The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

High mobility group proteins are chromatin binding factors with key roles in maintenance of nuclear homeostasis. The evidence indicates that extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promoting inflammation and participating to the pathogenesis of several disorders in peripheral organs. In this study, we have investigated the expression levels and relocation dynamics of HMGB1 in neural cells, as well as its neuropathological potential. We report that HMGB1 is released in the culture media of neurons and astrocytes challenged with necrotic but not apoptotic stimuli. Recombinant HMGB1 prompts induction of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2, interleukin-1b, and tumor necrosis factor a, and increases excitotoxic as well as ischemic neuronal death in vitro. Dexamethasone reduces HMGB1 dependent immune glia activation, having no effect on the protein's neurotoxic effects. HMGB1 is expressed in the nucleus of neurons and astrocytes of the mouse brain, and promptly (1 h) translocates into the cytoplasm of neurons within the ischemic brain. Brain microinjection of HMGB1 increases the transcript levels of proinflammatory mediators and sensitizes the tissue to the ischemic injury. Together, data underscore the neuropathological role of nuclear HMGB1, and point to the protein as a mediator of post-ischemic brain damage.

Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration.

HMGb1 is a nuclear protein playing a role in DNA architecture and transcription. This protein has also been shown to function as a cytokine and to stimulate keratinocyte scratch wound healing. Due to the importance of finding new wound healing molecules, we have studied the effects of HMGb1 on fibroblasts, another major skin cell type, using the NIH 3T3 line. HMGb1 expression in these cells was assessed by Western blot, while its nuclear localization was pointed out by confocal immunofluorescence. HMGb1-induced cell proliferation with a maximum at a concentration of 10 nM, and such a dose also stimulated cell migration and scratch wound healing. Western blot analysis showed that HMGb1 activates ERK1/2, while the use of an anti-RAGE receptor-blocking antibody and of the selective MEK1/2 inhibitor PD98059 blocked ERK1/2 activation and wound healing responses to HMGb1. Taken together data show that HMGb1 promotes 3T3 fibroblast wound healing by inducing cell proliferation and migration, and that this occurs through the activation of the RAGE/MEK/ERK pathway. In conclusion, HMGb1 seems a good candidate for the development of medical treatments to be used on chronic or severe wounds.

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [ 3 H]D-aspartate form gliosomes in a concentrationdependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [ 3 H]D-aspartate was independent of modifications of cytosolic Ca 2+ , but it was blocked by DL-threo-b-benzyloxyaspartate (DL-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca 2+ -independent and DL-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca 2+ . Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca 2+ . These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

HMGb1 is a DNA-binding protein whose role as an extracellular cytokine in inflammation and tissue regeneration has also been reported. Given the importance of keratinocytes in wound healing, we have studied the mechanism of action of HMGb1 on HaCaT keratinocytes during in vitro scratch wound repair. Western blot and confocal immunofluorescence microscopy showed that these cells express significant amounts of HMGb1, that the protein is prevalently localized in the nucleus, and that its release by cells is negligible. Western blot also showed that these cells express the HMGb1 receptor RAGE. Cell exposure to HMGb1 in the absence of serum resulted in a stimulation of cell proliferation and ERK1/2 activation. HMGb1 also accelerated the wound closure of scratch wounded cells and promoted cell migration, as evaluated by a transwell assay. The HMGb1-induced increases of cell proliferation, cell migration, and wound closure were abolished by the MEK inhibitor PD98059. Taken together, data show that, although HMGb1 is not released by HaCaT, when applied exogenously it can induce a marked increase of the wound repair of these cells. Data also suggest that HMGb1 acts via the RAGE/MEK/ERK pathway. These results bring scientific support to the potential application of HMGb1 in regenerative medicine.

HMGB1 (high mobility group box 1 protein) is a nuclear protein that can also act as an extracellular trigger of inflammation, proliferation and migration, mainly through RAGE (the receptor for advanced glycation end products); HMGB1-RAGE interactions have been found to be important in a number of cancers. We investigated whether HMGB1 is an autocrine factor in human glioma cells. Western blots showed HMGB1 and RAGE expression in human malignant glioma cell lines. HMGB1 induced a dose-dependent increase in cell proliferation, which was found to be RAGE-mediated and involved the MAPK/ERK pathway. Moreover, in a wounding model, it induced a significant increase in cell migration, and RAGE-dependent activation of Rac1 was crucial in giving the tumour cells a motile phenotype. The fact that blocking DNA replication with anti-mitotic agents did not reduce the distance migrated suggests the independence of the proliferative and migratory effects. We also found that glioma cells contain HMGB1 predominantly in the nucleus, and cannot secrete it constitutively or upon stimulation; however, necrotic glioma cells can release HMGB1 after it has translocated from the nucleus to cytosol. These findings provide the first evidence supporting the existence of HMGB1/RAGE signalling pathways in human glioblastoma cells, and suggest that HMGB1 may play an important role in the relationship between necrosis and malignancy in glioma tumours by acting as an autocrine factor that is capable of promoting the growth and migration of tumour cells.

Several evidences suggest that NK cells can patrol the body and eliminate tumors in their initial phases but may hardly control established solid tumors. Multiple factors, including the transition of tumor cells towards a proinvasive/prometastatic phenotype, the immunosuppressive effect of the tumor microenvironment, and the tumor structure complexity, may account for limited NK cell efficacy. Several putative mechanisms of NK cell suppression have been defined in these last years; conversely, the cross talk between NK cells and tumor cells undergoing different transitional phases remains poorly explored. Nevertheless, recent in vitro studies and immunohistochemical analyses on tumor biopsies suggest that NK cells could not only kill tumor cells but also influence their evolution. Indeed, NK cells may induce tumor cells to change the expression of HLA-I, PD-L1, or NKG2D-L and modulate their susceptibility to the immune response. Moreover, NK cells may be preferentially located in the borders of tumor masses, where, indeed, tumor cells can undergo Epithelial-to-Mesenchymal Transition (EMT) acquiring prometastatic phenotype. Finally, the recently highlighted role of HMGB1 both in EMT and in amplifying the recruitment of NK cells provides further hints on a possible effect of NK cells on tumor progression and fosters new studies on this issue.