Molecular insights into the multifaceted functions and therapeutic targeting of high mobility group box 1 in metabolic diseases

Abstract HMGB1 is a ubiquitously expressed protein localized in nucleus, cytoplasm, as well as secreted into extracellular space. Nuclear HMGB1 binds to DNAs and RNAs, regulating genomic stability and transcription. Cytoplasmic HMGB1 regulates autophagy through binding to core autophagy regulators. Secreted extracellular HMGB1 functions as a ligand to various receptors (RAGE and TLRs, etc.), regulating multiple signalling pathways, such as MAPK, PI3K and NF‐κB signallings. Trafficking and localization of HMGB1 across cellular compartments could be regulated by its posttranslational modifications, which fine‐tune its functions in metabolic diseases, inflammation and cancers. The current review examines the up‐to‐date findings pertaining to the biological functions of HMGB1, with focus on its posttranslational modifications and roles in downstream signalling pathways involved in metabolic diseases. This review also discusses the feasibility of targeting HMGB1 as a potential pharmacological intervention for metabolic diseases.


| INTRODUC TI ON
The high mobility group (HMG)-box (HMGB) family belongs to the HMG protein superfamily that plays important roles in modulating chromatin structures. The HMGB family consists of four chromosomal proteins: HMGB1, HMGB2, HMGB3 and HMGB4. HMGB1 is ubiquitously expressed in high abundance (roughly 1 × 10 6 molecules per mammalian cell), whereas HMGB2, 3 and 4 are expressed at lower levels with restricted expression patterns. [1][2][3] Herein, we summarize the literature reports on the roles of HMGB1 in metabolic diseases and discuss the perspectives of targeting HMGB1 as a potential therapeutic approach. HMGB1 protein is both a nuclear factor and a secreted protein. 4 In the nucleus, HMGB1 serves as a transcriptional regulator that binds or bends DNAs or RNAs and promotes transcriptional complex assembly on specific gene targets. 5,6 For example, HMGB1 binds to the promoter region of TNF and promotes the assembly of the repressor NF-κB factor RelB, thus suppressing TNFα expression. 7 Upon infection or injury, HMGB1 has been shown to translocate from nucleus to the cytoplasm and could also be secreted by activated immune cells. 8 In addition, HMGB1 can be secreted by various cells, including hepatocytes, keratinocytes and granulocytes, under necrosis, DNA damage or oxidative stress. [9][10][11][12] Once secreted, HMGB1 functions as a ligand and binds to the receptor for advanced glycation end products (RAGE), toll-like receptors (TLRs) and C-X-C Motif Chemokine Receptor 4 (CXCR4), playing important roles in regulation of inflammation and immune responses. 13 HMGB1 consists of 215 amino acids, organized into two high mobility group boxes, which function as DNA-binding domains and an acidic C-terminus, respectively ( Figure 1). It has been reported that several serine residues of HMGB1, including Ser39, Ser53 and Ser181, could be phosphorylated by protein kinase C zeta (PKC ζ), enhancing HMGB1 secretion. 14 HMGB1 is also hyperacetylated at multiple lysine residues near the nuclear localization sequence (NLS) (Figure 1), and such posttranslational modification of lysine residues is affected by JAK/STAT1 signalling. 11,15,16 The molecular mechanism responsible for dynamic acetylation and deacetylation of HMGB1 remains unclear and requires further investigation. Poly(ADPribose) polymerase-1 (PARP-1)-induced poly(ADP-ribosyl)ation of HMGB1 synergizes with acetylation and ultimately leads to HMGB1 secretion under LPS stimulation. 11 In addition, HMGB1 could be Nglycosylated at Asn37, Asn134 and Asn135, which is critical for promoting HMGB1 secretion. 17 However, inter-plays between different posttranslational modifications of HMGB1 have not been fully studied, which warrant further investigation.

