38 research outputs found
S100B protein accelerates the activation of quiescent myoblasts and muscle satellite cells.
No full textS100B, a Ca2+-binding protein of the EF-hand type that exerts both intracellular and extracellular regulatory activities (1), is expressed in mature skeletal myofibers (2). Extracellular S100B modulates myoblast differentiation by interfering with the activation of p38 MAPK, thereby inhibiting the expression of myogenin (3), a muscle-specific transcription factor essential for myogenesis. S100B also stimulates the proliferation of myoblasts and reduces their apoptosis (4). Thus, S100B might contribute to embryonic myogenesis and skeletal muscle regeneration by activating myoblasts and satellite cells (SCs), respectively, and stimulating their proliferation. However, effects of S100B appear to be dependent on myoblast density, the S100B concentration and the duration of exposure of myoblasts to the protein as well as on the differential engagement of RAGE (receptor for advanced glycation end products) and bFGF receptor 1 (FGFR1) in low-density (LD) and high-density (HD) cultures, respectively (submitted for publication). The differential effects of S100B in LD and HD myoblasts are dependent on S100B/bFGF complex formation and S100B/bFGF engagement of FGFR1, in HD, but not LD myoblasts. S100B also binds to RAGE in HD myoblasts, but this interaction results in the blockade of RAGE signaling. By contrast, S100B only engages RAGE in LD myoblasts, the protein being unable to interact with FGFR1-bound bFGF in this condition. We addressed the question whether S100B might affect skeletal muscle tissue in vivo. We show here that: 1) damaged muscles release S100B; 2) S100B in crushed-muscle extract stimulates myoblast proliferation; 3) intramuscular injection of S100B results in activation of SCs in wild-type, but not RAGE-/- mice; 4) S100B accelerates the activation of quiescent primary myoblasts thereby stimulating their migration and proliferation; and 5) when administered for 24 h to LD myoblast/myotube cultures three days after their switch to differentiation medium, S100B rapidly stimulates the proliferation of non-fused, quiescent myoblasts such that after S100B removal more numerous and hypertrophic myotubes form during the next few days, compared to controls. Our data strongly support the possibility that S100B might physiologically participate in the muscle regeneration process by activating SCs and stimulating their migration and expansion, and that clearance of S100B results in enhanced myoblast differentiation.
1. Donato R et al (2008) Biochim Biophys Acta, DOI: 10.1016/j.bbamcr.2008.11.009; 2. Arcuri C et al (2002) Neuroscience 109:371-88; 3. Sorci G et al (2003) Mol Cell Biol 23:4870-81; 4. Riuzzi F et al (2006) J Cell Physiol 207:461-70
S100B protein differentially regulates myoblast differentiation via direct binding to RAGE and bFGF-mediated activation of FGFR1 in low-density and high-density cultures, respectively
No full textS100B protein (1) promotes myoblast proliferation and survival and inhibits myoblast differentiation (2,3). We have addressed the questions: 1) whether S100B might participate in myogenesis by virtue of its ability to stimulate myoblast proliferation; and 2) what receptor transduces S100B’s effects in myoblasts. We show here that: 1) clearance of S100B results in enhanced myoblast differentiation in consequence of S100B-induced increase in the cell number; 2) the overall S100B effect on C2C12 and rat and mouse primary myoblasts in differentiation medium varies depending on cell density, the S100B concentration and the duration of treatment of myoblasts with the protein; 3) S100B differentially activates RAGE (receptor for advanced glycation end products) and bFGF receptor 1 (FGFR1) depending on the same parameters as above; 4) S100B interacts with bFGF in high-density (HD) but not low-density (LD) myoblasts; and 5) the S100B/bFGF adduct recruits RAGE and FGFR1 into a RAGE/S100B/bFGF/FGFR1 (trans)complex in apposed cells with resultant blockade of RAGE’s promyogenic signaling and enhancement of FGFR1’s anti-myogenic signaling. We also show that in RAGE-/- primary myoblasts S100B engages FGFR1 via prior interaction with bFGF irrespective of myoblast density, thereby stimulating myoblast proliferation and inhibiting differentiation. The complex regulatory effects of S100B on myoblast differentiation are highlighted by the S100B’s ability to upregulate FGFR1 expression in HD, but not LD myoblasts, by prior complex formation with bFGF, and enhancement of bFGF’s ability to upregulate FGFR1, and to upregulate RAGE expression in LD, but not HD myoblasts in a RAGE-dependent manner. We propose that S100B participates in the myogenic process by differentially activating RAGE and FGFR1in a cell density- and bFGF-dependent manner: whenever an S100B/bFGF complex can form, the resultant enhancement of FGFR1’s mitogenic activity and blockade of RAGE’s promyogenic activity might result in expansion of the myoblast population. By contrast, S100B-dependent RAGE activation in LD myoblasts might help to reduce the fraction of activated myoblasts undergoing quiescence and to increase the fraction of proliferating myoblasts; the myoblast’s final destiny would be dependent on whether sufficient amounts of S100B persist (in which case myoblasts keep proliferating) or S100B is being cleared (in which case myoblasts reduce their proliferation rate and differentiate and/or attain a quiescent status).
