The ability of a cell to respond specifically to various external and internal signals plays an essential role in regulating gene expression, differentiation, and cell death. Most often, the cellular responses to various stimuli involve transduction of signals received by specific receptors to defined targets that elicit specific responses in distinct cellular compartments. HMGB1 (High Mobility Group-B1), a member of the high-mobility group protein superfamily, with a molecular mass of ~27 kilodaltons and devoid of any enzymatic activity, has the ability to signal to many cellular targets in different cellular compartments and to affect several distinct and apparently unrelated biological pathways (Ref.1). HMGB1, also called Amphoterin, is almost ubiquitous and only 10 times less abundant than core histones, at 106 molecules per typical mammalian cell. It is a highly conserved component of eukaryotic nuclei and is rather known as a DNA binding protein involved in assembly of nucleoprotein complexes, maintenance of nucleosome structure and regulation of gene transcription (Ref.2). A series of recent discoveries have revealed the cytokine activity of HMGB1, that when secreted into the extracellular milieu, mediates downstream neurite outgrowth, smooth muscle cell chemotaxis, inflammatory responses and tumor metastasis (Ref.3).
Human HMGB1 has 219 residues in its primary amino acid sequence. Structurally, it has a tripartite structure composed of three domains: two homologous DNA-binding motifs termed A and B boxes, each made up of approximately 80 amino acids, and a negatively charged 30 amino acid-long, highly acidic COOH-terminus (Ref.4). Like many other nuclear proteins in living cells, HMGB1 molecules are in constant, rapid motion. The high intranuclear mobility of HMGB1 leads to frequent collisions with the chromatin fiber and facilitates interactions with other nuclear proteins. HMGB1 binds to DNA in a sequence-independent manner and modifies DNA structure to facilitate transcription, replication, and repair. The ability of the protein to affect many types of nuclear activities reflects its mode of binding to DNA and its ability to interact with a diverse set of proteins. Although HMGB1 binds to the minor groove of B-type DNA weakly and without apparent sequence specificity, upon binding it distorts the DNA sharply, inducing bends of >90° within a single turn of the DNA helix. The architectural changes induced in the DNA promote the assembly of multiprotein complexes at the distorted site (Ref.1).
HMGB1 is secreted by Macrophages, Monocytes, and Pituicytes after stimulation with endotoxins, LPS (Lipopolysaccharide),
IL-1 (Interleukin-1), TNF-Alpha (Tumor Necrosis Factor-Alpha) and
IFN-Gamma (Interferon-Gamma) (Ref.3), and can also be passively released by necrotic cells (Ref.5). In all cells, including resting inflammatory cells, HMGB1 shuttles between nucleus and cytoplasm; nuclear import is active, and the protein migrates back to the cytoplasm via passive diffusion and
XPO1 (Exportin-1)-mediated active export. When HMGB1 is underacetylated, the rate of nuclear import exceeds that of rediffusion plus export, and the protein appears predominantly or solely nuclear. Upon activation of inflammatory cells through binding of IL-1, TNF-Alpha, IFN-Gamma, LPS or HMGB1 itself to their own receptors (IL-1R, TNFR1, TLR4,
IFN-GammaR and RAGE respectively), the
(Nuclear Factor-KappaB) and MAPK (Mitogen-Activated Protein Kinase) pathways are activated. Phosphorylated
MAPKs and NF-KappaB migrate to the nucleus, where directly or via adaptor proteins they activate HATs (Histone Acetylases) or inhibit Deacetylases (Ref.6). This in turn promotes acetylation of HMGB1. Exported acetyl-HMGB1 cannot return to the nucleus. Myeloid cells are equipped with secretory Lysosomes, that can be secreted upon appropriate stimulation and that can accumulate HMGB1, presumably through specific transporters embedded in the Lysosomal membrane. Upon binding of LPC (Lysophosphatidylcholine), to its own receptor, the secretory lysosomes carrying HMGB1 fuse with the plasma membrane and secrete their cargo, that is, HMGB1 to the extracellular space. Necrotic cells release HMGB1 by simple diffusion, and thereby trigger inflammation; in contrast, apoptotic cells avidly retain HMGB1 bound to chromatin remnants even after their eventual lysis (Ref.2).
