Features and Resources

Summary from R. Kang et al.
"HMGB1 in Health and Disease", MolMed 2014;40:116



In 1879, German cytologist Walther Flemming discovered chromosomes. Today, we know that chromosomes contain all the genes that are necessary for the inheritance of well-defined characteristics from parents to their children. Chromosomes are actually packets of densely packed chromatin, a nucleoprotein complex.

The highly regulated interplay between DNA and chromosomal proteins regulates the function, structure and dynamics of chromosomes, which in turn enables genome stability and gene regulation. Chromosomal proteins can be divided into 3 classes. The first and most abundant group are histones, the second group are some relatively scarcely occurring tissue-specific, urea-insoluble proteins, and the third group (the second most abundant group) are the high-mobility group (HMG) proteins. In contrast to histones, HMG proteins are highly conserved evolutionarily, bind only weakly to chromatin and aim at DNA structures rather than at specific DNA sequences.

The group of HMG proteins is comprised of three sub-groups: HMGA proteins, HMGN proteins and HMGB proteins (the most common subgroup). They all share a common role of being used for DNA remodeling by interacting with nucleotides, histones, transcription factors and other chromosomal or nuclear proteins.

The HMGB protein group ultimately comprises 4 proteins: HMGB1, HMGB2, HMGB3 and HMGB4.

HMGB1 is the most widespread and abundant protein (about 1 million copies/cell) in this group. HMGB1 is evolutionarily conserved to the highest degree, and its origin can be dated to the time before the separation of protostomes and deuterostomes about 525 million years ago. Homologs to mammalian HMGB1 are found in yeast, fruit flies, non-biting midges (chironomids), echinoderms, bacteria, plants, fish, and Caenorhabditis elegans. The amino acid sequence homology in mammals is more than 99%.

To Top

Functions of HMGB1

Nuclear HMGB1

Nuclear HMGB1 acts as a chaperone with DNA binding and folding activity, and as a regulator of various key DNA processes. It ensures nucleosome stability by binding to histones. It regulates nucleosome organization as well as its development. Thus, HMGB1 knock-out cells have up to 30% less nucleosomes. In addition, HMGB1 also regulates the process of nucleosome release. Furthermore, HMGB1 plays a role in V(D)J recombination (also called somatic recombination), a rearrangement process of DNA in vertebrates, by being involved in the formation of essential protein complexes. HMGB1 is also able to influence the transcription rates and gene expression by different mechanisms. In the case of DNA replication, HMGB1 plays a role of regulating the activity of DNA polymerase. HMGB1 is also involved in DNA repair processes, such as the repair of DNA mismatches, base excision, nucleotide excision and dsDNA breaks.


Figure 1: Functions of HMGB1 intra- and extracellular
(click to enlarge) 

Cytosolic HMGB1

HMGB1 is translocated from the nucleus into the cytosol via acetylation. Normally, the ratio amounts to a 30:1 nucleus:cytosol. One of the main tasks of cytosolic HMGB1 is the positive regulation of autophagy. Autophagy stimuli ensure the translocation from the nucleus to the cytosol and subsequent binding to Beclin-1 to induce autophagy.

Membrane-bound HMGB1

Membrane-bound HMGB1 was shown to play a role in neurite outgrowth, platelet activation, the process of maturation of red blood cells, cell adhesion and the innate immune response. In platelet activation, HMGB1 is pro-actively translocated to the membrane and released to initiate NET formation and function.

Extracellular HMGB1

HMGB1 is actively released by the immunocompetent cells, but also passively by the dead, dying or damaged cells. Extracellular HMGB1 causes a variety of activities and is involved in many processes such as inflammation, immunity, cell migration, invasion, proliferation and differentiation, antimicrobial defense and tissue regeneration. To date, many studies have shown that HMGB1 binds to multiple receptors (e.g. RAGE and TLRs) of active macrophages, monocytes, neutrophils, fibroblasts and many others to induce cytokine production (e.g. TNF, IL-1α, IL-1β, IL- 1RA, IL-6, IL-8, IL-10 and many more). The molecular mechanisms underlying the pro-inflammatory activity of HMGB1 include, inter alia, p38, ERK, JNK, SIRT1 and NF-κB signaling pathway, to name but a few. Neutralization of HMGB1 release and/or activity (ST326052233) decreases the inflammatory response. The immune system consists of two parts, the innate and the adaptive immune response. Its role is to protect the organism against pathogens, cancer cells and foreign substances. HMGB1 plays an important role in the regulation of both immune responses. In the innate immune response, HMGB1 can activate or inhibit macrophages, depending on the localization and receptor pathway. Many other immunocompetent cells such as neutrophils, eosinophils and basophils, mast cells, dendritic cells, NK cells, T cells, B cells and myeloid suppressor cells also require HMGB1-mediated activation. Generally, HMGB1 mediates cellular migratory stimuli via the RAGE axis. It has already been shown that HMGB1 allows for the migration of neurites, smooth muscle cells, myoblasts, tumor cells, the hepatic stellate (Ito) cells, stem cells, endothelial and many others.

