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

Oxidative stress-induced S100B accumulation in myoblasts converts myoblasts into brown preadipocytes via an NF-κB/YY1/MIR-133 axis and NF-κB/YY1/BMP7 axis

Muscles of sarcopenic people show hypotrophic myofibers and infiltration with adipose and, at later stages, fibrotic tissue. The origin of infiltrating adipocytes resides in fibro-adipogenic precursors, nonmyogenic mesenchymal progenitor cells, and satellite cells, the adult stem cells of skeletal muscles. Myoblasts and brown adipocytes share a common Myf5+ progenitor cell, and cell fate decision depends on levels of BMP7, a TGF-β family member; high BMP7 levels cause Myf5+ progenitor cells to differentiate in brown adipocytes. When expressed at relatively high levels as observed in myoblasts from sarcopenic humans, intracellular S100B, a Ca2+-binding protein of the EF-hand type (1), exerts anti-myogenic effects that are reversed by S100B knockdown (2,3). We show that ROS-activated NF-κB induces accumulation of S100B that causes myoblasts to convert into brown preadipocytes via 1) an NF-κB/YY1 axis that negatively regulates the promyogenic and anti-brown adipogenic miR-133 with consequent accumulation of the pro-brown adipogenic transcription factor, PRDM16, and 2) an NF-κB/YY1/BMP7 axis with resultant BMP7 autocrine activity. Also, culturing L6C8 (S100b-overexpressing) myoblasts (2) in adipocyte differentiation medium causes NF-κB-dependent upregulation of S100B expression, which precedes and is required for lipid droplet formation. Lastly, S100B knockdown in myoblast-derived brown adipocytes reconvert them into fusion competent myoblasts. Thus, S100B is a major molecular determinant of cell fate decision of proliferating myoblasts; while modulating myoblast differentiation (2,3), at high levels S100B promotes myoblast-brown adipocyte transition, which might have pathophysiological implications in sarcopenia.This work was supported by grants from MIUR FIRB RBFR12BUMH_003 and Fondazione CRP 2016.0136.021

Proposed schematics of S100B/RAGE and RAGE/S100B/bFGF/FGFR1 interactions in LD and HD myoblasts and of action of released S100B following skeletal muscle injury.

<p>(<b>A</b>) (Left) In LD myoblast cultures S100B tetramers/octamers promote RAGE oligomerization and/or stabilization of RAGE oligomers resulting in the simultaneous stimulation of proliferation and activation of the myogenic program; this occurs irrespective of the absence or presence of bFGF. At high concentrations, high-order S100B oligomers bind bFGF and the S100B/bFGF complex cross-links RAGE and FGFR1 on the same cell. This results in enhancement of FGFR1 signaling and blockade of RAGE signaling likely due to lack of RAGE oligomerization and/or stabilization of RAGE oligomers. However, in the absence of bFGF either low or high S100B engages RAGE. (Right) In HD myoblast cultures, S100B tetramers/octamers bind bFGF and the S100B/bFGF complex cross-links RAGE and FGFR1 on apposed cells resulting in blockade of RAGE signaling likely due to lack of RAGE oligomerization and/or stabilization of RAGE oligomers and enhancement of FGFR1 signaling. This results in stimulation of proliferation and inhibition of differentiation. (<b>B</b>) Early after injury S100B mainly engages RAGE on activated satellite cells thereby stimulating their proliferation and activating the myogenic program. Whether S100B activates satellite cells remains to be investigated (?). During the proliferation phase S100B binds to both, RAGE and bFGF/FGFR1 and promotes the formation of a RAGE/S100B/bFGF/FGFR1 tetracomplex thereby stimulating myoblast proliferation and blocking RAGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028700#pone.0028700-Riuzzi1" target="_blank">[9]</a>. At this stage HMGB1 might compete with S100B for binding to RAGE thereby unlocking RAGE and stimulating its promyogenic activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028700#pone.0028700-Sorci2" target="_blank">[12]</a>. Later on, with diminishing S100B levels RAGE would be activated by HMGB1 thereby promoting myoblast differentiation and myocyte fusion into myofibers.</p

S100B Engages RAGE or bFGF/FGFR1 in Myoblasts Depending on Its Own Concentration and Myoblast Density. Implications for Muscle Regeneration

<div><p>In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density.</p> </div

S100B engages RAGE in LD wild-type myoblasts and activates FGFR1 in <i>Rage</i>^−/− myoblasts.

<p>(<b>A</b>) Primary myoblasts from WT and <i>Rage</i>^−/− mice in DM were treated for 30 min with 1 nM S100B and subjected to immunoprecipitation using an anti-S100B antibody. S100B immunoprecipitates were analyzed for RAGE, FGFR1, bFGF and c-Met levels. (<b>B</b>) LD WT and <i>Rage</i>^−/− myoblasts were cultivated in DM for 30 min in the absence or presence of either 1 nM S100B or 1 nM bFGF. Cells were lysed and cell lysates were subjected to Western blotting for detection of phosphorylated Tyr and FGFR1. (<b>C</b>) WT and <i>Rage</i>^−/− myoblasts cultivated in DM for 24 h in the absence or presence of 1 nM S100B were subjected to BrdU incorporation assay. (<b>D</b>) WT and <i>Rage</i>^−/− myoblasts were cultivated in DM in the absence or presence of 1 nM S100B for 24 or 72 h without medium renewal. One set of cells was washed and further cultivated for 48 h in DM without additions. At the end of treatments, cells were lysed and cell lysates were subjected to Western blotting to detect myogenin (as a myogenic differentiation marker). *Significantly different from control (first column from left). ^#Significantly different from internal control (second column from left).</p

S100B/RAGE and RAGE/FGFR1 complexes in regenerating skeletal muscle tissue.

