Guglielmo Sorci

S100B's double life: intracellular regulator and extracellular signal.

The Ca2+-binding protein of the EF-hand type, S100B, exerts both intracellular and extracellular functions. Recent studies have provided more detailed information concerning the mechanism(s) of action of S100B as an intracellular regulator and an extracellular signal. Indeed, intracellular S100B acts as a stimulator of cell proliferation and migration and an inhibitor of apoptosis and differentiation, which might have important implications during brain, cartilage and skeletal muscle development and repair, activation of astrocytes in the course of brain damage and neurodegenerative processes, and of cardiomyocyte remodeling after infarction, as well as in melanomagenesis and gliomagenesis. As an extracellular factor, S100B engages RAGE (receptor for advanced glycation end products) in a variety of cell types with different outcomes (i.e. beneficial or detrimental, pro-proliferative or pro-differentiative) depending on the concentration attained by the protein, the cell type and the microenvironment. Yet, RAGE might not be the sole S100B receptor, and S100Bs ability to engage RAGE might be regulated by its interaction with other extracellular factors. Future studies using S100B transgenic and S100B null mice might shed more light on the functional role(s) of the protein.

S100B expression in and effects on microglia.

We evaluated the intracellular and extracellular biological role of S100B protein with respect to microglia. S100B, which belongs to the multigenic family of Ca2+‐binding proteins, is abundant in astrocytes where it is found diffusely in the cytoplasm and is associated with membranes and cytoskeleton constituents. S100B protein is also secreted by astrocytes and acts on these cells to stimulate nitric oxide secretion in an autocrine manner. However, little is known about the relationship between S100B and microglia. To address this issue, we used primary microglia from newborn rat cortex and the BV‐2 microglial cell line, a well‐established cell model for the study of microglial properties. S100B expression was assessed by immunofluorescence in primary microglia and by RT‐PCR, Western blotting, and immunofluorescence in BV‐2 cells. S100B was found in microglia in the form of a filamentous network as well as diffusely in the cytoplasm and associated with intracellular membranes. S100B relocated around phagosomes during BV‐2 phagocytosis of opsonized Cryptococcus neoformans. Furthermore, interferon‐γ (IFN‐γ) treatment caused cell shape changes and redistribution of S100B, and downregulation of S100B mRNA expression in BV‐2 cells. Treatment of BV‐2 cells with nanomolar to micromolar amounts of S100B resulted in increased IFN‐γ–induced expression of inducible nitric oxide synthase mRNA as well as nitric oxide secretion. Taken together, these data suggest a possible role for S100B in the accomplishment/regulation of microglial cell functions. GLIA 33:131–142, 2001.

Amphoterin Stimulates Myogenesis and Counteracts the Antimyogenic Factors Basic Fibroblast Growth Factor and S100B via RAGE Binding

ABSTRACT The receptor for advanced glycation end products (RAGE), a multiligand receptor of the immunoglobulin superfamily, has been implicated in the inflammatory response, diabetic angiopathy and neuropathy, neurodegeneration, cell migration, tumor growth, neuroprotection, and neuronal differentiation. We show here that (i) RAGE is expressed in skeletal muscle tissue and its expression is developmentally regulated and (ii) RAGE engagement by amphoterin (HMGB1), a RAGE ligand, in rat L6 myoblasts results in stimulation of myogenic differentiation via activation of p38 mitogen-activated protein kinase (MAPK), up-regulation of myogenin and myosin heavy chain expression, and induction of muscle creatine kinase. No such effects were detected in myoblasts transfected with a RAGE mutant lacking the transducing domain or myoblasts transfected with a constitutively inactive form of the p38 MAPK upstream kinase, MAPK kinase 6, Cdc42, or Rac-1. Moreover, amphoterin counteracted the antimyogenic activity of the Ca2+-modulated protein S100B, which was reported to inhibit myogenic differentiation via inactivation of p38 MAPK, and basic fibroblast growth factor (bFGF), a known inhibitor of myogenic differentiation, in a manner that was inversely related to the S100B or bFGF concentration and directly related to the extent of RAGE expression. These data suggest that RAGE and amphoterin might play an important role in myogenesis, accelerating myogenic differentiation via Cdc42-Rac-1-MAPK kinase 6-p38 MAPK.

