Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.

The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.

Inhibition of inflammatory liver injury by the HMGB1-A box through HMGB1/TLR-4/NF-κB signaling in an acute liver failure mouse model.

We aimed to investigate the preventive effect of high mobility group box 1 (HMGB1)-A box and the mechanism by which it alleviates inflammatory injury in acute liver failure (ALF) by inhibiting the extracellular release of HMGB1. BALB/c mice were intraperitoneally (i.p.) administered LPS/D-GalN to establish an ALF mouse model. HMGB1-A box was administered (i.p.) 1 h before establishing the ALF mouse model. The levels of extracellularly released HMGB1, TLR-4/NF-κB signaling molecules, the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 and COX-2 were measured in the liver tissue and/or serum by Immunohistochemistry, Western blotting and Enzyme-linked immunosorbent assay (ELISA). The levels of extracellularly released HMGB1, TLR-4/NF-κB signaling molecules and proinflammatory cytokines were measured in Huh7 cells as well as LPS- and/or HMGB1-A box treatment by confocal microscopy, Western blotting and ELISA. In the ALF mouse model, the levels of HMGB1 were significantly increased both in the liver and serum, TLR-4/NF-κB signaling molecules and proinflammatory cytokines also was upregulated. Notably, HMGB1-A box could reverse these changes. HMGB1-A box could also cause these changes in LPS-induced Huh7 cells. HMGB1-A box played a protective role by inhibiting inflammatory liver injury via the regulation of HMGB1/TLR-4/NF-κB signaling in the LPS/D-GaIN-induced ALF mouse model, which may be related to inhibiting the extracellular release of HMGB1.

Extracellular HMGB1 promotes CD44 expression in hepatocellular carcinoma via regulating miR-21.

As a member of damage-associated molecular patterns (DAMPs), extracellular high-mobility group box 1 (HMGB1) plays a critical role in hepatocellular carcinoma (HCC) progression. Cluster differentiation 44 (CD44) has been demonstrated to participate in HCC progression. However, the relationship between extracellular HMGB1 and CD44 remains unclear. In this study, our results indicated that extracellular HMGB1 promoted the invasion, sphere formation and EMT process of HCC by increasing CD44 expression, which was dependent on miR-21. Moreover, miR-21 upregulated CD44 expression via activating OCT4/TGF-ß1 signaling. Finally, we demonstrated the activation of Rage/JNK signaling caused by extracellular HMGB1 was responsible for miR-21 overexpression. Together, these findings reveal an important role of extracellular HMGB1 in HCC progression through upregulating miR-21/CD44.

Blockade of extracellular high-mobility group box 1 attenuates inflammation-mediated damage and haze grade in mice with corneal wounds.

PURPOSE: To investigate the expression of extracellular high mobility group box 1 (HMGB1) and the effect of its inhibitor glycyrrhizin (GL) in corneal wound healing. METHODS: We treated C57BL/6J mice with GL or PBS before and after establishing a corneal injury model. Fluorescein staining, Ki-67 expression, haze grade, and haematoxylin/eosin (H&E) staining were used to assess treatment efficacy. The expression of HMGB1, NF-κB-p65, the NLRP3 inflammasome, IL-1ß, CCL2, CXCL2, TGF-ß1, α-SMA, fibronectin, and collagen III and neutrophil influx were examined by immunohistochemical staining, western blot, and RT-qPCR at various time points after corneal injury. RESULTS: After corneal injury, HMGB1 transferred from the nucleus to the cytoplasm and was passively released or actively secreted into the corneal stroma from epithelial cells and inflammatory cells; however, this increase was attenuated by GL treatment. Furthermore, GL indirectly attenuated the expression of IL-1ß by directly inhibiting extracellular HMGB1 functions, which activated the NF-κB-p65/NLRP3/IL-1ß signalling pathway. Moreover, application of GL alleviated the neutrophil infiltration that delays wound healing, accompanied by the downregulation of expression of the chemokines CCL2 and CXCL2. More interestingly, application of GL reduced the degree of haze grade through inactivating extracellular HMGB1 functions that induced TGF-ß1 release and myofibroblast differentiation. In addition, fluorescein and H&E staining and Ki-67 levels suggest that GL promotes regeneration of corneal epithelium. CONCLUSIONS: After corneal injury, extracellular HMGB1 can be an essential driver to trigger a neutrophil- and cytokine-mediated inflammatory injury amplification loop. The application of GL promotes the cornea to restore transparency and integrity, which may be related to the attenuation of extracellular HMGB1 levels and function.