1. Donato R et al (2008) Biochim Biophys Acta, DOI: 10.1016/j.bbamcr.2008.11.009; 2. Sorci G et al (2003) Mol Cell Biol 23:4870-81; 3. Riuzzi F et al (2006) J Cell Physiol 207:461-70
Involvement of a RAGE/p38MAPK/myogenin axis in cancer cachexia.
No full textCachexia is a highly debilitating multifactorial syndrome affecting more than 50% of patients with advanced
cancer, characterized by severe muscle wasting leading to pronounced weight loss, impaired quality of life,
reduced response to anti-cancer therapy, and premature death. Inflammatory cytokines, such as TNFα are
the main atrophy-inducing factors in cachexia causing excess catabolism of myofibrillary proteins through
activation of the ubiquitin-proteasome systems (UPS)1
.
An unexpected connection between the muscle-specific transcription factor, myogenin, and the induction of
atrogenes expression in different atrophying conditions (including TNFα-induced atrophy) has been
reported2,3. However, the receptor able to upregulate myogenin expression in atrophying conditions has not
been identified yet. We demonstrated that an appropriate recruitment of RAGE (receptor for advanced
glycation-end products) by its ligands, S100B and HMGB1, concurs to skeletal muscle development and
restoration of muscle homeostasis in physiological conditions and upon acute muscle injury4
. Here, we
investigated whether RAGE might up-regulate the expression of myogenin via p38 MAPK pathway in
atrophying conditions, as in the case of myoblasts, and lead to activation of the catabolic program. We found
that: i) Lewis lung carcinoma (LLC)-bearing mice re-express RAGE in myofibers and myogenin in myonuclei;
ii) muscles of LLC-bearing RAGE-null (Ager‒/‒) mice show reduced loss of mass and reduced Fbxo32
(atrogin1), Trim63 (MuRF1) and myogenin expression compared with LLC-bearing WT mice; iii) the
upregulation of Ager in atrophying C2C12 myotubes precedes the increase in Myog (myogenin), Fbxo32 and
Trim63 levels; iv) RAGE signaling is involved in the mechanism through which TNFα induces atrophy in vitro
(i.e., upregulation of myogenin via activation of the catabolic kinase, p38 MAPK); and v) high doses of S100B,
as found in the serum of cachectic mice, induce up-regulation of RAGE and myogenin expression with
concomitant activation of p38 MAPK and induction of the UPS in myotubes and in muscle tissue. Thus,
increased expression/activity of the RAGE/ p38 MAPK/myogenin axis in muscle tissue appears to concur to
cancer cachexia.
1. Porporato P.E., 2016, Oncogenesis 22(5): e200;
2. Moresi V. et al., 2010, Cell 143: 35-34;
3. Minetti G.C. et al., 2011, Sci Sign 4: ra80;
4. Riuzzi F. et al., 2012, J Cell Sci 125:1440-1454
Sertoli cell-secreted factors have promyogenic and antifibrotic properties on human DMD myoblasts with different mutations.
