Figure 1

Extracellular HMGB1 is highly PARylated in cells stimulated with LPS. (A) PARP-1 gene deletion inhibits LPS-induced HMGB1 release. WT or PARP-1^-/-MEFs were treated with 100 μg/mL LPS for 24 h at 37°C and then extracellular HMGB1 was secreted into the culture supernatant quantified by ELISA. **P < 0.01 compared with nontreated cells. Assay was done in triplicate. (B) Inhibition of PARP-1 reduces LPS-induced HMGB1 release. WT MEF cells were treated with LPS in the presence or absence of the noncompetitive PARP-1 inhibitor NU1025 for 24 h, after which extracellular HMGB1 was quantified by ELISA. **P < 0.01 compared with nontreated cells. (C) The decrease in HMGB1 release by LPS-stimulated PARP-1^-/- MEFs is reversed by reconstitution of expression of WT PARP-1 but not by enzymatically inactive PARP-1 (E988K). PARP-1^-/-MEFs were infected with an adenoviral vector expressing EYFP-WT PARP-1 (Ad-WT-PARP-1) or EYFP-E988K PARP-1 (Ad-E988K-PARP-1) or left uninfected. Forty-eight hours later, the cells were cultured for 24 h with 100 μg/mL LPS or left untreated (Con). Extracellular HMGB1 was quantified by ELISA. *P < 0.05 compared with uninfected PARP-1^-/- MEFs. **P < 0.01 compared with untreated WT MEFs. (D) HMGB1 secreted after TLR4 stimulation is highly PARylated. J447 mouse cells expressing Flag-HMGB1 were treated with 100 μg/mL LPS for 24 h or left untreated (Con). The culture supernatants and protein extracts prepared from the cells were then subjected to pull-down with anti-Flag agarose beads. The resulting precipitates were subjected to immunoblot analysis with antibodies to PAR and HMGB1. A representative gel is shown. Two additional independent experiments provided similar results. (E) Flag-HMGB1 pull-down under nondenaturing conditions and after PARP-1 and HMGB1 knockdown. J447 mouse cells expressing Flag-HMGB1 were treated with 100 μg/mL LPS for 24 h or left untreated (Con). The protein extracts were then subjected to pull-down with anti-Flag agarose beads under nondenaturing conditions. The resulting precipitates were subjected to immunoblot analysis with antibodies to PAR or HMGB1. (F) Flag-HMGB1 pull-down under denaturing conditions after PARP-1 and HMGB1 knockdown. The same experiment was conducted with J447-mouse cells expressing Flag-HMGB1 after PARP-1 or HMGB1 knockdown. The cells were transduced with lentiviruses encoding control shRNA (shRNA-Con), shRNA targeting HMGB1 (shRNA-HMGB1) or shRNA targeting PARP-1 (shRNA-PARP-1); expression of PARP-1, HMGB1 and actin was assessed by immunoblot analysis and compared with that in uninfected cells. (G) These infected or uninfected cells were treated with 100 μg/mL LPS for 24 h or left untreated. The protein extracts were then subjected to pull-down with anti-Flag agarose beads. The resulting precipitates were subjected to immunoblot analysis with antibodies to PAR or Flag. Results from representative experiments are shown. A second set of independent experiments provided similar findings.