DataSheet1_HMGB1 is a mediator of cuproptosis-related sterile inflammation.PDF

Cuproptosis is a recently recognized modality of cell death driven by intracellular copper-dependent mitochondrial stress. However, the mediators of the sterile inflammatory response to cuproptotic death are undetermined. Here, we report that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern, is released by cuproptotic cells to initiate inflammation. Mechanically, copper accumulation-induced adenosine triphosphate (ATP) depletion activates AMP-activated protein kinase (AMPK) to promote HMGB1 phosphorylation, resulting in increased extracellular release. In contrast, genetic (using RNAi) or pharmacologic (using dorsomorphin) inhibition of AMPK activation limits cuproptosis and HMGB1 release. Functionally, the ability of HMGB1-deficient cuproptotic cells to promote advanced glycosylation end product-specific receptor (AGER, also known as RAGE)-dependent inflammatory cytokine production is greatly reduced. Thus, HMGB1 is a key immune mediator of cuproptosis-initiated sterile inflammation.</p

Table2_HMGB1 is a mediator of cuproptosis-related sterile inflammation.XLSX

Cuproptosis is a recently recognized modality of cell death driven by intracellular copper-dependent mitochondrial stress. However, the mediators of the sterile inflammatory response to cuproptotic death are undetermined. Here, we report that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern, is released by cuproptotic cells to initiate inflammation. Mechanically, copper accumulation-induced adenosine triphosphate (ATP) depletion activates AMP-activated protein kinase (AMPK) to promote HMGB1 phosphorylation, resulting in increased extracellular release. In contrast, genetic (using RNAi) or pharmacologic (using dorsomorphin) inhibition of AMPK activation limits cuproptosis and HMGB1 release. Functionally, the ability of HMGB1-deficient cuproptotic cells to promote advanced glycosylation end product-specific receptor (AGER, also known as RAGE)-dependent inflammatory cytokine production is greatly reduced. Thus, HMGB1 is a key immune mediator of cuproptosis-initiated sterile inflammation.</p

Table1_HMGB1 is a mediator of cuproptosis-related sterile inflammation.XLSX

Cuproptosis is a recently recognized modality of cell death driven by intracellular copper-dependent mitochondrial stress. However, the mediators of the sterile inflammatory response to cuproptotic death are undetermined. Here, we report that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern, is released by cuproptotic cells to initiate inflammation. Mechanically, copper accumulation-induced adenosine triphosphate (ATP) depletion activates AMP-activated protein kinase (AMPK) to promote HMGB1 phosphorylation, resulting in increased extracellular release. In contrast, genetic (using RNAi) or pharmacologic (using dorsomorphin) inhibition of AMPK activation limits cuproptosis and HMGB1 release. Functionally, the ability of HMGB1-deficient cuproptotic cells to promote advanced glycosylation end product-specific receptor (AGER, also known as RAGE)-dependent inflammatory cytokine production is greatly reduced. Thus, HMGB1 is a key immune mediator of cuproptosis-initiated sterile inflammation.</p

Exogenous S100A8 regulate autophagy through RAGE receptor.

<p>K562 cells were transfected with control shRNA, RAGE shRNA or TLR4 shRNA for 48 hours, and then treated with S100A8 protein (1 µg/ml) for 24 hours. LC3, p62, RAGE and TLR4 were assayed by Western blot. All data were representatives of 3 independent experiments.</p

Overexpression of S100A8 increased the resistance of leukemia cells to chemotherapy.

<p>(<b>A</b>) K562 cells were transfected with control pLPCX or pLPCX-S100A8 plasmids. Protein level of S100A8 was assayed by Western blot. (<b>B</b>) K562 cells transfected with control pLPCX or pLPCX-S100A8 plasmids were treated with ADR (1 µg/mL) or VCR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (n = 3;^*<i>P</i><0.05). (<b>C</b>) K562 cells were treated as B, LC3-I/II and BECN1 levels were assayed by Western blot analysis. <b>UT,</b> untreated group of K562 cells transfected with S100A8 plasmids. <b>Control,</b> K562 cells were transfected with control pLPCX plasmids. (<b>D</b>) K562 cells were transfected with pLPCX control or pLPCX -S100A8 cDNA for 48 hours and then treated with ADR (1 µg/mL) for 24 hours in the presence or absence of bafilomycin A1 (Baf; 100 nmol/L). The protein levels of LC3 and p62 were assayed by Western blot. (<b>E</b>) K562 cells were transfected pLPCX or pLPCX-S100A8 cDNA with or without the indicated shRNA for 48 hours. Protein levels of S100A8, PI3KC3, BECN1, Atg7, LC3, and p62 were assayed by Western blots. (<b>F</b>) K562 cells transfected with control pLPCX or pLPCX-S100A8 cDNA were subjected to TEM analysis. Autophagosomes were highlighted by arrows. (<b>G</b>) K562 cells transfected with PLPCX-S100A8 cDNA were treated with bafilomycin A1 (Baf; 100 nmol/L) or 3-methyladenine (3-MA; 10 mmo/L) for 12 hours. LC3 were assayed by Western blot. <b>Control,</b> K562 cells were transfected with control pLPCX. (<b>H</b>) K562 cells transfected the indicated shRNA were treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours. Cell viability was analyzed by MTT assay (n = 3;^*<i>P</i><0.05). NS, not significant.</p

ULK1-mAtg13 regulated the fomation of S100A8-BECN1 complex formation in leukemia cells.

