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.

Serum Amyloid A Stimulates PKR Expression and HMGB1 Release Possibly through TLR4/RAGE Receptors

“…Primary peritoneal macrophages were isolated from Balb/C mice (Taconic; male, 7-8 wks, 20-25 g) at 2-3 d after intraperitoneal injection of 2 mL thioglycollate broth (4%), as previously described (36,37). RAW 264.7 macrophages, U937 monocytes and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS.…”

Carbenoxolone Blocks Endotoxin-Induced Protein Kinase R (PKR) Activation and High Mobility Group Box 1 (HMGB1) Release

“…GTA (also called enoxolone) can be chemically derivatized by esterification into a succinate ester termed "carbenoxolone." male, 7-8 wks, 20-25 g) at 2-3 d after intraperitoneal injection of 2 mL thioglycollate broth (4%) as described previously (35,36). Both RAW 264.7 cells and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS.…”

“…Both RAW 264.7 cells and primary macrophages were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. Adherent macrophages were gently washed with, and cultured in, DMEM before stimulating with LPS (0.5 μg/mL) in the absence or presence of CBX or purinergic P2X 7 R antagonists (oATP, 50 μmol/L; BBG, 0.5 μmol/L; KN62, 0.5 μmol/L) for 16 h. Subsequently, the cell-conditioned culture media were analyzed for levels of HMGB1, nitric oxide (NO) and other cytokines by Western blotting analysis, the Griess reaction, ELISA, and cytokine antibodies arrays as described previously (36)(37)(38).…”

“…For instance, mung bean extracts and green tea components (for example, EGCG) prevent extracellular HMGB1 release through stimulating HMGB1 aggregation and autophagic degradation (38,43). Danshen components (for example, tanshinone IIA sodium sulphonate) effectively stimulate endocytic HMGB1 uptake, thereby recycling extracellular HMGB1 back to cytoplasmic vesicles for eventual degradation (36). In a sharp contrast, CBX could neither induce autophagic HMGB1 degradation, nor stimulate endocytic HMGB1 uptake in macrophage cultures (data not shown).…”

High Mobility Group Box 1 (HMGB1) Phenotypic Role Revealed with Stress

“…Although autophagy inhibition prevents HMGB1 release, secretion and degradation, HMGB1 itself regulates autophagy at multiple levels (17)(18)(19)(20)(21)(22)(23)(24)(25). For example, nuclear HMGB1 functions as a transcriptional cofactor, regulating the expression of heat shock protein β-1 (26), which in turn sustains dynamic intracellular trafficking during autophagy and mitophagy (26).…”