RAGE in tissue homeostasis, repair and regeneration

RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in inflammation, diabetes, atherosclerosis, nephropathy, neurodegeneration, and cancer. Advanced glycation end-products, high mobility group box-1 (amphoterin), β-amyloid fibrils, certain S100 proteins, and DNA and RNA are RAGE ligands. Upon RAGE ligation, adaptor proteins (i.e., diaphanous-1, TIRAP, MyD88 and/or other as yet unidentified adaptors) associate with RAGE cytoplasmic domain resulting in signaling. However, RAGE activation may not be restricted to pathological statuses, the receptor being involved in tissue homeostasis and regeneration/repair upon acute injury, and in resolution of inflammation. RAGE effects are strongly dependent on the cell type and the context, which may condition therapeutic strategies aimed at reducing RAGE signaling.

The Amphoterin (HMGB1)/Receptor for Advanced Glycation End Products (RAGE) Pair Modulates Myoblast Proliferation, Apoptosis, Adhesiveness, Migration, and Invasiveness FUNCTIONAL INACTIVATION OF RAGE IN L6 MYOBLASTS RESULTS IN TUMOR FORMATION IN VIVO

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGEΔcyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mocktransfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21Waf1, and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGEΔcyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGEΔcyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation.

Association of S100B with intermediate filaments and microtubules in glial cells

Previous in vitro studies have shown that the Ca2+-regulated S100B protein modulates the assembly-disassembly of microtubules (MTs) and type III intermediate filaments (IFs). In the present report, by double immunofluorescence cytochemistry S 100B was localized to both GFAP/vimentin IFs and MTs as well as to centrosomes in U251 glial cells. In cells treated with the MT-depolymerizing agent, colchicine, S100B remained associated with the rearranged GFAP IFs throughout the cell and, at the cell periphery, vimentin IFs. In cells treated with the MT stabilizing agent, taxol, S100B followed partly the rearrangement of MTs and partly the rearrangement of IFs. Under the latter condition, bundles of MTs with their associated S100B appeared surrounded and/or flanked by rearranged IFs with their associated S100B. Colocalization of S100B with closely arranged IFs and MTs was best evident in cells manipulated with taxol and in triton-cytoskeletons. In these cases, MTs and their associated S100B appeared surrounded and/or flanked by and/or intermingled with IFs and their associated S100B. Also, a preferential association of S100B with GFAP vs. vimentin IFs could be observed near the nucleus where colocalization of S100B with MTs was also maximal. Condensation of IFs and alteration of the MT network caused by treatment of cells with the phosphatase inhibitor, okadaic acid, resulted in a concomitant condensation/alteration of the S100B immunoreactivity. The present results lend support to the possibility that S100B may be an important factor implicated in the regulation of the dynamics of MTs and IFs.

S100B Protein, a Damage-Associated Molecular Pattern Protein in the Brain and Heart, and Beyond

S100B belongs to a multigenic family of Ca2+-binding proteins of the EF-hand type and is expressed in high abundance in the brain. S100B interacts with target proteins within cells thereby altering their functions once secreted/released with the multiligand receptor RAGE. As an intracellular regulator, S100B affects protein phosphorylation, energy metabolism, the dynamics of cytoskeleton constituents (and hence, of cell shape and migration), Ca2+ homeostasis, and cell proliferation and differentiation. As an extracellular signal, at low, physiological concentrations, S100B protects neurons against apoptosis, stimulates neurite outgrowth and astrocyte proliferation, and negatively regulates astrocytic and microglial responses to neurotoxic agents, while at high doses S100B causes neuronal death and exhibits properties of a damage-associated molecular pattern protein. S100B also exerts effects outside the brain; as an intracellular regulator, S100B inhibits the postinfarction hypertrophic response in cardiomyocytes, while as an extracellular signal, (high) S100B causes cardiomyocyte death, activates endothelial cells, and stimulates vascular smooth muscle cell proliferation.

