Publications by authors named "Chiara Donati"

57 Publications

Phosphatidic Acid Stimulates Myoblast Proliferation through Interaction with LPA1 and LPA2 Receptors.

Int J Mol Sci 2021 Feb 1;22(3). Epub 2021 Feb 1.

Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, 48980 Bilbao, Spain.

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.
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http://dx.doi.org/10.3390/ijms22031452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867176PMC
February 2021

Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Department of Experimental and Clinical Biomedical Sciences "M. Serio", University of Florence, 50134 Florence, Italy.

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P and S1P and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.
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http://dx.doi.org/10.3390/ijms22031280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866171PMC
January 2021

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect.

Fertil Steril 2021 Jan 23. Epub 2021 Jan 23.

Department of Experimental and Clinical Biomedical Sciences "M. Serio," University of Florence, Florence, Italy.

Objective: To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A.

Design: Case-control laboratory study.

Setting: University institute and university hospital.

Patient(s): Patients with uterine fibroids (n = 26).

Interventions(s): Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy.

Main Outcome Measure(s): Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines.

Result(s): The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P, S1P, and S1P receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells.

Conclusion(s): This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.
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http://dx.doi.org/10.1016/j.fertnstert.2020.12.022DOI Listing
January 2021

Role of sphingosine 1-phosphate signalling in tissue fibrosis.

Cell Signal 2021 Feb 28;78:109861. Epub 2020 Nov 28.

Department of Experimental and Clinical Biomedical Sciences "M. Serio", University of Florence, viale GB Morgagni 50, 50134 Florence, Italy.

Fibrosis is characterized by the excessive accumulation of extracellular matrix components, leading to loss of tissue function in affected organs. Although the majority of fibrotic diseases have different origins, they have in common a persistent inflammatory stimulus and lymphocyte-monocyte interactions that determine the production of numerous fibrogenic cytokines. Treatment to contrast fibrosis is urgently needed, since some fibrotic diseases lead to systemic fibrosis and represent a major cause of death. In this article, the role of the bioactive sphingolipid sphingosine 1-phosphate (S1P) and its signalling pathway in the fibrosis of different tissue contexts is extensively reviewed, highlighting that it may represent an innovative and promising pharmacological therapeutic target for treating this devastating multifaceted disease. In multiple tissues S1P influences different aspects of fibrosis modulating the recruitment of inflammatory cells, as well as cell proliferation, migration and transdifferentiation into myofibroblasts, the cell type mainly involved in fibrosis development. Moreover, at the level of fibrotic lesions, S1P metabolism is profoundly influenced by multiple cross-talk with profibrotic mediators, such as transforming growth factor β, thus finely regulating the development of fibrosis. This article is part of a Special Issue entitled "Physiological and pathological roles of bioactive sphingolipids".
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http://dx.doi.org/10.1016/j.cellsig.2020.109861DOI Listing
February 2021

Sphingosine 1-phosphate receptors are dysregulated in endometriosis: possible implication in transforming growth factor β-induced fibrosis.

Fertil Steril 2021 Feb 6;115(2):501-511. Epub 2020 Sep 6.

Department of Experimental and Clinical Biomedical Sciences "M. Serio," University of Florence, Florence, Italy.

Objective: To study the molecular mechanisms involved in the appearance of the fibrotic trait in endometriosis by investigating whether the signaling pathway of the bioactive sphingolipid sphingosine 1-phosphate (S1P) was altered in endometriotic lesions.

Design: Case-control laboratory study.

Setting: University research institute and university hospital.

Patient(s): A total of 75 women, with and without endometriosis, were included in the study.

Interventions(s): Endometrial samples were obtained from women affected (n = 15 endometrioma [OMA]; n = 30 deep infiltrating endometriosis [DIE]) and not (n = 30) by endometriosis by means of laparoscopic surgery, followed by clinical and imaging investigation and checking for the expression of fibrosis markers and genes implicated in S1P metabolism and signaling by means of real-time polymerase chain reaction.

Main Outcome Measure(s): The role of the S1P signaling axis in endometriosis-associated fibrosis was studied in vitro, where RNA interference approaches were used to investigate if S1P synthesis by sphingosine kinases (SKs) and specific S1P receptors (S1PRs) are implicated in the profibrotic effect of the cytokine transforming growth factor (TGF) β1.