| REDOX REG UL ATI ON OF HMG B1
There are three conserved cysteine residues (Cys23, Cys45 and Cys106) on HMGB1 that have been implicated as redox-sensitive regulators of HMGB1's pro-inflammatory activity. In general, all protein Cys residues with functional thiol groups [containing a sulphur and hydrogen atom (-SH)] could be posttranslationally modified upon oxidative stress. In silico structural analysis of HMGB1 predicts that Cys23 and Cys45 could reversibly form intramolecular disulfide bonds 18  However, such claims have been found unreliable, and the reports were retracted recently. [19][20][21][22][23] Although independent proteomic analyses of HMGB1 support the notion that disulfide bond formation between Cys residues confers HMGB1's pro-inflammatory functions, 24 the overall strength of this body of literatures linking oxidized HMGB1 to pro-inflammatory responses remains low and unconvincing. There remains a critical need to rigorously assess the redox state(s) of HMGB1 in order to (re)-evaluate the signal transduction and biological activities stimulated by HMGB1 that occur under reducing and oxidizing conditions.
In summary, subcellular localization and secretion of HMGB1 direct its biological functions, which is tightly regulated by its posttranslational modifications, including phosphorylation, 14 acetylation, 12 N-linked glycosylation 17 and Poly(ADP-Ribosyl)ation. 11,16 The regulation of HMGB1 in inflammation and immunity has been systemically reviewed. 13 Therefore, the current review will focus on the role of HMGB1 in metabolic diseases, given the fact that inflammation also plays a critical in the initiation, propagation and development of metabolic diseases. 25

| N UCLE AR FUN C TI ON OF HMG B1 IN THE REG UL ATI ON OF ME TABOLI C D IS E A S E
In the nucleus, HMGB1 can bind to or bend with the DNAs or RNAs Warburg effect, also known as aerobic glycolysis, is a metabolic hallmark of most cancer cells, including HCC, characterized by an excessive conversion of glucose to lactate, despite sufficient oxygen supply. 28 HMGB1-YAP axis has been shown to be responsible for the Warburg effect in liver tumorigenesis via stabilization of YAP-HIF1α in the nucleus and enhancing HIF1α DNA-binding activity, leading to aerobic glycolysis and Warburg effect 26 (Figure 2A). In addition, HMGB1 regulates the expression of heat shock protein beta-1 (HSPB1), which is essential for maintaining quality control of mitochondria. 27 Mechanistically, HMGB1 increases HSPB1 expression, which controls actin cytoskeleton and delivers damaged mitochondria for degradation by autophagy (i.e., mitophagy). 27 Recent studies have demonstrated HMGB1 as a bona fide RNA-binding protein affecting splicing choices. 6 Together, HMGB1 functions as a DNA-or RNA-binding protein, modulating gene expression, splicing and translation, which are relevant in many pathological conditions. Further studies are warranted to specify which DNA or RNA sequences are bound with HMGB1 and their physiological and disease relevance.

| C Y TOPL A S MI C FUN C TI ON S OF HMG B1 IN ME TABOLI C D IS E A S E S
Cytoplasmic HMGB1 has been shown to associate with proteins involved in mitochondrial metabolism, such as mitochondrial degradation (mitophagy) and quality control, and autophagy. Dysregulation of these processes has been implicated in metabolic diseases. 29 For example, HMGB1 is responsible for the suppressive effects of p53 on autophagy in nonalcoholic fatty liver disease (NAFLD). It has been shown that translocation of HMGB1 from nucleus to cytoplasm, and subsequent induction of Beclin1 expression play important roles in NAFLD. 30 Mechanistically, HMGB1 interacts with Beclin1 and Atg5, which are key components for autophagic initiation and nucleation 31 ( Figure 2B). The complex of HMGB1-Beclin1 and HMGB1-Atg5 are resistant to degradation by calpain, inhibiting mitochondria-dependent cell death. 32,33 As autophagy is a process