S100B inhibits myogenic differentiation and myotube formation in a RAGE-independent manner.

ABSTRACT S100B is a Ca2+-modulated protein of the EF-hand type with both intracellular and extracellular roles. S100B, which is most abundant in the brain, has been shown to exert trophic and toxic effects on neurons depending on the concentration attained in the extracellular space. S100B is also found in normal serum, and its serum concentration increases in several nervous and nonnervous pathological conditions, suggesting that S100B-expressing cells outside the brain might release the protein and S100B might exert effects on nonnervous cells. We show here that at picomolar to nanomolar levels, S100B inhibits myogenic differentiation of rat L6 myoblasts via inactivation of p38 kinase with resulting decrease in the expression of the myogenic differentiation markers, myogenin, muscle creatine kinase, and myosin heavy chain, and reduction of myotube formation. Although myoblasts express the multiligand receptor RAGE, which has been shown to transduce S100B effects on neurons, S100B produces identical effects on myoblasts overexpressing either full-length RAGE or RAGE lacking the transducing domain. This suggests that S100B affects myoblasts by interacting with another receptor and that RAGE is not the only receptor for S100B. Our data suggest that S100B might participate in the regulation of muscle development and regeneration by inhibiting crucial steps of the myogenic program in a RAGE-independent manner.

The danger signal S100B integrates pathogen- and danger-sensing pathways to restrain inflammation.

Humans inhale hundreds of Aspergillus conidia without adverse consequences. Powerful protective mechanisms may ensure prompt control of the pathogen and inflammation. Here we reveal a previously unknown mechanism by which the danger molecule S100B integrates pathogen– and danger–sensing pathways to restrain inflammation. Upon forming complexes with TLR2 ligands, S100B inhibited TLR2 via RAGE, through a paracrine epithelial cells/neutrophil circuit that restrained pathogen-induced inflammation. However, upon binding to nucleic acids, S100B activated intracellular TLRs eventually resolve danger-induced inflammation via transcriptional inhibition of S100B. Thus, the spatiotemporal regulation of TLRs and RAGE by S100B provides evidence for an evolving braking circuit in infection whereby an endogenous danger protects against pathogen–induced inflammation and a pathogen–sensing mechanism resolves danger–induced inflammation.

S100B causes apoptosis in a myoblast cell line in a RAGE-independent manner

S100B, a Ca2+‐modulated protein with both intracellular and extracellular regulatory roles, is most abundant in astrocytes, is expressed in various amounts in several non‐nervous cells and is also found in normal serum. Astrocytes secrete S100B, and extracellular S100B exerts trophic and toxic effects on neurons depending on its concentration, in part by interacting with the receptor for advanced glycation end products (RAGE). The presence of S100B in normal serum and elevation of its serum concentration in several non‐nervous pathological conditions suggest that S100B‐expressing cells outside the brain might release the protein and S100B might affect non‐nervous cells. Recently we reported that at picomolar to nanomolar doses S100B inhibits rat L6 myoblast differentiation via inactivation of p38 kinase in a RAGE‐independent manner. We show here that at ≥5 nM in the absence of and at >100 nM in the presence of serum S100B causes myoblast apoptosis via stimulation of reactive oxygen species (ROS) production and inhibition of the pro‐survival kinase, extracellular signal‐regulated kinase (ERK)1/2, again in a RAGE‐independent manner. Together with our previous data, the present results suggest that S100B might participate in the regulation of muscle development and regeneration by two independent mechanism, i.e., by inhibiting crucial steps of the myogenic program at the physiological levels found in serum and by causing elevation of ROS production and myoblast apoptosis following accumulation in serum and/or muscle extracellular space. Our data also suggest that RAGE has no role in the transduction of S100B effects on myoblasts, implying that S100B can interact with more than one receptor to affect its target cells. J. Cell. Physiol. 199: 274–283, 2004© 2003 Wiley‐Liss, Inc.