Result(s): mRNA expression analysis of S1PR demonstrated a deep dysregulation of S1P signaling in endometriosis, characterized by increased expression of fibrosis markers: S1P was transcriptionally more expressed in OMA, and S1P and S1P mRNA levels were significantly augmented in both OMA and DIE. SK1 and its activating protein calcium- and integrin-binding protein 1 (CIB1) were significantly up-regulated in OMA and DIE. A crucial role for the SK/S1PR axis in the profibrotic effect elicited by TGFβ1 was highlighted in vitro.

Conclusion(s): The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis.
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http://dx.doi.org/10.1016/j.fertnstert.2020.08.012DOI Listing
February 2021

Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P receptor axis.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 09 19;1865(9):158759. Epub 2020 Jun 19.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Florence, Italy.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.
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http://dx.doi.org/10.1016/j.bbalip.2020.158759DOI Listing
September 2020

Adenosine A receptors inhibit K currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Biochem Pharmacol 2020 07 3;177:113956. Epub 2020 Apr 3.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, Italy.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A receptors inhibit cell maturation by reducing voltage-dependent K currents. No data are available to date about the A receptor (AR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P) are also crucial modulators of OPC development. An interaction between this pathway and the AR is reported in peripheral cells. We studied the role of ARs in modulating K currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the AR agonist BAY60-6583 and its new analogue P453 inhibit K currents in cultured OPC and the effect was prevented by the AR antagonist MRS1706, by K channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, AR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, AR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that ARs inhibit K currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.
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http://dx.doi.org/10.1016/j.bcp.2020.113956DOI Listing
July 2020

β3-adrenoreceptor blockade reduces tumor growth and increases neuronal differentiation in neuroblastoma via SK2/S1P modulation.

Oncogene 2020 01 2;39(2):368-384. Epub 2019 Sep 2.

Department of Paediatric Haematology-Oncology, A. Meyer University Children's Hospital, Florence, Italy.

Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P signaling.
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http://dx.doi.org/10.1038/s41388-019-0993-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949192PMC
January 2020

Cadmium effects on superoxide dismutase 1 in human cells revealed by NMR.

Redox Biol 2019 02 8;21:101102. Epub 2019 Jan 8.

Magnetic Resonance Center - CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. Electronic address:

Cadmium is a toxic pollutant that in recent decades has become more widespread in the environment due to anthropogenic activities, significantly increasing the risk of exposure. Concurrently, a continually growing body of research has begun to enumerate the harmful effects that this heavy metal has on human health. Consequently, additional research is required to better understand the mechanism and effects of cadmium at the molecular level. The main mechanism of cadmium toxicity is based on the indirect induction of severe oxidative stress, through several processes that unbalance the anti-oxidant cellular defence system, including the displacement of metals such as zinc from its native binding sites. Such mechanism was thought to alter the in vivo enzymatic activity of SOD1, one of the main antioxidant proteins of many tissues, including the central nervous system. SOD1 misfolding and aggregation is correlated with cytotoxicity in neurodegenerative diseases such as amyotrophic lateral sclerosis. We assessed the effect of cadmium on SOD1 folding and maturation pathway directly in human cells through in-cell NMR. Cadmium does not directly bind intracellular SOD1, instead causes the formation of its intramolecular disulfide bond in the zinc-bound form. Metallothionein overexpression is strongly induced by cadmium, reaching NMR-detectable levels. The intracellular availability of zinc modulates both SOD1 oxidation and metallothionein overexpression, strengthening the notion that zinc-loaded metallothioneins help maintaining the redox balance under cadmium-induced acute stress.
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http://dx.doi.org/10.1016/j.redox.2019.101102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348768PMC
February 2019

Sphingosine 1-phosphate-mediated activation of ezrin-radixin-moesin proteins contributes to cytoskeletal remodeling and changes of membrane properties in epithelial otic vesicle progenitors.

Biochim Biophys Acta Mol Cell Res 2019 04 3;1866(4):554-565. Epub 2019 Jan 3.