No full textDuchenne muscular dystrophy (DMD) is a recessive X-linked lethal disease affecting one over 5,000 live male births in which mutations in the dystrophin gene (DMD) lead to lack of a functional protein resulting in susceptibility of myofibers to rupture during contraction. Muscles of DMD patients or experimental models of DMD show progressive necrosis of the myofibers, chronic inflammation and reactive regeneration, which lead to exhaustion of muscle precursor cell pool and replacement of myofibers with fibrous and fatty tissues.1 Although multiple therapeutic approaches have been explored in the last decades and are still under investigation, the standard therapy to DMD remains the use of glucocorticoids despite their limited efficacy and undesired side effects.2 We set up a preclinical approach based on the peculiar properties of Sertoli cells (SeC)3,4 a cell type of the seminiferous tubules of the testis in which they favor the maturation of developing germ cells and protect them against the host immune system. Besides creating a physical barrier (the blood-testis barrier), SeC secrete a plethora of trophic and immunomodulatory factors that confer to these cells the ability to survive long time after engraftment, and protect allo- and xenogenic engraftments of tissues and organs.5 SeC isolated from specific pathogens free (SPF) pre-pubertal pigs were encapsulated in highly biocompatible alginate-based microcapsules (MC-SeC) and injected into the peritoneal cavity of mdx mice, an experimental model of DMD, in the absence of pharmacological immunosuppression.3,4A single i.p. injection of MC-SeC resulted in amelioration of muscle morphology and performance as a consequence of the secretion by SeC of anti-inflammatory factors and heregulin β1, an inducer of the dystrophin paralogue, utrophin, opening new routes in the treatment of DMD. However, information is lacking about possible direct effects of SeC on myoblasts/myotubes. Here, we show that SeC secrete factors able to stimulate cell proliferation in the early phase of the myogenic differentiation process in murine C2C12 and human (healthy and DMD) myoblasts. In DMD myoblasts, SeC-derived factors delayed the expression of the muscle-specific terminal differentiation markers, myogenin and myosin heavy chain (MyHC)-II in the early phase (24h) of the differentiation process; nevertheless, SeC stimulated terminal differentiation (6 days) in these cells, suggesting that the promitogenic activity of SeC does not affect the myogenic potential. Moreover, SeC inhibit the expression of the myofibroblast transdifferentiation markers, COL1A1, FN1 and CTGF/CCN2 in DMD myoblasts, suggesting an antifibrotic effect of the SeC-derived factors. Finally, SeC induced utrophin expression in preformed DMD myotubes regardless of the mutation, with the same mechanism reported in dystrophic mice. Altogether, these results further support the use of MC-SeC or SeC-derived factors in the treatment of DMD patients, and suggest that SeC-based approaches might be useful also in improving the early phase of muscle regeneration, during which myoblasts have to proliferate in order to reach the critical amount required to replace the damaged muscle mass.
Keywords: Duchenne muscular dystrophy; Sertoli cell; muscle differentiation; fibrosis.
References
1. Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol. 2006;10:762-73. doi: 10.1038/nrm 2024
2. Muntoni F, Fisher I, Morgan, JE, Abraham D. Steroids in Duchenne muscular dystrophy: from clinical trials to genomic research. Neuromuscul. Disord. 2002;1:S162-5. doi: 10.1016/s0960-8966 (02)00101-3
3. Chiappalupi S, Luca G, Mancuso F, Madaro L, Fallarino F, Nicoletti C, Calvitti M, Arato I, Falabella F, Salvadori L, Di Meo A, Bufalari A, Giovagnoli S, Calafiore R, Donato R, Sorci G. Intraperitoneal injection of microencapsulated Sertoli cells restores muscle morphology and performance in dystrophic mice. Biomaterials. 2016;75:313-26. doi:10.1016/j.biomaterials. 2015. 10.029
4. Chiappalupi S, Luca G, Mancuso F, Madaro L, Fallarino F, Nicoletti C, Calvitti M, Arato I, Falabella F, Salvadori L, Di Meo A, Bufalari A, Giovagnoli S, Calafiore R, Donato R, Sorci G. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice. Data in Brief. 2015;5:1015-21. doi:10.1016/j.dib.2015.11.016
5. Chiappalupi S, Salvadori L, Luca G, Riuzzi F, Calafiore R, Donato R, Sorci G. Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy? Cell Proliferation. 2019;26:e12599; doi: 10.1111/cpr.12599
S100B inhibits myoblast differentiation via activation of a Ras-MEK-ERK1/2 signaling pathway
No full textWe have recently reported that the Ca2+-modulated protein of the EF-hand type, S100B, inhibits rat L6 myoblast differentiation by interacting with an unknown receptor with a high affinity (Kd ~40 pM) and causing inactivation of p38 MAPK (1) and counteracts the promyogenic effect of RAGE activation by amphoterin (2). We show here that the S100B’s ability to inhibit myoblast differentiation depends on activation of the Ras-
MEK-ERK1/2 pathway as S100B is unable to inhibit myogenesis in the presence of the MEK inhibitor, PD98059, or in myoblasts transfected with a dominant-negative mutant of Ras. Also, S100B stimulates NF-κB transcriptional activity dose-dependently up to 200 pM, this latter effect being negated in the presence of PD98059, NF-κB inhibitors (PDTC or the NF-κB super-repressor, I-κBαSR) or the antioxidant N-acetylcysteine. Yet, no obvious correlation could be ascertained between S100B effects on NF-κB activity and the S100B inhibitory effect on myogenesis. In
fact, S100B is unable to stimulate NF-κB transcriptional activity at >200 pM (i.e., at doses that are still antimyogenic), suggesting that S100B mainly inhibits myogenesis via inactivation of p38 MAPK likely through persistent stimulation of Ras-MEK-ERK1/2. Finally, extracellular S100B inhibits the induction of the anti-proliferative factor, p21WAF1, the activity of which is required for myoblasts to exit from the cell cycle
(3). Analyses are in progress to identify the factor(s) linking inhibition of p38 MAPK to inhibition of p21WAF1 induction in myoblasts exposed to pM doses of S100B under differentiation conditions, and functional correlates of stimulation of NF-κB transcriptional activity in myoblasts by low pM levels of S100B.
