High mobility group protein B1 (HMGB1) interacts with receptor for advanced glycation end products (RAGE) to promote airway smooth muscle cell proliferation through ERK and NF- κ B pathways

Abstract

Background: High-mobility graoup box protein 1 (HMGB1) has been shown to mediate a wide range of pathologic responses by interacting with RAGE (receptor for advanced glycation endproducts) and TLRs (Toll-like receptors). Our previous study showed that HMGB1 has been involved in pathogenesis of airway remodeling in an allergen-induced chronic mice asthma model. Increased airway smooth muscle (ASM) mass is a vital feature of airway remodeling.

Objective: To evaluate the effect of HMGB1 on proliferation of ASMs and the underlying mechanisms.

Methods: Rat airway smooth muscle cells (RASMs) were obtained by primary explant techniques. We investigated the effect of HMGB1 on the proliferation of RASMs. To identify which receptors and signaling pathways be involved in proliferation of RASMs, we performed western blot and CCK-8 assay by specific receptor blockade and inhibition of MAPK (p38, JNK and ERK) and NF-κB signaling pathways.

Results: HMGB1 stimulated RASMs proliferation in a dose- and time-dependent manner and also increased proliferating cell nuclear antigen (PCNA) and RAGE expression of RASMs. The inhibitor of RAGE, but not TLR2 and TLR4, reversed HMGB1-induced RASM proliferation and PCNA expression. Incubation of RASMs with HMGB1 caused a rapid increase in P65 and ERK phosphorylation. RASM proliferation and PCNA expression toward HMGB1 were significantly inhibited by the inhibitors of ERK and NF-κB.

Conclusion: HMGB1 induces proliferation of RASMs through a RAGE-dependent activation of ERK and NF-κB signaling pathways.

Keywords: HMGB1; RAGE; RASM cells; cell remodeling.

Figure 1

Effect of HMGB1 on proliferation of RASMCs. A. Representative western blot analysis for PCNA protein. The primary RASMCs were treated with the indicated concentrations of HMGB1 (100-1000 ng/ml) for 48 h. GAPDH was selected to normalize the PCNA level. *P < 0.05 vs. Control cells; #P < 0.05 vs the RASMCs treated with HMGB1 100 ng/ml. B. Effect of HMGB1 on RASMCs proliferation detected by the CCK-8 assay. The primary RASMCs were treated with HMGB1 for 48 h. *P < 0.05 vs. Control cells; #P < 0.05 vs. the RASMCs treated with HMGB1 100 ng/ml. C. The RASMCs were treated with HMGB1 1000 ng/ml for 0, 12, 24, 48 h. *: P < 0.05 vs. unstimulated cells at 0 h; #: P < 0.05 vs the RASMCs treated with HMGB1 for 12 h; &: P < 0.05 vs the RASMCs treated with HMGB1 for 24 h. D. Cell viability was detected by trypan blue staining. Data are expressed as mean ± SEM from four independent experiments.

Figure 2

Effect of HMGB1 on receptors expression of RASMCs. A. The primary RASMCs were treated with the indicated concentrations of HMGB1 (100-1000 ng/ml) for 48 h, then these cells were assessed for TLR2, TLR4 and RAGE mRNA expression by RT-PCR. B. The primary RASMCs were treated with HMGB1 for 48 h, then the cell total proteins were extracted, and the TLR2, TLR4, and RAGE protein expression were determined by western blotting. *: P < 0.05 vs. Control cells; #: P < 0.05 vs. the RASMCs treated with HMGB1 100 ng/ml; &: P < 0.05 vs. the RASMCs treated with HMGB1 500 ng/ml. Data were expressed as mean ± SEM from four independent experiments.

Figure 3

The effects of related receptors on HMGB1-induced proliferation of RASMCs. The primary RASMCs were pretreated with Cu-CPT22 (50 nM), CLI-095 (1 ug/ml) and FPS-ZMI (230 nM) for 1 hour, followed by stimulation with HMGB1 1000 ng/ml for 48 h. A. Cell proliferation was evaluated by a CCK-8 assay. B. The PCNA protein expression was determined by western blot and GAPDH was used as the internal control. *: P < 0.05 vs. unstimulated cells; #: P < 0.05 vs. the RASMCs treated with HMGB1. Data are expressed as mean ± SEM from four independent experiments.

Figure 4

Effects of specific signaling pathway inhibitors on HMGB1-induced proliferation of RASMCs. The primary RASMCs were incubated for 1 h with or without the protein kinase inhibitors BAY11-7082 (10 µM), U0126 (10 µM), SB203580 (10 µM) or SP600125 (50 µM) before stimulation with HMGB1 (1,000 ng/ml) for 48 h. A. The cell proliferation was evaluated by a CCK-8 assay. B. The PCNA protein expression was determined by western blot and GAPDH was used as the internal control. *: P < 0.05 vs. unstimulated cells; #: P < 0.05 vs. the RASMCs treated with HMGB1. Data are expressed as mean ± SEM from four independent experiments.

Figure 5

HMGB1 activated NF-ĸB and ERK1/2 signaling pathways. Primary RASMCs were treated with the indicated concentrations of HMGB1 (100-1000 ng/ml) for 12 h, then 50 µg of protein of cell lysates were analyzed for phosphorylation of p65 (A) and extracellular signal-regulated kinase 1/2 (ERK1/2) (B). GAPDH was used as internal control. *: P < 0.05 vs. Control cells; #: P < 0.05 vs. RASMCs treated with HMGB1 100 ng/ml; &: P < 0.05 vs. the RASMCs treated with HMGB1 500 ng/ml. Data are expressed as mean ± SEM from four independent experiments.

Figure 6

RAGE mediated HMGB1-induced phosphorylation of p65 and ERK1/2. Primary RASMCs were incubated for 1 h with or without FPS-ZMI (230 nM), BAY11-7082 (10 µM), U0126 (10 µM) before stimulation with HMGB1 (1,000 ng/ml) for 12 h. then 50 µg of protein of cell lysates were analyzed for phosphorylation of p65 (A) and extracellular signal-regulated kinase 1/2 (ERK1/2) (B). GAPDH was used as internal control. *: P < 0.05 vs. Control cells; #: P < 0.05 vs. the RASMCs treated with HMGB1 1000 ng/ml. Data are expressed as mean ± SEM from four independent experiments.