Department of Experimental and Clinical Biomedical Sciences "M. Serio", University of Florence, viale GB Morgagni 50, 50134 Florence, Italy. Electronic address:

Hearing loss is among the most prevalent sensory impairments in humans. Cochlear implantable devices represent the current therapies for hearing loss but have various shortcomings. ERM (ezrin- radixin -moesin) are a family of adaptor proteins that link plasma membrane with actin cytoskeleton, playing a crucial role in cell morphology and in the formation of membrane protrusions. Recently, bioactive sphingolipids have emerged as regulators of ERM proteins. Sphingosine 1-phosphate (S1P) is a pleiotropic sphingolipid which regulates fundamental cellular functions such as proliferation, survival, migration as well as processes such as development and inflammation mainly via ligation to its specific receptors S1PR (S1P). Experimental findings demonstrate a key role for S1P signaling axis in the maintenance of auditory function. Preservation of cellular junctions is a fundamental function both for S1P and ERM proteins, crucial for the maintenance of cochlear integrity. In the present work, S1P was found to activate ERM in a S1P-dependent manner in murine auditory epithelial progenitors US/VOT-E36. S1P-induced ERM activation potently contributed to actin cytoskeletal remodeling and to the appearance of ionic currents and membrane passive properties changes typical of more differentiated cells. Moreover, PKC and Akt activation was found to mediate S1P-induced ERM phosphorylation. The obtained findings contribute to demonstrate the role of S1P signaling pathway in inner ear biology and to disclose potential innovative therapeutical approaches in the field of hearing loss prevention and treatment.
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http://dx.doi.org/10.1016/j.bbamcr.2018.12.007DOI Listing
April 2019

S1P promotes migration, differentiation and immune regulatory activity in amniotic-fluid-derived stem cells.

Eur J Pharmacol 2018 Aug 7;833:173-182. Epub 2018 Jun 7.

Department of Experimental Medicine, University of Perugia, Italy. Electronic address:

Stem cells have high potential for cell therapy in regenerative medicine. We previously isolated stem cell types from human amniotic fluid, derived from prenatal amniocentesis. One type, characterized by a fast doubling time, was designated as fast human amniotic stem cells (fHASCs). These cells exhibited high differentiation potential and immunoregulatory properties. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that influences stem-cell pluripotency, differentiation, mobility, and regulates immune functions. In this study, we investigated the influence of S1P on fHASC migration, proliferation, differentiation and immune regulatory functions. We found that fHASC stimulation with S1P potentiated their migratory and proliferative activity in vitro. Notably, short fHASC exposure to S1P enhanced their differentiation towards multiple lineages, including adipocytes, osteocytes and endothelial cells, an effect that was associated with downregulation of the main transcription factors involved in the maintenance of a stem-cell undifferentiated state. A specific crosstalk between S1P and tumor growth factor β1 (TGF-β1) has recently been demonstrated. We found that fHASC exposure to S1P in combination with TGF-β1 promoted the expression of the immune regulatory pathway of indoleamine 2,3-dioxygenase 1 (IDO1). In addition, human peripheral blood mononuclear cells, co-cultured with fHASCs treated with S1P and TGF-β1, expanded regulatory T-cells, via a mechanism requiring IDO1. Overall, this study demonstrates that S1P potentiates several properties in fHASCs, an effect that may be critical for exploiting the therapeutic potential of fHASCs and might explain the specific effects of S1P on stem cells during pregnancy.
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http://dx.doi.org/10.1016/j.ejphar.2018.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086338PMC
August 2018

Bradykinin mediates myogenic differentiation in murine myoblasts through the involvement of SK1/Spns2/S1P axis.

Cell Signal 2018 05 3;45:110-121. Epub 2018 Feb 3.

Dipartimento di Scienze Biomediche Sperimentali e Cliniche "Mario Serio", Università di Firenze, Viale GB Morgagni 50, 50134 Firenze, Italy; Istituto interuniversitario di Miologia, IIM, Padova, Italy.

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.
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http://dx.doi.org/10.1016/j.cellsig.2018.02.001DOI Listing
May 2018

Lysophosphatidic Acid Signaling Axis Mediates Ceramide 1-Phosphate-Induced Proliferation of C2C12 Myoblasts.

Int J Mol Sci 2018 Jan 4;19(1). Epub 2018 Jan 4.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, Viale GB Morgagni 50, 50134 Firenze, Italy.

