Marta Farnararo

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.

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.

Characterization of sphingomyelinase activity released by thrombin-stimulated platelets

In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.

Tumor necrosis factor‐α exerts pro‐myogenic action in C2C12 myoblasts via sphingosine kinase/S1P2 signaling

In this study, we report that low doses of tumor necrosis factor‐α (TNFα) promote myogenesis in C2C12 myoblasts. Moreover, the cytokine increased sphingosine kinase (SphK) activity and induced SphK1 translocation to membranes. The inhibition of SphK functionality by various approaches abrogated the pro‐myogenic effect of TNFα. Moreover, silencing of S1P2 impaired the positive action of TNFα on myogenesis. These results represent the first evidence that SphK/S1P2 axis is required for the regulation of myogenesis by TNFα. In view of the physiological role of TNFα in muscle regeneration, the present finding reinforces the notion that SphK/S1P2 signaling is critically implicated in myogenesis.

Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D

The bioactive lipid sphingosine 1-phosphate (S1P) is known to exert powerful biological effects through the interaction with various members of the endothelial differentiation gene (EDG) receptor family, recently renamed S1P receptors. In the present study, evidence is provided that differentiation of C2C12 myoblasts into myotubes was accompanied by profound changes of EDG/S1P receptor expression. Indeed, in differentiated cells a significant increase of EDG3/S1P3 together with a large decrease of EDG5/S1P2 expression at mRNA as well as protein level was detected. Moreover, S1P was capable to initiate the signalling pathways downstream to cytosolic Ca(2+) increase in myotubes, similarly to that observed in myoblasts, whereas the signalling of the bioactive lipid to phospholipase D (PLD), but not that of bradykinin (BK) or lysophosphatidic acid (LPA), was found impaired in differentiated cells. Intriguingly, overexpression of EDG5/S1P2, but not EDG1/S1P1 or EDG3/S1P3, potentiated the efficacy of S1P to stimulate PLD, strongly suggesting a role for EDG5/S1P2 in the signalling to PLD. This view was also supported by the marked reduction of S1P-induced PLD activity in myoblasts loaded with antisense oligodeoxyribonucleotides (ODN) to EDG5/S1P2. Furthermore, overexpression of EDG5/S1P2 rescued the coupling of S1P signalling to PLD in C2C12 myotubes. Experimental evidence here provided supports the notion that EDG5/S1P2 plays a dominant role in the coupling of S1P to PLD in myoblasts and that the down-regulation of the receptor subtype is responsible for the specific uncoupling of S1P signalling to PLD in myotubes.

Regulation by phosphate of alkaline phosphatase in rat kidney

Abstract The mechanism by which inorganic phosphate regulates the level of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney has been investigated: changes in the level of inorganic phosphate in the renal cell were produced by dietary treatment. The regulatory effect appears to be specific. Out of 10 enzymes studied only alkaline phosphatase increases when phosphate decreases. The decrease in the level of phosphate, under our conditions, is not accompanied by a general change in the pattern of phosphorylated metabolites. The regulation by phosphate is abolished by inhibitors of protein or nucleic acid synthesis. These features of the regulatory system are consistent with a mechanism of genic repression, that could be fundamentally similar to that described for Escherichia coli . The above conclusion has been confirmed by studying the time course of derepression and repression of alkaline phosphatase by following successive changes in phosphate level in the kidney. The half-life of enzyme breakdown during repression was found to be 0.9 day, and this value corresponds (in the same order of magnitude) with the known half-lives for other proteins of the rat.

Synthesis of diacylglycerol de novo is responsible for permanent activation and down-regulation of protein kinase C in transformed cells

We measured the synthesis of diacylglycerol de novo in normal NIH/3T3 fibroblasts and in cells transformed by ras, src, sis and abl oncogenes. Analysis of the incorporation of glucose-derived 14C into diacylglycerol indicated that neosynthesis of diacylglycerol was constitutively active in the transformed cell lines. Elevated levels of diacylglycerol and persistent activation/down-regulation of protein kinase C reduced the binding of phorbol dibutyrate to transformed cells. This phenomenon could be reversed by blocking the glycolytic pathway, thus indicating that neosynthesized diacylglycerol was responsible for persistent activation and down-regulation of protein kinase C. In transformed cells, protein kinase C activity could not be stimulated by the addition of diolein; however, inhibition of glycolysis restored the ability of transformed cells to respond to diolein. Taken together these data indicate that constitutive synthesis of diacylglycerol de novo is responsible for activation and down-regulation of protein kinase C in transformed cells, and it may play a role in altered mitogenic signalling.

Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin‐enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.

Evidence for the identity of alkaline phosphatase and inorganic pyrophosphatase in rat kidney

Abstract Alkaline phosphatase, extensively purified from rat kidney, was found to hydrolyze inorganic pyrophosphate. The following experimental results suggest that alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase. 1. 1. The purification of rat kidney alkaline phosphatase was accompanied by a concomitant increase in inorganic pyrophosphatase activity and an almost constant ratio between the two enzyme activities at different purification steps was found. Moreover, both enzymatic activities were eluted together as a single peak after DEAE-Sephadex A-25 chromatography of microsomal proteins solubilized by deoxycholate and lipase. 2. 2. A decrease in the level of inorganic phosphate in the renal cell produces concomitant and significant increases of both enzymatic activities. 3. 3. The amount of phosphate released in the mixed substrate experiments was less than would be expected for independent hydrolysis of the substrates. 4. 4. Both enzyme activities were affected similarly by cysteine, a noncompetitive inhibitor, and by heating in the absence of substrate.