HMGB1 secreted from stimulated Monocytes, or Macrophages or released from necrotic cells can activate cell surface receptors on various cell types through
ERK1/2 (Extracellular Signal-Regulated Kinases) and two stress-activated MAPKs: JNK (c-Jun terminal Kinase) and p38;
PI3K (Phosphatidylinositde-3 Kinase) and Akt
; the transcription factors
and SP1 as well as the small GTPases: Rac1 and CDC42 (Cell Division Cycle-42) (Ref.5). Receptor signal transduction of HMGB1 occurs through the receptor RAGE (Receptor for Advanced Glycation End-products), a multiligand receptor of the immunoglobulin superfamily, expressed on Monocytes and Macrophages. Cell activation by HMGB1 results in the release of a proinflammatory cytokines and chemokines: TNF-Alpha, IL-1Alpha, IL-1Beta, IL-1RA (Interleukin-1 Receptor Antagonist), IL-6, IL-8, and MCP1 (Monocyte Chemotactic Protein-1); upregulation of adhesion molecules: ICAM1 (Intercellular Adhesion Molecule-1) and VCAM1 (Vascular Cell Adhesion Molecule-1), the HMGB1 receptor: RAGE, and HMGB1 itself. HMGB1 signaling through RAGE also occurs in Endothelial cells, Neurons and smooth-muscle cells (Ref.2). TNF-Alpha acts locally to amplify responses initiated by HMGB1. HMGB1-mediated proinflammatory responses have multiple effects at various sites. In the Brain, HMGB1-mediated proinflammatory responses are manifested by Fever, Anorexia and pain sensation. In the lungs, these facilitate Neutrophil Infiltration, Endothelia Activation, and may also culminate in Inflammation, Edema and injury. In the Intestine, HMGB1 signaling results in Loss of Epithelia barrier function and Bactericidal effects. HMGB1 signaling also plays a significant role in Arthritis and inflammation of bone joints. Administration of HMGB1 via intracerebroventricular, intratracheal, intraperitoneal and intraarticular routes induces marked inflammatory responses in an organ-specific manner, and activates various innate immune cells (Ref.7).
HMGB1-RAGE interaction recruits the plasminogen activation system and also signals
internally to actin filaments and other components involved in cell migration.
Secretion of regulators of fibrinolysis: tPA (tissue-type Plasminogen
Activator) and PAI1 (Plasminogen
Activator Inhibitor-1) results in the activation of plasmin in Macrophages
that may limit HMGB1 responses
by proteolytic degradation of HMGB1
(Ref.3). HMGB1 signals to the cell motility system by activating CDC42, Rac1 and Rho.
Convergence of HMGB1 binding
to RAGE on CDC42-Rac1-MKK6-p38 MAPK to upregulate Myogenin and MHC (Myosin Heavy Chain) expression, that accelerate myotube formation. At the lamellopodia, HMGB1 interacts with the ECM (Extracellular Matrix) and membrane receptors, affecting cell motility
and metastasis. In the developing nervous system, membrane-associated HMGB1 localizes to growth
cones and promotes neurite outgrowth and extension by binding to RAGE. In neural tissue and malignant cells, RAGE
activation by HMGB1 leads to MAPK activation and is associated with enhanced tumor growth, Metastases, and
release of MMPs
(Matrix Metalloproteinases) (Ref.8). Extracellular HMGB1 and RAGE induce both migration and proliferation of vessel-associated stem cells, and thus may play a
role in muscle tissue regeneration (Ref.2).
Release of HMGB1 from necrotic cells triggers the release of various cytokines, thereby propagating cell death and release of nuclear HMGB1. The massive release of HMGB1 from the nuclei leads to extensive cell death and septic shock (Ref.7). High serum HMGB1 levels in patients with sepsis are associated with increased mortality (Ref.3). The discovery of HMGB1 as a potent cytokine mediator of Endotoxemia and Sepsis and the widespread expression of RAGE on endothelium has initiated a new field of investigation for the development of therapeutics in the treatment of Sepsis (Ref.4). Intratracheal administration of HMGB1 produces Acute Lung Injury as manifested by Neutrophil accumulation. Administration of anti-HMGB1 antibodies inhibit systemic inflammation, even in established cases, because HMGB1 activity is elevated at significantly later time points than TNF or IL-1. HMGB1 plays a pivotal role in the pathogenesis of chronic arthritis and it may mediate strong, direct bactericidal effects. (Ref.1). The interaction between HMGB1 and RAGE has also been found to be important in tumor formation. Targeting the HMGB1 ligand or its receptor represents an important potential application in Cancer therapeutics. HMGB1 and its counter-receptor, RAGE, represent suitable targets for investigation, integrating many aspects of modern biology, particularly that associated with chronic diseases involving inflammation, dysregulated cell death and Cancer (Ref.8).