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. S100B, a member of a multigenic family of Ca 2ϩ -regulated proteins of the EF-hand type, is highly abundant in astrocytes and is expressed in relatively large amounts in a variety of nonneural cell types (for reviews, see references 12, 13, and 36). Besides being implicated in the Ca 2ϩ -dependent regulation of several intracellular activities (12, 13, 36), S100B is released by astrocytes into the extracellular space (45) and is also found in serum (13). S100B has been shown to interact with neurons, astrocytes, and microglia and to exert various effects on these cells depending on its concentration. S100B has also been shown to enhance neuronal survival and stimulate neurite outgrowth and astrocyte proliferation at nanomolar concentrations (2,3,6,7,16,20,21,23,39,47) and to cause neuronal and astrocyte apoptosis and stimulate interleukin-6 secretion by neurons and nitric oxide release by astrocytes and microglia at micromolar concentrations (1, 18-20, 25, 27, 31). Therefore, S100B has been hypothesized to play roles in brain development and neuronal protection (2,3,6,7,16,20,21,23,39,47) and in the pathophysiology of neurodegenerative disorders (15,20,30,40,41), depending on the concentration attained in the extracellular space. Trophic effects of S100B on neurons have been shown to depend on activation of the transcription factor NF-B (3). The receptor for advanced glycation end products (RAGE), a multiligand receptor of the immunoglobulin superfamily (for reviews, see references 37 and 38), has been shown to bind S100B (17) and to mediate the effects of both low and high levels of S100B on a neuronal cell line (20...

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

“…While in other cell types responsive to S100B such as neurons and microglia the receptor for advanced glycation end products (RAGE) was demonstrated to bind S100B and to transduce the regulatory effects of S100B (20,23) and while myoblasts were observed to express RAGE (mRNA and protein) constitutively (51), no such role for RAGE could be documented in the case of the inhibitory effects of S100B on myoblasts. In fact, S100B inhibited myoblast differentiation and myotube formation in L6 myoblasts stably overexpressing either full-length RAGE (L6/RAGE myoblasts) or a RAGE mutant lacking the cytoplasmic and transducing domain (L6/ RAGE⌬cyto myoblasts) (51). Collectively, these data suggested that S100B negatively regulated myogenesis in vitro by binding to a receptor other than RAGE.…”

“…Total cytoplasmic RNA was isolated from L6 myoblasts by the Trizol reagent method. Subsequent steps were done as described previously (51). After amplification, samples (10 l) of each PCR mixture were electrophoresed on a 1.2% agarose gel and reverse transcription-PCR (RT-PCR) products were revealed by ethidium bromide staining.…”

“…Rat L6 myoblasts (clone L6C31) were cultured for 24 h in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (FBS; Life Technologies), 100 U of penicillin/ml, and 100 g of streptomycin/ml, in an H 2 O-saturated 5% CO 2 atmosphere at 37°C before decreasing FBS to 2% to induce myoblast differentiation and myotube formation. L6 myoblast clones stably overexpressing RAGE or RAGE⌬cyto were selected and characterized as described previously (51). Experiments were performed on wild-type (WT), mock-transfected, L6/RAGE, and L6/RAGE⌬cyto myoblasts in 2% FBS unless stated otherwise.…”

“…Recently, we found that S100B, a member of a multigenic family of Ca 2ϩ -modulated proteins of the EF-hand type with both intracellular and extracellular regulatory activities (11,19), inhibited myoblast differentiation and myotube formation when administered to the rat myoblast cell line L6 (51). Inhibition of myogenesis was registered at picomolar doses of S100B and was reversible, pointing to S100B binding to a cell surface receptor with a relatively high affinity.…”

“…While the cell surface receptor transducing the effects of S100B on myoblasts remains to be identified, these data indicate that RAGE may not transduce the activity of myoblast differentiation inhibitory factors. Actually, in the course of these studies (51) we ob-served that overexpression of RAGE, but not of RAGE⌬cyto, enhanced myoblast differentiation and myotube formation, suggesting the possibility that RAGE, likely activated by a factor contained in the culture medium, might play a role in myogenesis, transducing a promyogenic signal.…”