.lastname: Li
authors.forename: Huan
authors.initials: H
authors.lastname: Yang
authors.forename: Saijun
authors.initials: S
authors.lastname: Fan
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Tracey
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Sama
authors.forename: Haichao
authors.initials: H
authors.lastname: Wang
mesh_terms: D045784:Abietanes / Q000494:pharmacology*; D015153:Blotting, Western; D004705:Endocytosis / Q000187:drug effects*; D024243:HMGB1 Protein / Q000378:metabolism*; D006801:Humans
chemical_list: D045784:Abietanes; D024243:HMGB1 Protein; C021751:tanshinone