To Top

HMGB1 Modifications

Intranuclear modifications

The most well-known HMGB1 modification is acetylation. HMGB1 contains two so-called "nuclear localisation sites" (NLS). Hyper-acetylation in these positions provides for translocation from the nucleus out into the cytoplasm.
HMGB1 ADP ribosylation is mainly observed in cancer cells. Hyper-ADP-ribosylation of HMGB1 down-regulates gene transcription. However, poly-ADP-ribosylation mediated by PARP1 is also necessary to orchestrate HMGB1 release from the dying cells, especially in necrosis. Various mono-methylations of HMGB1 were observed in neutrophils and renal cell tumors. Phosphorylation of HMGB1 determines its ability to bind and/or fold DNA. Furthermore, phosphorylation determines its intracellular localization in a similar way as acetylation. HMGB1 can also be glycosylated. However, the scope is extremely minor, and the function of glycosylation hitherto unknown.


HMGB1 current knowledge
Figure 2: HMGB1 biology by the current state of scientific knowledge
(click to enlarge) 

The oxidative status of HMGB1 is crucial for its function. In contrast to the nucleus, the cytoplasm and the extracellular milieu are oxidative environments. The three cysteines at positions 23, 45 and 106 with their thiol groups are crucial for the oxidation of HMGB1. Immediately after release, all 3 cysteines are in the reduced thiol state. This HMGB1 isoform is referred to as "fully-reduced HMGB1" (REHM114-116).
Because they are located opposite each other, cysteines C23 and C45 in are particularly susceptible to oxidation -- they rapidly build a disulfide bond, while C106 remains relatively stable. This HMGB1 isoform is referred to as "disulfide HMGB1" (REHM120-122) . Complete oxidation of HMGB1 causes sulfonylation of all three cysteines. This HMGB1 isoform is referred to as "terminally-oxidized HMGB1".
The oxidative state of HMGB1 determines its mechanism of action. The fully-reduced HMGB1 has a chemotactic function and causes migration of immune cells to the danger zone. The disulfide HMGB1, on the other hand, acts as a pro-inflammatory cytokine inducing production of further cytokines by binding to i.e. TLR4, such as TNFα, IL6, IL-10. The exact function of the terminally-oxidized HMGB1 has, however, not yet been adequately studied, but is generally associated with inflammatory process termination.

To Top

HMGB1 and Disease

In 1999, Haichao Wang and colleagues were the first in the world to describe the occurrence of HMGB1 outside the cell and its function in sepsis. They have demonstrated that HMGB1 is released very late by the immune cells (here macrophages) to induce the innate immune response, namely approximately 20 hours after LPS activation. This has been confirmed in many subsequent studies. At the same time, Wang and colleagues showed that further administration of HMGB1 was lethal for the sepsis-model mice, while neutralization of HMGB1 (ST326052233) ensured survival, and that HMGB1 is actually found in the circulation of sepsis patients (ST51011).

In reperfusion injury of various organs, it has been shown that HMGB1 is one of the main triggering agents responsible for this injury. Reperfusion injury is defined as the process of re-establishing blood supply following tissue ischemia, which brings about a sterile inflammatory process.

In the mouse model of reperfusion injury, HMGB1 levels (ST51011) are elevated as early as one hour after reperfusion and persist at this level for up to 24 hours. Analogously to the situation in sepsis, neutralization of HMGB1 (ST326052233) leads to a significant reduction of reperfusion injury, while the administration of recombinant HMGB1 (REHM120-122) greatly increases said damage.

In the central nervous system (CNS), HMGB1 is responsible for neurite formation. HMGB1 expression is greatest in the brain of young adults and then declines with age. As a general inflammatory mediator, HMGB1 is of importance in neuroinflammatory processes as well. This applies to a large number of CNS disorders such as the entire range of neurodegenerative diseases, motor neuron diseases, autoimmune diseases of the central nervous system, neuropathic pain, and many others.

In the domain of the cardiovascular system, HMGB1 plays a role in various vascular disorders. Myocardial infarction exemplarily displays the dual role played by HMGB1. On the one hand, it is needed for the regeneration of the heart muscle (REHM114-116), and on the other hand it acts as a cytokine (REHM120-122) which promotes the inflammatory process and hence injury.

There are many other disorders characterized by HMGB1 playing a crucial role in the disease process. Cancers with their diverse tumor types deserve special mention here. Again, please refer to the publication by R. Kang et al. "HMGB1 in Health and Disease" (Mol Aspects Med 2014 Dec; 40: 1-116.).

To Top