Sphingolipids are not only crucial for membrane architecture but act as critical regulators of cell functions. The bioactive sphingolipid ceramide 1-phosphate (C1P), generated by the action of ceramide kinase, has been reported to stimulate cell proliferation, cell migration and to regulate inflammatory responses via activation of different signaling pathways. We have previously shown that skeletal muscle is a tissue target for C1P since the phosphosphingolipid plays a positive role in myoblast proliferation implying a role in muscle regeneration. Skeletal muscle displays strong capacity of regeneration thanks to the presence of quiescent adult stem cells called satellite cells that upon trauma enter into the cell cycle and start proliferating. However, at present, the exact molecular mechanism by which C1P triggers its mitogenic effect in myoblasts is lacking. Here, we report for the first time that C1P stimulates C2C12 myoblast proliferation via lysophosphatidic acid (LPA) signaling axis. Indeed, C1P subsequently to phospholipase A2 activation leads to LPA₁ and LPA₃ engagement, which in turn drive Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinases 1/2) activation, thus stimulating DNA synthesis. The present findings shed new light on the key role of bioactive sphingolipids in skeletal muscle and provide further support to the notion that these pleiotropic molecules might be useful therapeutic targets for skeletal muscle regeneration.
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http://dx.doi.org/10.3390/ijms19010139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796088PMC
January 2018

Ablation of S1P receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

Am J Physiol Cell Physiol 2017 Jul 26;313(1):C54-C67. Epub 2017 Apr 26.

Department of Biomedical Sciences, University of Padova, Padua, Italy;

We investigated the effects of S1P deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.
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http://dx.doi.org/10.1152/ajpcell.00027.2017DOI Listing
July 2017

NMR metabolomics highlights sphingosine kinase-1 as a new molecular switch in the orchestration of aberrant metabolic phenotype in cancer cells.

Mol Oncol 2017 05 30;11(5):517-533. Epub 2017 Mar 30.

CERM and Department of Chemistry, University of Florence, Italy.

Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR-based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO production. Additionally, SK1-expressing cells displayed a significant increase in glucose uptake paralleled by GLUT3 transporter upregulation. The role of SK1 is not limited to the induction of aerobic glycolysis, affecting metabolic pathways that appear to support the biosynthesis of macromolecules. These findings highlight the role of SK1 signaling axis in cancer metabolic reprogramming, pointing out innovative strategies for cancer therapies.
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http://dx.doi.org/10.1002/1878-0261.12048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527469PMC
May 2017

Sphingosine 1-phosphate signaling axis mediates fibroblast growth factor 2-induced proliferation and survival of murine auditory neuroblasts.

Biochim Biophys Acta Mol Cell Res 2017 May 7;1864(5):814-824. Epub 2017 Feb 7.

Dipartimento di Scienze Biomediche Sperimentali e Cliniche "M. Serio", viale G B Morgagni 50, 50134 Firenze, Italy.

Hearing loss affects millions of people in the world. In mammals the auditory system comprises diverse cell types which are terminally differentiated and with no regenerative potential. There is a tremendous research interest aimed at identifying cell therapy based solutions or pharmacological approaches that could be applied therapeutically alongside auditory devices to prevent hair cell and neuron loss. Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid that plays key role in the regulation of many physiological and pathological functions. S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P. In this study, murine auditory neuroblasts named US/VOT-N33 have been used as progenitors of neurons of the spiral ganglion. We demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action was dependent on SK1, SK2 as well as S1P and S1P. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. Additionally, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively. Taken together, these findings demonstrate a crucial role for S1P signaling axis in the proliferation and the survival of otic vesicle neuroprogenitors, highlighting the identification of possible novel therapeutical approaches to prevent neuronal degeneration during hearing loss.
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http://dx.doi.org/10.1016/j.bbamcr.2017.02.004DOI Listing
May 2017

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

J Appl Physiol (1985) 2016 Jun 30;120(11):1288-300. Epub 2015 Dec 30.

Department of Biomedical Sciences, University of Padova, Padova, Italy; IIM, Interuniversity Institute of Myology, Italy;

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.
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http://dx.doi.org/10.1152/japplphysiol.00345.2015DOI Listing
June 2016

Sphingosine 1-phosphate signaling pathway in inner ear biology. New therapeutic strategies for hearing loss?

