HMGB1 Increases IL-1β Production in Vascular Smooth Muscle Cells via NLRP3 Inflammasome

Abstract

Vascular smooth muscle cells (VSMCs) are the major cell type in the blood vessel walls, and their phenotypic modulation is a key cellular event driving vascular remodeling. Although high mobility group box-1 (HMGB1) plays a pivotal role in inflammatory processes after vascular injuries, the importance of the links between VSMCs, HMGB1 and vascular inflammation has not been clarified. To prove the hypothesis that VSMCs might be active players in vascular inflammation by secreting inflammatory cytokines, we investigated the proinflammatory effects of HMGB1 and its intermediary signaling pathways in VSMCs. When cultured human VSMCs were stimulated with HMGB1 (10-500 ng/ml), IL-1β production was markedly increased. HMGB1 also increased the expression of NLRP3 inflammasome components including NLRP3, ASC and caspase-1. Among these components, HMGB1-induced expressions of NLRP3 and caspase-1 were markedly attenuated in TLR2 siRNA-transfected cells, whereas ASC and caspase-1 expressions were reduced in RAGE-deficient cells. In TLR4-deficient cells, HMGB1-induced caspase-1 expression was significantly attenuated. Moreover, IL-1β production in HMGB1-stimulated cells was significantly reduced in cells transfected with caspase-1 siRNA as well as in cells treated with monoclonal antibodies or siRNAs for TLR2, TLR4 and RAGE. Overall, this study identified a pivotal role for NLRP3 inflammasome and its receptor signaling involved in the production of IL-1β in VSMCs stimulated with HMGB1. Thus, targeting HMGB1 signaling in VSMCs offers a promising therapeutic strategy for treating vascular remodeling diseases.

Keywords: HMGB1; IL-1β; NLRP3 inflammasome; RAGE; TLRs; VSMC.

Figure 1

Effects of HMGB1 on IL-1β expression and its release from VSMCs. VSMCs were treated with HMGB1 (100 ng/ml) for 0–24 h, and were also treated with HMGB1 (0–500 ng/ml) for 6 h. (A,B) The mRNA levels of IL-1β were determined by RT-PCR. GAPDH was used as a control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (C,D) The protein levels of active IL-1β were determined by Western blot. β-Actin expression served as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. (E,F) VSMCs were treated with HMGB1 (100 ng/ml) for 0–48 h, and were also treated with HMGB1 (0–500 ng/ml) for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control (untreated cells).

Figure 2

Effects of HMGB1 on the expression of NLRP3 inflammasome in VSMCs. (A,B) VSMCs were treated with HMGB1 (100 ng/ml) for 0–24 h, and also treated with HMGB1 (0–500 ng/ml) for 12 h. The mRNA levels of NLRP3 were determined by RT-PCR using GAPDH as a control. (C–E) VSMCs were treated with HMGB1 (100 ng/ml) for 0–48 h. The protein levels NLRP3, ASC, and Caspase-1 were determined by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. ^*P < 0.05 and ^**P < 0.01 vs. control (untreated cells).

Figure 3

Involvement of TLR2, TLR4, and RAGE in HMGB1-induced NLRP3 inflammasome expression in VSMCs. (A–C) VSMCs were transfected with siRNA for TLR2, TLR4, and RAGE, and knockdown efficacies were determined by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 3 independent experiments. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control (untreated cells). (D–F) VSMCs were transfected with siRNA (200 nM) for TLR2, TLR4, and RAGE, and then stimulated with HMGB1 for 24 h. The expressions of NLRP3, ASC, and Caspase-1 were determined by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 to 6 independent experiments. ^*P < 0.05, ^**P < 0.01 vs. corresponding value in untreated cells. ^#P < 0.05 and ^##P < 0.01 vs. corresponding value in negative control.

Figure 4

Functional role of HMGB1 receptors on the production of IL-1β in HMGB1-stimulated VSMCs. (A) VSMCs were transfected with siRNA (200 nM) for TLR2, TLR4, and RAGE, and then incubated with HMGB1 for 24 h. The levels of IL-1β in the culture media were quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. ^**P < 0.01 vs. non-treated control. ^##P < 0.01 vs. corresponding value in negative control. (B) VSMCs were pretreated with anti-IgG antibody (10 μg/ml), anti-TLR2 antibody (10 μg/ml), anti-TLR4 antibody (10 μg/ml), or anti-RAGE antibody (10 μg/ml) for 30 min, and then stimulated with HMGB1 (100 ng/ml) for 24 h. IL-1β release into culture media was quantified by ELISA. Data are expressed as means ± SEMs of triplicates pooled from 4 independent experiments. (C) Protein levels of active IL-1β were assessed by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. ^**P < 0.01 vs. non-treated control. ^#P < 0.05 and ^##P < 0.01 vs. control in HMGB1-treated cells.

Figure 5

Functional role of NLRP3 inflammasome on the production of IL-1β in HMGB1-stimulated VSMCs. (A) VSMCs were treated with HMGB1 (100 ng/ml) for 0–48 h. The protein levels of caspase-1, active caspase-1 (p20), pro- IL-1β, and active IL-1β were assessed by Western blot using β-actin as an internal control. (B) VSMCs were transfected with caspase-1 siRNA (200 nM), and then stimulated with HMGB1 for 24 h. The expression of active IL-1β was determined by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. ^*P < 0.05 vs. non-treated control. ^##P < 0.01 vs. corresponding value in negative control. (C) VSMCs were pretreated with MCC-950 (100 nM; a NLRP3 inhibitor) for 30 min and then stimulated with HMGB1 for 24 h. The protein levels of active IL-1β were determined by Western blot using β-actin as an internal control. Data are expressed as means ± SEMs of duplicates pooled from 4 independent experiments. ^**P < 0.01 vs. non-treated control in vehicle. ^###P < 0.001 vs. corresponding value in HMGB1-treated cells.