Publications by authors named "Caterina Bernacchioni"

34 Publications

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

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

Cancer cell death induced by ferritins and the peculiar role of their labile iron pool.

Oncotarget 2018 Jun 15;9(46):27974-27984. Epub 2018 Jun 15.

Center for Magnetic Resonance, University of Florence, Florence, Italy.

Cellular uptake of human H-ferritin loaded with 50 or 350 iron ions results in significant cytotoxicity on HeLa cells at submicromolar concentrations. Conversely, Horse Spleen Ferritin, that can be considered a model of L-cages, as it contains only about 10% of H subunits, even when loaded with 1000 iron ions, is toxic only at >1 order of magnitude higher protein concentrations. We propose here that the different cytotoxicity of the two ferritin cages originates from the presence in H-ferritin of a pool of non-biomineralized iron ions bound at the ferroxidase catalytic sites of H-ferritin subunits. This iron pool is readily released during the endosomal-mediated H-ferritin internalization.
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http://dx.doi.org/10.18632/oncotarget.25416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021343PMC
June 2018

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

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Chemistry 2017 Jul 27;23(41):9879-9887. Epub 2017 Jun 27.

Istituto per lo Studio delle Macromolecole, CNR, Via Corti 12, 20133, Milano, Italy.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.
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http://dx.doi.org/10.1002/chem.201701164DOI 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

Chemistry at the protein-mineral interface in L-ferritin assists the assembly of a functional (μ-oxo)Tris[(μ-peroxo)] triiron(III) cluster.

Proc Natl Acad Sci U S A 2017 03 15;114(10):2580-2585. Epub 2017 Feb 15.

Centro di Risonanze Magnetiche, University of Florence, Sesto Fiorentino, Florence 50019, Italy;

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ-oxo)Tris[(μ-peroxo)(μ-glutamato-κ:κ')](glutamato-κ)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.
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http://dx.doi.org/10.1073/pnas.1614302114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347543PMC
March 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

Ferroxidase Activity in Eukaryotic Ferritin is Controlled by Accessory-Iron-Binding Sites in the Catalytic Cavity.

Chemistry 2016 Nov 21;22(45):16213-16219. Epub 2016 Sep 21.

CERM and Department of Chemistry, University of Florence, Via Sacconi 6 Sesto Fiorentino, Firenze, 50019, Italy.

Ferritins are iron-storage nanocage proteins that catalyze the oxidation of Fe to Fe at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild-type cages.
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http://dx.doi.org/10.1002/chem.201602842DOI Listing
November 2016

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels.

J Biol Chem 2016 Dec 18;291(49):25617-25628. Epub 2016 Oct 18.

From the Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa,

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H' protein and some of its variants as case studies. We describe the structural features that determine Fe translocation with atomistic detail, and we propose a putative mechanism for Fe transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.
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http://dx.doi.org/10.1074/jbc.M116.748046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207259PMC
December 2016

Solid-State NMR of PEGylated Proteins.

Angew Chem Int Ed Engl 2016 Feb 12;55(7):2446-9. Epub 2016 Jan 12.

Magnetic Resonance Center (CERM) and Department of Chemistry "Ugo Schiff", University of Florence, and Magnetic Resonance Consortium (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino, FI, Italy.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.
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http://dx.doi.org/10.1002/anie.201510148DOI Listing
February 2016

Iron binding to human heavy-chain ferritin.

Acta Crystallogr D Biol Crystallogr 2015 Sep 25;71(Pt 9):1909-20. Epub 2015 Aug 25.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via Aldo Moro 2, 53100 Siena, Italy.

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein from Rana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with its R. catesbeiana counterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.
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http://dx.doi.org/10.1107/S1399004715013073DOI Listing
September 2015

Is His54 a gating residue for the ferritin ferroxidase site?

Biochim Biophys Acta 2015 Sep 26;1854(9):1118-22. Epub 2015 Feb 26.

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:

Ferritin is a ubiquitous iron concentrating nanocage protein that functions through the enzymatic oxidation of ferrous iron and the reversible synthesis of a caged ferric-oxo biomineral. Among vertebrate ferritins, the bullfrog M homopolymer ferritin is a frequent model for analyzing the role of specific amino acids in the enzymatic reaction and translocation of iron species within the protein cage. X-ray crystal structures of ferritin in the presence of metal ions have revealed His54 binding to iron(II) and other divalent cations, with its imidazole ring proposed as "gate" that influences iron movement to/from the active site. To investigate its role, His54 was mutated to Ala. The H54A ferritin variant was expressed and its reactivity studied via UV-vis stopped-flow kinetics. The H54A variant exhibited a 20% increase in the initial reaction rate of formation of ferric products with 2 or 4 Fe²⁺/subunit and higher than 200% with 20 Fe²⁺/subunit. The possible meaning of the increased efficiency of the ferritin reaction induced by this mutation is proposed taking advantage of the comparative sequence analysis of other ferritins. The data here reported are consistent with a role for His54 as a metal ion trap that maintains the correct levels of access of iron to the active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
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http://dx.doi.org/10.1016/j.bbapap.2015.02.011DOI Listing
September 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

Loop electrostatics modulates the intersubunit interactions in ferritin.