Front Aging Neurosci 2015 23;7:60. Epub 2015 Apr 23.

Department Scienze Biomediche Sperimentali e Cliniche "Mario Serio", University of Florence Firenze, Italy.

Hearing loss is one of the most prevalent conditions around the world, in particular among people over 60 years old. Thus, an increase of this affection is predicted as result of the aging process in our population. In this context, it is important to further explore the function of molecular targets involved in the biology of inner ear sensory cells to better individuate new candidates for therapeutic application. One of the main causes of deafness resides into the premature death of hair cells and auditory neurons. In this regard, neurotrophins and growth factors such as insulin like growth factor are known to be beneficial by favoring the survival of these cells. An elevated number of published data in the last 20 years have individuated sphingolipids not only as structural components of biological membranes but also as critical regulators of key biological processes, including cell survival. Ceramide, formed by catabolism of sphingomyelin (SM) and other complex sphingolipids, is a strong inducer of apoptotic pathway, whereas sphingosine 1-phosphate (S1P), generated by cleavage of ceramide to sphingosine and phosphorylation catalyzed by two distinct sphingosine kinase (SK) enzymes, stimulates cell survival. Interestingly S1P, by acting as intracellular mediator or as ligand of a family of five distinct S1P receptors (S1P1-S1P5), is a very powerful bioactive sphingolipid, capable of triggering also other diverse cellular responses such as cell migration, proliferation and differentiation, and is critically involved in the development and homeostasis of several organs and tissues. Although new interesting data have become available, the information on S1P pathway and other sphingolipids in the biology of the inner ear is limited. Nonetheless, there are several lines of evidence implicating these signaling molecules during neurogenesis in other cell populations. In this review, we discuss the role of S1P during inner ear development, also as guidance for future studies.
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http://dx.doi.org/10.3389/fnagi.2015.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407579PMC
May 2015

Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts.

J Mol Med (Berl) 2015 Oct 9;93(10):1145-57. Epub 2015 May 9.

Dipartimento di Scienze Biomediche Sperimentali e Cliniche "Mario Serio", Università di Firenze, Viale G.B. Morgagni 50, 50134, Florence, Italy.

Unlabelled: The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions.

Key Message: • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB. • S1P1/3 were involved on CM-induced morphogenesis and migration of MAB. • Matrigel plug assay showed the role of S1P axis in MAB-endothelial cell interaction.
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http://dx.doi.org/10.1007/s00109-015-1292-0DOI Listing
October 2015

CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFβ.

Biochim Biophys Acta 2015 Feb 29;1851(2):194-202. Epub 2014 Nov 29.

Dipartimento di Scienze Biomediche Sperimentali e Cliniche "Mario Serio," Università di Firenze, Viale G.B. Morgagni 50, Firenze 50134, Italy; Istituto Interuniversitario di Miologia, Italy. Electronic address:

The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype.
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http://dx.doi.org/10.1016/j.bbalip.2014.11.011DOI Listing
February 2015

Lysophosphatidic acid stimulates cell migration of satellite cells. A role for the sphingosine kinase/sphingosine 1-phosphate axis.

FEBS J 2014 Oct 25;281(19):4467-78. Epub 2014 Aug 25.

Dipartimento di Scienze Biomediche Sperimentali e Cliniche 'Mario Serio', Università di Firenze, Italy; Istituto Interuniversitario di Miologia, Italy.

Regulation of the motility of skeletal muscle precursor cells, such as satellite cells, is critically important for their proper recruitment at the site of tissue damage, and ultimately for its correct repair. Here we show that lysophosphatidic acid (LPA), which is well-recognized as a powerful bioactive agent, strongly stimulates cell migration of activated murine satellite cells. The biological effect exerted by LPA was found to be induced via activation of LPA1 and LPA3 , being abolished by cell treatment with the antagonist Ki16425, and severely impaired by siRNA-mediated down-regulation of the two receptor isoforms. In contrast, silencing of LPA2 potentiated the stimulation of cell motility by LPA, suggesting that it is negatively coupled to cell migration. Pharmacological inhibition of both sphingosine kinase (SK) isoforms using VPC96047, or the selective blocking of SK1 using VPC96091, abolished cell responsiveness to LPA; in agreement, gene silencing of SK1 or SK2 significantly reduced the biological effect of LPA. Moreover, the LPA-dependent stimulation of cell chemotaxis was found to be impaired by down-regulation of the sphingosine 1-phosphate (S1P) receptors S1P1 or S1P4 by specific siRNAs. In summary, the results obtained support the notion that the sphingosine kinase/sphingosine 1-phosphate (SK/S1P) axis is critically involved in the mechanism by which LPA elicits its pro-migratory action. This study provides compelling new information on the regulatory mechanisms of satellite cell motility, and reinforces the view that the SK/S1P signaling pathway plays a crucial role in the control of skeletal muscle precursor cell biology.
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http://dx.doi.org/10.1111/febs.12955DOI Listing
October 2014