| E X TR ACELLUL AR FUN C TION OF HMG B1 IN ME TABOLI C D IS E A S E S
Previously, the functions of extracellular HMGB1 in metabolic diseases have been widely studied. Secreted HMGB1 could bind to receptors, such as RAGE, toll-like receptor 2 and 4 (TLR2/4), and CXCR, regulating downstream signalling, including MAPK, PI3K and NF-κB ( Figure 2C). 13,35,36 All these regulatory events are highly relevant to metabolic diseases, such as diabetes and associated compli- and glycated haemoglobin (HbA1c), and negatively correlated with lung functions in subjects with both T2DM and COPD. 37 Interestingly, clinical data indicate that the serum HMGB1 levels are not associated with gestational diabetes mellitus (GDM), although HMGB1 was correlated with maternal age, a risk factor of GDM. 38 In addition, HMGB1 plays a critical role in diabetes-related dysfunctions of bone marrow stromal cells (BMSCs) and impaired osteointegration. 39 The regulatory role of secreted HMGB1 in diabetes and associated complications, as well as liver disease and cardiovascular diseases, has been shown to be through its binding to different receptors.
Obstructive sleep apnoea (OSA), a common T2DM complication, leads to exacerbated intermittent hypoxia (IH), which could severely affect cognitive functions. 40 IH induces HMGB1-TLR4 signalling in hippocampal tissue, concomitant with suppressed autophagy and enhanced apoptosis. 41 Silencing of HMGB1 suppressed TLR4 signalling, and restored autophagy and suppressed apoptosis in hippocampal neurons of animal model of T2DM with OSA complication. 41 Activation of microglial cells may account for neuronal apoptosis and cognitive deficits. 42 For example, in one recent study, diabetic KK-Ay mice exhibited increased cognitive dysfunction, microglial activation and hippocampal neuronal apoptosis, compared with C57 control mice. 42 Activation of BV2 microglia leads to active secretion of HMGB1 from microglial cells and the secreted HMGB1 functions as an inflammatory factor and sustains the activation of these microglial cells in a positive feedback loop, leading to deteriorated neuronal damage. Mechanistically, HMGB1 activates NF-κB-p65 in microglia, resulting in the secretion of TNFα and IL-1β, and excessive ROS, which mediates the apoptosis of HT22 cells via the PI3K/ Akt/GSK-3β signalling pathway ( Figure 2C). 42 Moreover, extracellular HMGB1 is involved in streptozotocin-induced diabetic nephropathy (DN) via its activation of TLR2, TLR4 and RAGE, while blockade of HMGB1 signalling attenuates streptozotocin-induced DN. 43 Furthermore, HMGB1 plays a crucial role in diabetic neuropathy accompanied neuroinflammation, characterized by the upregulation of HMGB1 and its receptors (TLR4 and CXCR4) ( Figure 2C). 35 In addition, periodontal inflammation in diabetic patients was regulated by HMGB1-RAGE-TNFα/IL6 signalling. 44  Taken together, extracellular HMGB1 is involved in the development of metabolic diseases, mainly through its cytokine-like effects.
Thus, targeting extracellular HMGB1 might provide potential therapeutic agents for metabolic diseases.

| PHARMACOLOG IC AL INTERVENTIONS OF HMG B1 IN ME TABOLI C D IS E A S E
As HMGB1 plays important roles in metabolic diseases, significant efforts have been directed towards the discovery of specific HMGB1 inhibitors. 53 A recent report has expatiated the findings of preclinical HMGB1-directed therapies. Such therapeutic approaches involve direct binding of small molecule/peptide-like antagonists to HMGB1 and ultimately result in the inhibition of posttranslational modifications and/or neutralization of extracellular HMGB1. 53 In addition, colonic epithelial hyperplasia is detected in the small intestines of DM mice, which is an underlying risk factor for colorectal cancer development. 54  and its receptors (TLR4 and CXCR4) in mice with diabetic neuropathy, improving mechanical and thermal pain threshold in these animals. Mechanistically, glycyrrhizin inhibits the release of HMGB1. 35 Moreover, in vitro and in vivo studies of glycyrrhizin demonstrate that it improves osteogenic differentiation, attenuates lipid peroxide, restores hyperglycaemia-induced impairment of trabecular structure and osteointegration, via inhibiting HMGB1-RAGE cascade. 39 Together, the therapeutic interventions on HMGB1 are mainly related to their targeting of extracellular HMGB1. However, it would be also critical to design specific drugs targeting extracellular or nuclear HMGB1 for different utilities.

| CON CLUS I ON S AND PER S PEC TIVE
HMGB1 plays multifaced roles in metabolic diseases through its differential functions at different subcellular locations. In the nucleus, HMGB1 functions as transcriptional regulator, bending and binding with DNAs or RNAs. In the cytoplasm, HMGB1 is an autophagy or mitophagy regulator through its binding to autophagic components.
In the extracellular space, HMGB1 plays distinct roles via its binding writing -original draft (equal); writing -review and editing (equal).

ACK N OWLED G EM ENT
The figures were created with BioRe nder.com. This work is funded in part by Melanoma Research Alliance (MRA) postdoctoral fellowship (812717 to Z. T).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.