Bianca Sparatore

Cutting Edge: Extracellular High Mobility Group Box-1 Protein Is a Proangiogenic Cytokine

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 box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo

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‐1β, and tumor necrosis factor α, 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 pro‐inflammatory 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.

Cytosolic calcium dependent proteinase of human erythrocytes: Formation of an enzyme-natural inhibitor complex induced by Ca2+ ions

Abstract A calcium dependent soluble neutral proteinase has been purified to homogeneity from human erythrocytes. The proteinase is composed of two different polypeptide chains of approximate molecular weight of 80 k and 30 k daltons. Maximum activity is expressed at 50 μM Ca 2+ . The enzyme is regulated by reversible binding to a natural inhibitor, also present in the cytosolic compartment. The formation of the enzyme-inhibitor complex is dependent on high Ca 2+ concentrations and is reversed by chelating agents. The proteinase is inhibited by leupeptin, chymostatin, antipain and free hemin and has a marked specificity for native or denatured human globin chains.

Selective Proinflammatory Activation of Astrocytes by High-Mobility Group Box 1 Protein Signaling

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.

Stimulation of excitatory amino acid release from adult mouse brain glia subcellular particles by high mobility group box 1 protein

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 [3H]d‐aspartate form gliosomes in a concentration‐dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1‐evoked release of [3H]d‐aspartate was independent of modifications of cytosolic Ca2+ , but it was blocked by dl‐threo‐β‐benzyloxyaspartate (dl‐TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca2+‐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 Ca2+. 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 Ca2+. These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

HMGB1 as an autocrine stimulus in human T98G glioblastoma cells: role in cell growth and migration

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.

The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.

Binding to erythrocyte membrane is the physiological mechanism for activation of Ca2+-dependent neutral proteinase

In the presence of micromolar concentrations of Ca2+ the catalytic 80 kDa subunit of human erythrocyte procalpain binds to the cytosolic surface of the erythrocyte membrane. Binding is rapid, highly specific and is reversed by the removal of Ca2+. In the bound form the 80 kDa catalytic subunit undergoes a rapid conversion to calpain, the active 75 kDa Ca2+-requiring proteinase. The activated proteinase produces extensive degradation of membrane components, particularly of band 4.1 and 2.1 proteins. Binding to membranes may represent an obligatory physiological mechanism for the conversion of procalpain to calpain.

Acyl-CoA-binding Protein Is a Potent m-Calpain Activator

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed inEscherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca2+] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of μ-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca2+-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

The calpain-calpastatin system in mammalian cells: properties and possible functions

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell. Both calpain forms are heterodimers composed of a heavy subunit (80 kDa) that contains the catalytic site and a smaller (regulatory?) subunit (30 kDa). Calpain I expresses full activity at 10-50 microM Ca2+, whereas calpain II requires calcium concentrations in the millimolar range. The removal by autoproteolysis of a fragment from the N-terminus of both calpain subunits generates a proteinase form that can express catalytic activity at concentrations of Ca2+ close to the physiological range. This process is significantly accelerated in the presence of cell membranes or phospholipid vesicles. Calpastatin, the specific inhibitor of calpain, prevents activation and the expression of catalytic activity of calpain. It is in itself a substrate of the proteinase and undergoes a degradation process which correlates with the general mechanism of regulation of the intracellular proteolytic system. The natural calpain activator specifically acts on calpain II isoform, by reducing the Ca2+ required for the autoproteolytic activation process. Based on the general properties of the calpain-calpastatin system and on the substrate specificity, its role in the expression of specific cell functions can be postulated.