Cardiomyopathy in a male patient with neutropenia and growth delay.

Ital J Pediatr 2014 May 12;40:45. Epub 2014 May 12.

Department of Pediatrics, Pediatrics Clinic, Spedali Civili of Brescia, Brescia, Italy.

Neutropenia encompasses a family of neutropenic disorders, both permanent and intermittent, ranging from severe (<500 neutrophils/mm(3)) to mild (500-1500 neutrophils/mm(3)), which may also affect other organ systems such as the pancreas, central nervous system, heart, muscle and skin. Neutropenia can lead to life-threatening pyogenic infections whose severity is roughly inversely proportional to the circulating neutrophil counts.When neutropenia is detected, an attempt should be made to establish the etiology, and to distinguish acquired forms (the most frequent, including post viral neutropenia and autoimmune neutropenia) and congenital forms (rare disorders) that may be either isolated or part of a complex rare genetic disease. We report on a male patient initially diagnosed with isolated neutropenia who later turned out to be affected with Barth syndrome, a rare complex inherited disorder.
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http://dx.doi.org/10.1186/1824-7288-40-45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046054PMC
May 2014

Sphingosine 1-phosphate axis: a new leader actor in skeletal muscle biology.

Front Physiol 2013 Nov 25;4:338. Epub 2013 Nov 25.

Dipartimento di Scienze Biomediche, Sperimentali e Cliniche, University of Florence Florence, Italy ; Istituto Interuniversitario di Miologia Italy.

Sphingosine 1-phosphate (S1P) is a bioactive lipid involved in the regulation of biological processes such as proliferation, differentiation, motility, and survival. Here we review the role of S1P in the biology and homeostasis of skeletal muscle. S1P derives from the catabolism of sphingomyelin and is produced by sphingosine phosphorylation catalyzed by sphingosine kinase (SK). S1P can act either intracellularly or extracellularly through specific ligation to its five G protein-coupled receptors (GPCR) named S1P receptors (S1PR). Many experimental findings obtained in the last 20 years demonstrate that S1P and its metabolism play a multifaceted role in the regulation of skeletal muscle regeneration. Indeed, this lipid is known to activate muscle-resident satellite cells, regulating their proliferation and differentiation, as well as mesenchymal progenitors such as mesoangioblasts that originate outside skeletal muscle, both involved in tissue repair following an injury or disease. The molecular mechanism of action of S1P in skeletal muscle cell precursors is highly complex, especially because S1P axis is under the control of a number of growth factors and cytokines, canonical regulators of skeletal muscle biology. Moreover, this lipid is crucially involved in the regulation of skeletal muscle contractile properties, responsiveness to insulin, fatigue resistance and tropism. Overall, on the basis of these findings S1P signaling appears to be an appealing pharmacological target for improving skeletal muscle repair. Nevertheless, further understanding is required on the regulation of S1P downstream signaling pathways and the expression of S1PR. This article will resume our current knowledge on S1P signaling in skeletal muscle, hopefully stimulating further investigation in the field, aimed at individuating novel molecular targets for ameliorating skeletal muscle regeneration and reducing fibrosis of the tissue after a trauma or due to skeletal muscle diseases.
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http://dx.doi.org/10.3389/fphys.2013.00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839259PMC
November 2013

Adenosine is present in rat brain synaptic vesicles.

