Chiara Donati

Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor

In this study a novel biological activity of sphingosine 1‐phosphate (S1P) in C2C12 myoblasts was identified. In these cells the bioactive lipid profoundly regulated myogenesis exerting an antimitogenic activity, by reducing serum‐induced cell proliferation, and acting as powerful prodifferentiating agent by enhancing the expression of myogenic differentiation markers such as myogenin, myosin heavy chain, and caveolin‐3. The S1P‐dependent diminution of serum‐induced labeled thymidine incorporation was abrogated by antisense oligodeoxyribonucleotides (ODN) to S1P2, but not to S1P1 or S1P3 receptor, also expressed in C2C12 cells, implicating S1P2 in the biological response. Using antisense ODN and short interfering RNA treatment, we highlighted the key role played by S1P2 in the S1P‐dependent induction of muscle‐specific gene products. Notably, S1P2 overexpression increased the content of myogenic markers and hastened the onset of differentiated muscle phenotype in comparison with control cells. Cell treatment with pertussis toxin did not affect the biological responses to S1P, ruling out the involvement of Gi‐mediated events in the signaling promoted by the sphingolipid. Among the various signaling pathways activated by S1P, the activation of ERK1/ERK2 and p38 MAPK, both identified as downstream effectors of S1P2, was required for the inhibition of cell proliferation and the stimulation of myogenic differentiation, respectively.

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.

An active form of sphingosine kinase-1 is released in the extracellular medium as component of membrane vesicles shed by two human tumor cell lines.

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischaemia. During ischaemia, adenosine is in a concentration range (muM) that stimulates all four adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)). In recent years, evidence has indicated that the A(2A) receptor subtype is of critical importance in stroke. We have previously shown that 24 h after medial cerebral artery occlusion (MCAo), A(2A) receptors up-regulate on neurons and microglia of ischaemic striatum and cortex and that subchronically administered adenosine A(2A) receptor antagonists protect against brain damage and neurological deficit and reduce activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells. The mechanisms by which A(2A) receptors are noxious during ischaemia still remain elusive. The objective of the present study was to investigate whether the adenosine A(2A) antagonist SCH58261 affects JNK and MEK1/ERK MAPK activation. A further aim was to investigate cell types expressing activated JNK and MEK1/ERK MAPK after ischaemia. We hereby report that the selective adenosine A(2A) receptor antagonist, SCH58261, administered subchronically (0.01 mg/kg i.p) 5 min, 6 and 20 h after MCAo in male Wistar rats, reduced JNK MAPK activation (immunoblot analysis: phospho-JNK54 isoform by 81% and phospho-JNK46 isoform by 60%) in the ischaemic striatum. Twenty-four hours after MCAo, the Olig2 transcription factor of oligodendroglial progenitor cells and mature oligodendrocytes was highly expressed in cell bodies in the ischaemic striatum. Immunofluorescence staining showed that JNK MAPK is maximally expressed in Olig2-stained oligodendrocytes and in a few NeuN stained neurons. Striatal cell fractioning into nuclear and extra-nuclear fractions demonstrated the presence of Olig2 transcription factor and JNK MAPK in both fractions. The A(2A) antagonist reduced striatal Olig 2 transcription factor (immunoblot analysis: by 55%) and prevented myelin disorganization, assessed by myelin-associated glycoprotein staining. Twenty-four hours after MCAo, ERK1/2 MAPK was highly activated in the ischaemic striatum, mostly in microglia, while it was reduced in the ischaemic cortex. The A(2A) antagonist did not affect activation of the ERK1/2 pathway. The efficacy of A(2A) receptor antagonism in reducing activation of JNK MAPK in oligodendrocytes suggests a mechanism of protection consisting of scarring oligodendrocyte inhibitory molecules that can hinder myelin reconstitution and neuron functionality.

Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum

In the central nervous system (CNS) ATP and adenosine act as transmitters and neuromodulators on their own receptors but it is still unknown which part of extracellular adenosine derives per se from cells and which part is formed from the hydrolysis of released ATP. In this study extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under in vivo physiological conditions and after focal ischemia induced by medial cerebral artery occlusion. Under physiological conditions, adenosine and ATP concentrations were in the range of 130 nmol/L and 40 nmol/L, respectively. In the presence of the novel ecto-ATPase inhibitor, PV4 (100 nmol/L), the extracellular concentration of ATP increased 12-fold to ~360 nmol/L but the adenosine concentration was not altered. This demonstrates that, under physiological conditions, adenosine is not a product of extracellular ATP. In the first 4h after ischemia, adenosine increased to ~690 nmol/L and ATP to ~50 nmol/L. In the presence of PV4 the extracellular concentration of ATP was in the range of 450 nmol/L and a significant decrease in extracellular adenosine (to ~270 nmol/L) was measured. The contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia onset. Furthermore we demonstrated, by immunoelectron microscopy, the presence of the concentrative nucleoside transporter CNT2 on plasma and vesicle membranes isolated from the rat striatum. These results are in favor that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions. Early after ischemia, extracellular ATP is hydrolyzed by ecto-nucleotidases which significantly contribute to the increase in extracellular adenosine. To establish the contribution of extracellular ATP to adenosine might constitute the basis for devising a correct putative purinergic strategy aimed at protection from ischemic damage.

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells A role for protein kinase C

The present study showed that sphingosine 1‐phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor‐mediated since it was fully inhibited by pertussis toxin. All known SPP‐specific receptors, Edg‐1, Edg‐3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP‐induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCα, PKCδ and PKCλ, however, only PKCα and PKCδ played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCδ, prevented PLD stimulation.

Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the formation of sphingosine 1‐phosphate (S1P), an important lipid mediator, which regulates fundamental biological processes. Here, we provide evidence that SphK is required for the achievement of cell growth arrest as well as myogenic differentiation of C2C12 myoblasts. Indeed, SphK activity, SphK1 protein content and S1P formation were found to be enhanced in myoblasts that became confluent as well as in differentiating cells. Enforced expression of SphK1 reduced the myoblast proliferation rate, enhanced the expression of myogenic differentiation markers and anticipated the onset of differentiated muscle phenotype. Conversely, down‐regulation of SphK1 by specific silencing by RNA interference or overexpression of the catalytically inactive SphK1, significantly increased cell growth and delayed the beginning of myogenesis; noticeably, exogenous addition of S1P rescued the biological processes. Importantly, stimulation of myogenesis in SphK1‐overexpressing myoblasts was abrogated by treatment with short interfering RNA specific for S1P2 receptor. This is the first report of the role of endogenous SphK1 in myoblast growth arrest and stimulation of myogenesis through the formation of S1P that acts as morphogenic factor via the engagement of S1P2. J. Cell. Physiol. 214:210–220, 2008.

Pleiotropic effects of sphingolipids in skeletal muscle

Abstract.Studies of the last two decades have demonstrated that sphingolipids are important signalling molecules exerting key roles in the control of fundamental biological processes including proliferation, differentiation, motility and survival. Here we review the role of bioactive sphingolipids such as ceramide, sphingosine, sphingosine 1-phosphate, ganglioside GM3, in the regulation of skeletal muscle biology. The emerging picture is in favour of a complex role of these molecules, which appear implicated in the activation of muscle resident stem cells, their proliferation and differentiation, finalized at skeletal muscle regeneration. Moreover, they are involved in the regulation of contractile properties, tissue responsiveness to insulin and muscle fiber trophism. Hopefully, this article will provide a framework for future investigation into the field, aimed at establishing whether altered sphingolipid metabolism is implicated in the onset of skeletal muscle diseases and identifying new pharmacological targets for the therapy of multiple illnesses, including muscular dystrophies and diabetes.

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.