Caterina Bernacchioni

Transforming Growth Factor-β1 Induces Transdifferentiation of Myoblasts into Myofibroblasts via Up-Regulation of Sphingosine Kinase-1/S1P3 Axis

Transforming growth factor-β1 induces Smad-dependent transdifferentiation of myoblasts into myofibroblasts via up-regulation of sphingosine kinase-1/S1P3 axis with a mechanism involving Rho/Rho kinase activation.

Sphingosine 1‐Phosphate Mediates Proliferation and Survival of Mesoangioblasts

Mesoangioblasts are stem cells capable of differentiating in various mesodermal tissues and are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases, as well as myocardial infarction. The enhancement of their proliferation and survival after injection in vivo could greatly improve their ability to repopulate damaged tissues. In this study, we show that the bioactive sphingolipid sphingosine 1‐phosphate (S1P) regulates critical functions of mesoangioblast cell biology. S1P evoked a full mitogenic response in mesoangioblasts, measured by labeled thymidine incorporation and cell counting. Moreover, S1P strongly counteracted the apoptotic process triggered by stimuli as diverse as serum deprivation, C2‐ceramide treatment, or staurosporine treatment, as assessed by cell counting, as well as histone‐associated fragments and caspase‐3 activity determinations. S1P acts both as an intracellular messenger and through specific membrane receptors. Real‐time polymerase chain reaction analysis revealed that mesoangioblasts express the S1P‐specific receptor S1P3 and, to a minor extent, S1P1 and S1P2. By using S1P receptor subtype‐specific agonists and antagonists, we found that the proliferative response to S1P was mediated mainly by S1P2. By contrast, the antiapoptotic effect did not implicate S1P receptors. These findings demonstrate an important role of S1P in mesoangioblast proliferation and survival and indicate that targeting modulation of S1P‐dependent signaling pathways may be used to improve the efficiency of muscle repair by these cells.

A protective role for lipid raft cholesterol against amyloid-induced membrane damage in human neuroblastoma cells

Increasing evidence supports the idea that the initial events of Abeta oligomerization and cytotoxicity in Alzheimers disease involve the interaction of amyloid Abeta-derived diffusible ligands (ADDLs) with the cell membrane. This also indicates lipid rafts, ordered membrane microdomains enriched in cholesterol, sphingolipids and gangliosides, as likely primary interaction sites of ADDLs. To shed further light on the relation between ADDL-cell membrane interaction and oligomer cytotoxicity, we investigated the dependence of ADDLs binding to lipid rafts on membrane cholesterol content in human SH-SY5Y neuroblastoma cells. Confocal laser microscopy showed that Abeta1-42 oligomers markedly interact with membrane rafts and that a moderate enrichment of membrane cholesterol prevents their association with the monosialoganglioside GM1. Moreover, anisotropy fluorescence measurements of flotillin-1-positive rafts purified by sucrose density gradient suggested that the content of membrane cholesterol and membrane perturbation by ADDLs are inversely correlated. Finally, contact mode atomic force microscope images of lipid rafts in liquid showed that ADDLs induce changes in raft morphology with the appearance of large cavities whose size and depth were significantly reduced in similarly treated cholesterol-enriched rafts. Our data suggest that cholesterol reduces amyloid-induced membrane modifications at the lipid raft level by altering raft physicochemical features.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells

Abstract.Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as α-smooth muscle actin (αSMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated αSMA. More importantly, S1P challenge was responsible for the functional appearance of Ca2+ currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P2 turned out to be critical for the pro-differentiating effect of S1P, while S1P3 appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

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 Gi-coupled C1P receptor as pertussis toxin, which maintains Gi 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.

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

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.

Sphingosine Kinase Mediates Resistance to the Synthetic Retinoid N-(4-Hydroxyphenyl)retinamide in Human Ovarian Cancer Cells

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

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

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.—Cencetti, F., Bernacchioni, C., Tonelli, F., Roberts, E., Donati, C., Bruni, P. TGFβ1 evokes myoblast apoptotic response via a novel signaling pathway involving S1P4 transactivation upstream of Rho‐kinase‐2 activation. FASEB J. 27, 4532–4546 (2013). www.fasebj.org

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

BackgroundInsulin-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.MethodsRNA 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.ResultsWe 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.ConclusionsWe 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.

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

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.