Neuroreport 2013 Dec;24(17):982-7

Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Evidences in the central nervous system are in favor that adenosine under basal conditions is released by a direct excitation-secretion modality. However, till now, there is no direct evidence that adenosine is contained in synaptic vesicles. Eight synaptic vesicle fractions were recovered on a discontinuous sucrose gradient after ultracentrifugation of the crude synaptosomal fraction (pellet P2) of rat brain. The adenosine content in each fraction was measured by high-performance liquid chromatography coupled to a fluorescence detector (minimum sensitivity 10 femtomoles). The immunoblot analysis, to detect synaptophysin, a molecular marker for the vesicle membrane, showed that fractions from 3 to 8 were rich in synaptophysin. The sum of adenosine found in fractions 3-8 was (mean ± SEM, n = 4) 3325.6 ± 94.6 pmol/mg of tissue protein. We proved that adenosine measured in synaptic vesicle fractions was not contaminated by cytosolic adenosine, as adenosine exogenously added to the P2 preferentially distributed in fractions 1 and 2 that are synaptophysin-free and did not contaminate the vesicle pellet P3. Data provide direct demonstration that adenosine is present in rat brain synaptic vesicle fractions. This information is consistent with the notion that adenosine is stored in synaptic vesicles and is released under normoxic physiological conditions by an excitation-secretion mechanism typical of neuronal cells.
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http://dx.doi.org/10.1097/WNR.0000000000000033DOI Listing
December 2013

TGFβ1 evokes myoblast apoptotic response via a novel signaling pathway involving S1P4 transactivation upstream of Rho-kinase-2 activation.

FASEB J 2013 Nov 2;27(11):4532-46. Epub 2013 Aug 2.

2Dipartimento di Scienze Biomediche, Sperimentali e Cliniche, Università di Firenze, Viale G.B. Morgagni 50, 50134 Florence, Italy.

In view of its multiple detrimental effects, transforming growth factor β1 (TGFβ1) is recognized as critical negative regulator of skeletal muscle repair. Apoptosis of skeletal muscle precursor cells driven by TGFβ1 contributes to the negative role exerted by the cytokine in tissue repair, although the underlying molecular mechanisms are still elusive. Herein we report the identification of a new signaling pathway, relying on Rho kinase-2 stimulation, subsequent to SMAD-dependent S1P4 up-regulation and transactivation via sphingosine kinase (SK)-2, that accounts for TGFβ1-induced apoptosis in cultured myoblasts. S1P4-specific gene silencing reduced by almost 50% activation of caspase-3 and poly-ADP ribosyl transferase cleavage elicited by TGFβ1. Moreover, the selective S1P4 antagonist CYM50358 also reduced the TGFβ1 proapoptotic effects. By employing pharmacological and molecular biological approaches, the involvement of SK2 and ROCK2 in the transmission of the TGFβ1 apoptotic action was also demonstrated. These results reinforce the notion that the SK/S1P axis plays a fundamental role in TGFβ1 mode of action in skeletal muscle cells and, by disclosing a novel mechanism by which TGFβ1 exerts its harmful action, pinpoint new molecular targets that in principle could be beneficial in the treatment of several skeletal muscle disorders or aging-dependent muscle atrophy.
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http://dx.doi.org/10.1096/fj.13-228528DOI Listing
November 2013

Role of sphingosine 1-phosphate in skeletal muscle cell biology.

Handb Exp Pharmacol 2013 (216):457-67

Dipartimento di Scienze Biochimiche, Università di Firenze, Firenze, Italy.

Studies performed in the last fifteen years have clearly established that the bioactive sphingolipid sphingosine 1-phosphate (S1P) affects various different biological properties of myogenic precursor cells as well as physiological features of adult skeletal muscle. Noticeably, in myogenic precursor cells multiple growth factors and cytokines cross-communicate with S1P axis and the engagement of distinct S1P receptor subtypes appears to be crucially implicated in transmitting specific biological effects. This paper summarizes current research findings and discloses the potential for new therapeutics designed to alter S1P signaling with the aim of improving skeletal muscle repair.
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http://dx.doi.org/10.1007/978-3-7091-1511-4_23DOI Listing
July 2013

New insights into the role of sphingosine 1-phosphate and lysophosphatidic acid in the regulation of skeletal muscle cell biology.

Biochim Biophys Acta 2013 Jan 2;1831(1):176-84. Epub 2012 Aug 2.

Dipartimento di Scienze Biochimiche, Università di Firenze, Firenze, Italy.

Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
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http://dx.doi.org/10.1016/j.bbalip.2012.06.013DOI Listing
January 2013

Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation.

Skelet Muscle 2012 Jul 12;2(1):15. Epub 2012 Jul 12.

Department of Biochemical Sciences, University of Florence, GB Morgagni 50, 50134, Florence, Italy.

Background: Insulin-like growth factor-1 (IGF-1) is the most important physiological regulator of skeletal muscle progenitor cells, which are responsible for adult skeletal muscle regeneration. The ability of IGF-1 to affect multiple aspects of skeletal muscle cell biology such as proliferation, differentiation, survival and motility is well recognized, although the molecular mechanisms implicated in its complex biological action are not fully defined. Since sphingosine 1-phosphate (S1P) has recently emerged as a key player in skeletal muscle regeneration, we investigated the possible involvement of the sphingosine kinase (SK)/S1P receptor axis on the biological effects of IGF-1 in murine myoblasts.

Methods: RNA interference, chemical inhibition and immunofluorescence approaches were used to assess the role of the SK/S1P axis on the myogenic and mitogenic effects of IGF-1 in C2C12 myoblasts.

Results: We show that IGF-1 increases SK activity in mouse myoblasts. The effect of the growth factor does not involve transcriptional regulation of SK1 or SK2, since the protein content of both isoforms is not affected; rather, IGF-1 enhances the fraction of the active form of SK. Moreover, transactivation of the S1P2 receptor induced by IGF-1 via SK activation appears to be involved in the myogenic effect of the growth factor. Indeed, the pro-differentiating effect of IGF-1 in myoblasts is impaired when SK activity is pharmacologically inhibited, or SK1 or SK2 are specifically silenced, or the S1P2 receptor is downregulated. Furthermore, in this study we show that IGF-1 transactivates S1P1/S1P3 receptors via SK activation and that this molecular event negatively regulates the mitogenic effect elicited by the growth factor, since the specific silencing of S1P1 or S1P3 receptors increases cell proliferation induced by IGF-1.

Conclusions: We demonstrate a dual role of the SK/S1P axis in response to myoblast challenge with IGF-1, that likely is important to regulate the biological effect of this growth factor. These findings add new information to the understanding of the mechanism by which IGF-1 regulates skeletal muscle regeneration.
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http://dx.doi.org/10.1186/2044-5040-2-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439699PMC
July 2012

S1P2 receptor promotes mouse skeletal muscle regeneration.

J Appl Physiol (1985) 2012 Sep 28;113(5):707-13. Epub 2012 Jun 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Sphingosine 1-phosphate is a bioactive lipid that modulates skeletal muscle growth through its interaction with specific receptors localized in the cell membrane of muscle fibers and satellite cells. This study analyzes the role of S1P(2) receptor during in vivo regeneration of soleus muscle in two models of S1P(2) deficiency: the S1P(2)-null mouse and wild-type mice systemically treated with the S1P(2) receptor antagonist JTE-013. To stimulate regeneration, muscle degeneration was induced by injecting into soleus muscle the myotoxic drug notexin. Both ablation of S1P(2) receptor and its functional inactivation delayed regeneration of soleus muscle. The exogenous supplementation of S1P or its removal, by a specific antibody, two conditions known to stimulate or inhibit, respectively, soleus muscle regeneration, were without effects when the S1P(2) receptor was absent or inactive. The delayed regeneration was associated with a lower level of myogenin, a muscle differentiation marker, and reduced phosphorylation of Akt, a key marker of muscle growth. Consistently, silencing of S1P(2) receptor abrogated the pro-myogenic action of S1P in satellite cells, paralleled by low levels of the myogenic transcription factor myogenin. The study indicates that S1P(2) receptor plays a key role in the early phases of muscle regeneration by sustaining differentiation and growth of new-forming myofibers.
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http://dx.doi.org/10.1152/japplphysiol.00300.2012DOI Listing
September 2012

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors.

Biochim Biophys Acta 2012 Feb 8;1823(2):439-50. Epub 2011 Dec 8.

Dipartimento di Scienze Biochimiche, Università di Firenze, 50134 Firenze, Italy.

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.
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http://dx.doi.org/10.1016/j.bbamcr.2011.11.016DOI Listing
February 2012