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

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

Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through receptor for advanced glycation end products (RAGE) activation.

Amphoterin is a protein enhancing process extension and migration in embryonic neurons and in tumor cells through binding to receptor for advanced glycation end products (RAGE), a multiligand transmembrane receptor. S100 proteins, especially S100B, are abundantly expressed in the nervous system and are suggested to function as cytokines with both neurotrophic and neurotoxic effects. However, the cell surface receptor for the cytokine function of S100B has not been identified. Here we show that two S100 family proteins, S100B and S100A1, activate RAGE in concert with amphoterin inducing neurite outgrowth and activation of transcription factor NF-kappaB. Furthermore, activation of RAGE by amphoterin and S100B promotes cell survival through increased expression of the anti-apoptotic protein Bcl-2. However, whereas nanomolar concentrations of S100B induce trophic effects in RAGE-expressing cells, micromolar concentrations of S100B induce apoptosis in an oxidant-dependent manner. Both trophic and toxic effects are specific for cells expressing full-length RAGE since cells expressing a cytoplasmic domain deletion mutant of RAGE are unresponsive to these stimuli. These findings suggest that activation of RAGE by multiple ligands is able to promote trophic effects whereas hyperactivation of RAGE signaling pathways promotes apoptosis. We suggest that RAGE is a signal-transducing receptor for both trophic and toxic effects of S100B

S100B's double life: Intracellular regulator and extracellular signal

AbstractThe 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 S100B's 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

Absence of RAGE in an animal experimental model of Duchenne muscular dystrophy results in reduced muscle necrosis and inflammation

Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder characterized by progressive muscle degeneration due to lack of dystrophin, a protein essential for the integrity of sarcolemma during contraction. Chronic inflammation is a hallmark of muscles in DMD subjects, and contributes to progressive muscle wasting. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in physiological and pathological processes including inflammation and myogenesis [1]. While absent in healthy adult muscle tissue, RAGE is expressed in regenerating myofibers during muscle regeneration [2,3], in dystrophic muscles and activated immune cells. To have information about the role of RAGE in the pathophysiology of DMD we generated a double mutant mouse lacking dystrophin and RAGE (mdx/Ager–/– mouse) by cross-breeding dystrophic (mdx) mice with RAGE-null (Ager-/-) mice. Comparison of Quadriceps femoris of mdx and mdx/Ager–/– mice at different ages (i.e., 2, 3, 4 and 5 weeks, and 6 and 12 months of age) showed that the absence of RAGE in dystrophic mice did not affect the onset of the pathology. However, compared with age-matched mdx mice, muscles of 5 week- and 6 and 12 month-old mdx/Ager–/– mice showed i) significantly reduced numbers of necrotic myofibers, ii) a shift towards higher values of the cross-sectional areas (CSA) of myofibers, which was also evident in regenerating (centrally-nucleated) myofibers, and iii) reduced areas of immune cell infiltrate. The expression of MAC3, a marker of activated macrophages, was strongly reduced in muscles of mdx/Ager–/– mice compared with mdx mice. Moreover, muscles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell numbers, suggesting a reduced recruitment of muscle precursor cells and more efficient regeneration in dystrophic mice lacking RAGE. Our results suggest that RAGE may sustain inflammatory and degenerative processes in dystrophic muscles, and the inhibition of its expression/activity might represent a potential therapeutic approach in DMD patients.This work was supported by grants from MIUR 2012N8YJC3, AFM-Téléthon 16812 and Fondazione CRP 2015.0325.021

S100B protein regulates myoblast and macrophage functions in skeletal muscle regeneration

Regeneration of acutely injured skeletal muscles relies on a tightly controlled chain of cellular and molecular events, but a complete picture of factors concurring to the regeneration process is still missing. Extracellular S100B protein inhibits myoblast differentiation and stimulates myoblast proliferation by activating its canonical receptor, RAGE (receptor for advanced glycation endproducts), or bFGF/FGFR1 depending on myoblast density (1-4). S100B is released by damaged muscle tissue early after injury in advance of bFGF release, with declining release thereafter (4). We show that S100B is required for correct timing of skeletal muscle regeneration after acute injury. S100B expands the myoblast population, attracts macrophages to damage sites, promotes macrophage polarization into M2 (pro-regenerative) phenotype and reduces fibroblast proliferation. Also, S100B is transiently induced in and released by infiltrating macrophages under the action of proinflammatory and antiinflammatory cytokines, and effects of macrophage-derived S100B sum up with those of myofiber-released S100B. S100B’s effects are mediated by RAGE during the first 3 days after injury, however during the myoblast proliferation phase/macrophage M2 phase (i.e. at days 4-6 post-injury) S100B also activates bFGF-FGFR1 to stimulate myoblast proliferation and macrophage M1/M2 transition. Thus, S100B is a major molecular determinant of timed muscle regeneration after acute injury by virtue of its regulatory effects on myoblasts and macrophages.This work was supported by grants from MIUR PRIN-2010R8JK2X_004, AFM-Téléthon 16260 and Fondazione CRP 2012.0241.021

THE CALCIUM-MODULATED PROTEINS, S100A1 AND S100B, AS POTENTIAL REGULATORS OF THE DYNAMICS OF TYPE III INTERMEDIATE FILAMENTS

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities

An ultrastructural study of Sertoli cells inside alginate microcapsules

Sertoli cells (SeC) are the main components of the blood-testis barrier, are essential for spermatogenesis and are long known for their ability to secrete trophic, anti-inflammatory and immunomodulatory factors [1]. For these reasons, SeC have been encapsulated in sodium alginate microcapsules and then used to create an ectopic immune-privileged environment to prolong survival of co-transplanted cells or modulate the immune responses [2]. Encapsulation has represented an improvement for the use of SeC. In fact, it has been reported that inside the microcapsules SeC (SeC-MC) act as a “micro-biofactory” and drug delivery system. By secreting immunomodulatory and trophic factors once injected into the peritoneal cavity of dystrophic mice [3], they can ameliorate muscle morphology and function. Since the manipulation of the microcapsules is rather complicated, we performed for the first time, an ultrastructure study on SeC-MC. The good cell morphology, along with viability of organellar compartment, was demonstrated

The many faces of S100B protein: when an extracellular factor inactivates its own receptor and activates another one

The Ca2+-binding protein of the EF­hand type, S100B, is an intracellular regulator and an extracellular signal. Within cells S100B interacts with several proteins thereby regulating energy metabolism, Ca2+ homeostasis, protein phosphorylation and degradation, and cell locomotion, proliferation and differentiation. Once secreted/released, S100B exerts autocrine and paracrine effects on responsive cells by engaging the receptor for advanced glycation end products. However, recent evidence suggests that S100B might also activate basic fibroblast growth factor receptor 1 via prior binding to basic fibroblast growth factor