ACS Chem Biol 2014 Nov 29;9(11):2517-25. Epub 2014 Aug 29.

Magnetic Resonance Center CERM, University of Florence , Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.

Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.
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http://dx.doi.org/10.1021/cb500431rDOI Listing
November 2014

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

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C4 cage axes.

J Biol Inorg Chem 2014 Jun 7;19(4-5):615-22. Epub 2014 Feb 7.

Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA, 94609, USA,

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3·H2O minerals from Fe(2+) for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe(2+) transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe(2+)/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe(2+) + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3·H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.
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http://dx.doi.org/10.1007/s00775-014-1103-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083054PMC
June 2014

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

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

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

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts.

Biochimie 2012 Mar 17;94(3):597-607. Epub 2011 Sep 17.

Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.
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http://dx.doi.org/10.1016/j.biochi.2011.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314975PMC
March 2012

Sphingosine 1-phosphate induces differentiation of mesoangioblasts towards smooth muscle. A role for GATA6.

PLoS One 2011 24;6(5):e20389. Epub 2011 May 24.

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

Different cells can contribute to repair following vascular injury by differentiating into smooth muscle (SM) cells; however the extracellular signals involved are presently poorly characterized. Mesoangioblasts are progenitor cells capable of differentiating into various mesoderm cell types including SM cells. In this study the biological action exerted by the pleiotropic sphingolipid sphingosine 1-phosphate (S1P) in human mesoangioblasts has been initially investigated by cDNA microarray analysis. Obtained data confirmed the anti-apoptotic action of this sphingolipid and identified for the first time a strong differentiating action toward SM cells. Quantitative mRNA and protein analysis corroborated the microarray results demonstrating enhanced expression of myogenic marker proteins and regulation of the expression of transcription factor GATA6 and its co-regulator, LMCD1. Importantly, GATA6 up-regulation induced by S1P was responsible for the enhanced expression of SM-specific contractile proteins. Moreover, by specific gene silencing experiments GATA6 was critical in the pro-differentiating activity of the cytokine TGFβ. Finally, the pharmacological inhibition of endogenous S1P formation in response to TGFβ abrogated GATA6 up-regulation, supporting the view that the S1P pathway plays a physiological role in mediating the pro-myogenic effect of TGFβ. This study individuates GATA6 as novel player in the complex transcriptional regulation of mesoangioblast differentiation into SM cells and highlights a role for S1P to favour vascular regeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020389PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101247PMC
September 2011

Sphingosine kinase-1/S1P1 signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts.

Cell Signal 2010 Nov 1;22(11):1688-99. Epub 2010 Jul 1.

Dipartimento di Scienze Biochimiche, Universita; di Firenze, 50134 Firenze, Italy.

PDGF is known to be critically implicated in skeletal muscle repair; however its molecular mechanism of action has been only marginally investigated. In this study we show that in mouse myoblasts PDGF transactivates S1P(1) receptor via sphingosine kinase (SK)-1 activation and that this molecular event exerts a negative regulation of the mitogenic effect elicited by this growth factor. Indeed, pharmacological inhibition of S1P(1), or its specific silencing increased PDGF-dependent cell proliferation, whereas S1P(1) overexpression diminished the biological effect. Moreover, the mitogenic response to PDGF was enhanced by pharmacological inhibition of SK activity as well as specific silencing of SK1 but not SK2. Furthermore, ERK1/2 signalling pathway was found to be upstream of the observed attenuation of PDGF-induced cell proliferation. Interestingly, PDGF-directed engagement of S1P(1) exerted also a positive modulatory action of the growth factor-dependent cell motility. The here highlighted dual role of S1P(1)-mediated signalling in response to myoblast challenge with PDGF is likely important to guarantee the fine control of the biological response to this growth factor, finalized to efficient repopulation of skeletal muscle after damage, where a tight balance between proliferation and migration of tissue progenitor cells is required.
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http://dx.doi.org/10.1016/j.cellsig.2010.06.009DOI Listing
November 2010