<p>(<b>A</b>) Uninjured and injured skeletal muscle tissue was subjected to in situ PLA using anti-S100B/anti-RAGE antibody pairs (left) or anti-RAGE/anti-FGFR1 antibody pairs (right). Myofiber boundaries in transverse sections are marked by dotted lines. No S100B/RAGE or RAGE/FGFR1 complexes can be detected in uninjured skeletal muscle tissue. Arrows point to RAGE/FGFR1 complexes at apposed myoblasts. RAGE/FGFR1 complexes actually represent RAGE/S100B/bFGF/FGFR1 - see text). Bars = 200 µm. (<b>B</b>) Quantitative analysis of the distribution of S100B/RAGE and RAGE/FGFR1 complexes in uninjured skeletal muscle tissue and at the indicated days post-injury. In situ complexes were counted in 10 fields (×20 magnification)/section in a total of 10 sections/time point from 2 animals. *Significantly different.</p

S100B, bFGF and HMGB1 are released from injured muscles.

<p>(<b>A</b>) CM from uninjured or BaCl2-injured <i>Tibialis anterior</i> muscles were processed for detection of S100B, bFGF and HMGB1 by Western blotting. S100B, bFGF and HMGB1 in CM were quantified by comparing the density of individual western blot bands in the CM with that of increasing amounts of each purified protein in parallel western blots. (<b>B</b>) After collection of CM, muscle tissue was homogenized and subjected to Western blotting for detection of RAGE and FGFR1 (20 µg protein loaded/lane). For experiments in A and B three animals/time point were used and the whole experiment was performed two times. Results in A and B are expressed as means ± SD. C, Freshly excised uninjured or BaCl₂-injured <i>Tibialis anterior</i> muscles were processed for detection of Pax7 and RAGE by double-immunofluorescence. Nuclei were counterstained with DAPI. RAGE is not expressed in uninjured tissue. Arrow points to a quiescent satellite (Pax7⁺) cell (a,a′) and to a Pax7⁺/RAGE⁺ myoblast outside regenerating myofibers (b,b′). Arrowhead points to a Pax7⁺/RAGE⁺ myoblast within a regenerating myofiber (b,b′). Bars = 100 µm.</p

S100B effects on differentiation vary depending on myoblast density and the duration of treatment with S100B.

<p>(<b>A</b>) LD myoblasts were cultivated in DM in the absence or presence of S100B for 24 h, washed and cultivated for another 24 h in DM with no additions before MyHC immunostaining. (<b>B</b>) Same as in A except that LD myoblasts were subjected to Western blotting for detection of MyHC, M-cadherin and caveolin-3 at 24 h after washout (<i>n</i> = 3). (<b>C</b>) LD C2C12 myoblasts were cultivated for 1 h in DM with the p38 inhibitor, SB203580 (2 µM) or the MEK-ERK1/2 inhibitor, PD90059 (20 µM) and then for 24 h in the absence or presence of S100B, and subjected to Western blotting at 24 h after washout using anti-myogenin antibody (<i>n</i> = 3). (<b>D</b>) Same as in A except that LD myoblasts were treated with bFGF for 24 h and immunostained for MyHC at 24 and 48 h after bFGF washout. (<b>E</b>) LD myoblasts were cultivated in DM for 1, 24 or 72 h in the absence or presence of S100B and analyzed for ERK1/2 and p38 phosphorylation by Western blotting (<i>n</i> = 3). (<b>F</b>) LD myoblasts were cultivated in DM for 72 h in the absence or presence of S100B and subjected to MyHC immunostaining. *Significantly different from control. ^#Significantly different from internal control. Bars = 100 µm (A,F).</p

RAGE, but not bFGF/FGFR1 co-immunoprecipitates with S100B in LD myoblasts at early differentiation stages.

<p>(<b>A</b>) LD myoblasts cultivated in DM for 30 min in the presence of 1 nM S100B were subjected to immunoprecipitation using anti-S100B, anti-FGFR1, anti-bFGF or anti-RAGE antibody. The immunoprecipitates were subjected to Western blotting for detection of S100B, FGFR1, bFGF, or RAGE. In control samples non-immune IgG (IP-IgG) were used. Twenty µg protein were loaded in input lanes. (<b>B</b>) LD myoblasts were cultivated in DM for 30 min in the absence or presence of either 1 nM S100B or 1 nM bFGF after pretreatment with IgG (10 µg/ml) or anti-RAGE antibody (10 µg/ml). Cells were lysed and cell lysates were subjected to Western blotting for detection of phosphorylated Tyr and FGFR1. (<b>C</b>) LD myoblasts cultivated in DM for 2 h in the presence of either non-immune IgG (10 µg/ml) or anti-RAGE antibody (10 µg/ml). Cultures were then added with 1 nM S100B for 30 min and subjected to immunoprecipitation using anti-S100B antibody. Immunoprecipitates were probed by Western blotting with anti-S100B, anti-FGFR1, anti-bFGF or anti-RAGE antibody. The WB:FGFR1 filter was reprobed with anti- phosphorylated Tyr antibody (marked by the “[” symbol). (<b>D</b>) LD myoblasts were cultivated in DM for 3 days in the presence of 1 nM S100B without medium renovation and subjected to immunoprecipitation using anti-S100B antibody. The immunoprecipitates were probed by Western blotting with anti-S100B, anti-FGFR1, anti-bFGF or anti-RAGE antibody. (<b>E</b>) LD myoblasts were cultivated in DM for 3 days in the presence of 1 nM S100B without medium renovation and counted. *Significantly different from control.</p