High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed. We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-£\, IL-6 and TGF-£]) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 £gg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

Asthma is a chronic disorder highlighted by intermittent airway inflammation and characterized by paroxysmal dyspnea and airway hyperresponsiveness (AHR). A key feature of severe asthma is the development of airway wall remodeling, which is thought to occur through repeated rounds of inflammation and tissue repair. Remodeling includes structural changes such as increased mass of airway smooth muscle (ASM), and excessive collagen deposition. ASM cells contribute to airway remodeling via the expression and secretion of extracellular matrix (ECM) proteins. This is particularly driven by inflammatory processes, which include mediators such as transforming growth factor (TGF)-β1 and damage associated molecular pattern (DAMP) proteins, such as high mobility group box 1 (HMGB1). HMGB1 is ubiquitously expressed as a non-histone DNA-binding protein that can regulate gene expression, but can also be released in response to stress to underpin inflammation and tissue repair. In this study we tested the hypothesis that extracellular HMGB1 induces signaling pathways that control responses linked to progression of airway inflammation, remodeling and hyperresponsiveness in human ASM cells. We used primary cultured ASM cells as well as hTERT-immortalized human ASM cells. With immunoblotting we demonstrate that exogenous HMGB1 (10 ng/mL) can induce rapid and sustained phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) that is comparable to that induced by a potent mitogen, platelet derived growth factor (PDGF-BB, 10 ng/mL). We also found that TGF-β1 (2.5 ng/mL) promotes the accumulation of secreted HMGB1 in culture medium in a time line concomitant with expression of ECM proteins, collagen and fibronectin, suggesting a role for HMGB1 in pro-fibrotic effects of TGF-β1. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) that silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for HMGB1-induced cell responses through one of its receptors, receptor for advanced glycation end products (RAGE). Immunoblot analyses revealed that silencing of mDia1 was associated with markedly decreased induction of p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis and secretion of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). However, exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGF-β1 in HMGB1-silenced cells - this suggests that intracellular, but not necessarily secreted HMGB1, regulates ECM expression and secretion in response to TGF-β1. Consistent with this interpretation, exogenous HMGB1 alone was not sufficient to induce ECM synthesis or secretion in primary cultured ASM cells. In conclusion, we show that though in human ASM cells extracellular HMGB1 alone can activate MAPK signaling, likely via mDia1-dependent pathways involving RAGE. it is not capable of prompting ECM protein expression. Recombinanat exogenous HMGB1 does not appear to directly affect ECM synthesis, rather intracellular (nuclear) HMGB1 likely modulates activity of genes that are affected by TGF-β1. Overall, HMGB1 has potential to regulate tissue repair processes involving ASM through intracellular and extracellular mechanisms, thus our findings support further work to elucidate the role of HMGB1 in pathogenesis of obstructive airway disease.