<p>(<b>A-C</b>) K562 cells were transfected with S100A8 shRNA (A and B) or ULK1 shRNA (C) for 48 hours and then were treated with ADR (1 µg/mL) for 24 hours. Cells were then processed for immunoprecipitation (IP) or Western blotting (IB) as described in Materials and Methods. All data are representative of 3 experiments. (<b>D</b>) K562 cells transfected with S100A8 shRNA (A and B) or ULK1 shRNA (C) for 48 hours were treated with ADR (1 µg/mL) or VCR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (n = 3;^*<i>P</i><0.05).</p

Suppression of S100A8 sensitized drug resistance leukemia cells to chemotherapy.

<p><b>(A)</b> HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours. Protein and mRNA level of S100A8 was assayed by Western blot and real time RT-PCR, respectively. (<b>B</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, then treated with adriamycin (ADR) and vincristine (VCR) for an additional 24 hours. Cell viability was analyzed by MTT. (<b>C and D</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for additional 24 hours. Apoptosis was analyzed by measuring positive percentage of Annexin V cells via flow cytometry (<b>C</b>; n = 3;^*<i>P</i><0.05); cleaved PARP was analyzed by Western blotting (<b>D</b>). (<b>E</b>) HL60/ADR and K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours, and then treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for additional 24 hours with or without ZVAD-FMK (20 µmol/L). Activation of caspase-3 was analyzed (n = 3;^*<i>P</i><0.05). (<b>F</b>) HL60/ADR cells were transfected with control shRNA or S100A8 shRNA (from Gene Pharma, China) for 48 hours and then treated with ADR (12.5 µg/mL), VCR (12.5 µg/mL) for 24 hours. S100A8 protein was determined by Western blot; Cell viability was analyzed by MTT; apoptosis was analyzed by flow cytometry (n = 3;^*<i>P</i><0.05).</p

S100A8 regulated the chemotherapy–induced autophagy in leukemia cells.

<p>(<b>A and B)</b> K562 cells were transfected with control shRNA or S100A8 shRNA for 48 hours and then treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours in the presence or absence of bafilomycin A1 (Baf; 100 nmol/L). The protein levels of LC3 and p62 were assayed by Western blot (<b>A</b>); LC3 puncta were analyzed by LC3 antibody or mRFP–GFP–LC3 (Magnification is 10×60 oil) (<b>B</b>) (n = 3;^*<i>P</i><0.05). (<b>C</b>) K562 cells were transfected with control shRNA or S100A8 shRNA for 48 hours and then treated with ADR (1 µg/mL) and VCR (1 µg/mL) for 24 hours. Autophagosome-like structures (indicated by the red arrows) were assayed by TEM (n = 3;^*<i>P</i><0.05). Bar = 2 µm. (<b>D and E</b>) K562/A02 cells were transfected with control shRNA or S100A8 shRNA for 48 hours. After pretreatment with rapamycin (Rap; 100 nmol/L) for 6 hours, cells were treated with ADR (1 µg/mL) for 24 hours. Apoptosis was analyzed by measuring Annexin V–positive cells with flow cytometry (<b>D</b>). Autophagy was analyzed by measuring LC3 puncta formation (<b>E</b>; n = 3;^*<i>P</i><0.05).</p

S100A8 was elevated in drug resistance leukemia cells and chemotherapy agents induced S100A8 expression in leukemia cells.

<p>(<b>A</b>) Protein level of S100A8 was analyzed by Western blotting in Jurkat, HL-60, K562 and MV4-11 cells (n = 3, ^*<i>P</i><0.05). (<b>B and C</b>) S100A8 mRNA level in leukemia cells was analyzed by real time RT-PCR (n = 3, ^*<i>P</i><0.05 versus Jurkat cells in <b>B</b> and ^*<i>P</i><0.05 versus HL-60 or K562 in <b>C</b>, Jurkat group set as 1). (<b>D</b>) Basal LC3-I/II level was analyzed by Western blotting in leukemia cells (n = 3, ^*<i>P</i><0.05 <i>versus</i> Jurkat cells ). (<b>E and F</b>) IC50 levels of adriamycin (ADR) in Jurkat, K562, HL-60, MV-4-11, K562/A02, and HL-60/ADR cells (n = 3, ^*<i>P</i><0.05 versus Jurkat cells in <b>E</b>, ^*<i>P</i><0.05 versus HL-60 or K562 in <b>F</b>). (<b>G</b>) Jurkat, HL-60, K562 and MV4-11 cells were treated with ADR (1 µg/ml), VCR (1 µg/ml) or As2O3 (5 µM) for 24 hours and S100A8 protein level was analyzed by Western blotting (n = 3, *P<0.05 vs. UT, untreated group). AU, arbitrary unit. (<b>H</b>) Jurkat, HL-60, K562 and MV4-11 cells were treated with ADR (1 µg/ml), vincristine (VCR, 1 µg/ml) or arsenic trioxide (As2O3, 5 µM) for 24 hours and S100A8 mRNA level was analyzed by real time RT-PCR (n = 3, *P<0.05 versus control group, control group set as 1).</p

Statistically significant miRs from microRNA profiling data after F test of δC_t values.

1<p>False discovery rate.</p>a<p>LPS-stimulated cultures.</p>b<p>MEF HMGB^−/− lysate exposed cultures.</p>c<p>MEF HMGB1^+/+ lysate exposed cultures.</p>d<p>Untreated cultures.</p