1. Sorci G et al (2003) Mol Cell Biol 23:4870-4881; 2. Sorci G et al (2004) Mol Cell Biol 24, 4880-4894; 3. Lee J et al (2002) BBRC 298:765-771
Absence of RAGE in an animal experimental model of Duchenne muscular dystrophy results in reduced muscle necrosis and inflammation
No full textDuchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder character-ized 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 ad-vanced 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 myofi-bers 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 myofi-bers, 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, mus-cles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell num-bers, suggesting a reduced recruitment of muscle precursor cells and more efficient regen-eration in dystrophic mice lacking RAGE. Our results suggest that RAGE may sustain in-flammatory and degenerative processes in dystrophic muscles, and the inhibition of its ex-pression/activity might represent a potential therapeutic approach in DMD patients.
This work was supported by grants from MIUR 2012N8YJC3, AFM 16812 and Fonda-zione CRP 2015.0325.021.
References
[1] Sorci G. et al. (2004) Mol. Cell. Biol. 24:4880-94
[2] Riuzzi F. et al. (2012) J. Cell Sci. 125:1440-54
[3] Haslbeck K.M. et al. (2005) Acta Neuropathol. 110:247-5
Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy?
No full textSertoli cells (SeC) are responsible for the immunoprivileged status of the testis thanks
to which allogeneic or xenogeneic engraftments can survive without pharmacological
immune suppression if co‐injected with SeC. This peculiar ability of SeC is dependent
on secretion of a plethora of factors including maturation factors, hormones,
growth factors, cytokines and immunomodulatory factors. The anti‐inflammatory
and trophic properties of SeC have been largely exploited in several experimental
models of diseases, diabetes being the most studied. Duchenne muscular dystrophy
(DMD) is a lethal X‐linked recessive pathology in which lack of functional dystrophin
leads to progressive muscle degeneration culminating in loss of locomotion and premature
death. Despite a huge effort to find a cure, DMD patients are currently
treated with anti‐inflammatory steroids. Recently, encapsulated porcine SeC (MCSeC)
have been injected ip in the absence of immunosuppression in an animal model
of DMD resulting in reduction of muscle inflammation and amelioration of muscle
morphology and functionality, thus opening an additional avenue in the treatment of
DMD. The novel protocol is endowed with the advantage of being potentially applicable
to all the cohort of DMD patients regardless of the mutation. This mini‐review
addresses several issues linked to the possible use of MC‐SeC injected ip in dystrophic
people
Multivariate modelling of milk fatty acid profile to discriminate the forages in dairy cows’ ration
No full textAlthough there are many studies on the importance of fatty acids (FA) in our diet and on the influence of dairy diets on FA metabolism, only a few investigate their predictive capacity to discriminate the type, amount and conservation method of farm forages. This research quantifies differences in milk FA concentrations and, using a supervised factorial discriminant analysis, assesses potential biomarkers when replacing maize with other silages, grass/lucerne hays or fresh grass. The statistical modelling identified three main clusters of milk FA profiles associated with silages, hays and fresh grass as dominant roughages. The main implication of a dairy cow feeding system based on poliphytic forages from permanent meadows is enhancing milk’s nutritional quality due to an increase in beneficial omega-3 polyunsaturated FA, conjugated linoleic acids and odd chain FA, compared to feeding maize silage. The study also identified a small but powerful and reliable pool of milk FA that can act as biomarkers to authenticate feeding systems: C16:1 c-9, C17:0, C18:0, C18:3 c-9, c-12, c-15, C18:1 c-9, C18:1 t-11 and C20:0
Socio-economic analysis of the EU citizens’ attitudes toward farmed animal welfare from the 2023 Eurobarometer polling survey
No full textBackground and methods: Europeans’ expectations and opinions regarding the conditions and welfare of farmed animals have evolved continuously. Since 2005, the Eurobarometer (Eb) polling instrument has been used to monitor EU citizens’ attitudes towards farmed animal welfare (FAW). Using the last Eb survey (2023), this study categorized respondents into clusters according to their answers to 12 selected questions on FAW. The ultimate goal was to highlight trends useful to stakeholders and policymakers within the animal food supply chain to design and implement activity planning, progress, and information campaigns. Results and discussion: As the Eb data came from a stratified multi-stage, random (probability) sample design, the seven clusters sorted through our statistical approach reflected the opinions of the EU population in 2023. These clusters could be further merged into three macro-clusters with two main opposite levels of concern (>80% positive answers) about FAW: concerned abou..
