Edon Melloni

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.

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.

MOLECULAR AND FUNCTIONAL PROPERTIES OF A CALPAIN ACTIVATOR PROTEIN SPECIFIC FOR MU -ISOFORMS

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ fromHaemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the μ-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on μ-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re‐sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia‐specific proteins glial fibrillary acidic protein (GFAP) and S‐100, but not the neuronal proteins 95‐kDa postsynaptic density protein (PSD‐95), microtubule‐associated protein 2 (MAP‐2) and β‐tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin‐αM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1–5 µm) efficiently stimulated the release of tritium from gliosomes pre‐labelled with [3H]d‐aspartate and of endogenous glutamate in a Ca2+‐dependent and bafilomycin A1‐sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca2+‐dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d‐aspartate release in a concentration‐ (0.1–3 mm) and Ca2+‐dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin‐1, vesicular‐associated membrane protein type 2 (VAMP‐2), 23‐kDa synaptosome‐associated protein (SNAP‐23) and 25‐kDa synaptosome‐associated protein (SNAP‐25)] co‐existing with GFAP immunoreactivity. Moreover, GFAP or VAMP‐2 co‐expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several ∼30‐nm non‐clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate‐accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

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.

Stimulated astrocytes release high-mobility group 1 protein, an inducer of LAN-5 neuroblastoma cell differentiation

Stimulated astrocytes specifically release large amounts of high-mobility group 1 protein into the extracellular medium. The identity of the released protein has been established on the basis of its biological activity on murine erythroleukaemia cells and by its immunoreactivity against a specific monoclonal antibody. High-mobility group 1 protein also plays an essential role in differentiation of LAN-5 neuroblastoma cells which, following stimulation with retinoic acid, express high-mobility group 1 protein on to the external surface of the plasma membrane. In retinoic acid-induced LAN-5 cells, high-mobility group 1 protein is not secreted but is accumulated in a membrane-bound form, particularly at the level of neurite outgrowths. These cells can also be induced to differentiate by high-mobility group 1 protein coated on the surface of the cell culture vessels. The specific function of the protein in this process is indicated by inhibition of cell differentiation by an anti-high-mobility group 1 protein antibody. The data are consistent with a role of high-mobility group 1 protein in promoting cell-cell interactions and in the development of nerve tissues.

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.

Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization

The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E‐64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca2+, membrane‐bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+‐induced translocation to the inner surface of plasma membranes. In the membrane‐bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

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.