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.

We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in A Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxylterminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.

Transient receptor potential canonical (TRPC) channels provide cation and Ca2+ entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca2+ influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

Objective-Tumor necrosis factor ␣ (TNF-␣), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. Methods and Results-In human endothelial cells, we found that eNOS activation by TNF-␣ is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-␣. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P 1 and S1P 3 , either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-␣ via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-␣. Key Words: endothelial NO synthase Ⅲ TNF-␣ Ⅲ neutral sphingomyelinase 2 Ⅲ sphingosine kinase 1 Ⅲ sphingosine 1 phosphate N itric oxide (NO) generated in the endothelium by the endothelial NO synthase (eNOS) plays crucial roles not only in physiology but also in regulating specific inflammatory events involving the endothelium, such as the expression of adhesion molecules and leukocyte adhesion. 1,2 Indeed, eNOS Ϫ/Ϫ animals, when exposed to inflammatory stimuli, display significant increases in extravasated neutrophils and poor wound healing and are more prone to diseases in which inflammation is significant, including atherosclerosis and diabetes. 1,2 eNOS regulates the effects on migration, angiogenesis, vasodilation, endothelial barrier permeability, apoptosis, and expression of inflammatory and adhesion molecules exerted by tumor necrosis factor ␣ (TNF-␣), a key proinflammatory cytokine acting on the endothelium. [3][4][5][6][7][8][9] In some cases, the effects of NO appeared directly associated with eNOS activity; 8,9 in others, they were consistent with the involvement of this enzyme, because they were too rapid to be explained by synthesis of the inducible NO synthase (NOS) 1,2 or because they took place in human cells 4,7 that do not express inducible NOS when exposed to TNF-␣ alone. 8,10 Despite the relevance to TNF-␣ action, eNOS activation by the cytokine in endothelial cells had been investigated in only 2 studies 8,11 that did not provide evidence of the pathway involved, apart from the identification of a phosphatidylinositol 3-kinase (PI3K)/Aktdependent step, 11 a signaling event common to many eNOSactivating stimuli. 2 In a recent study in HeLa cells, we reported that eNOS activation by TNF-␣ is mediated by the activation of neutral sphingomyelinase (N-SMase), with generation of the sphingolipid ceramid...

Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the ␣L␤2

In the post-infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM-1-,VCAM-positive). C2C12 myoblasts did not trans-differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin-producing C2C12 myoblasts displayed greater efficacy to engraft the post-ischaemic scar and to induce extracellular matrix re-modelling and angiogenesis as compared with the control cells. By echocardio-graphy, C2C12-engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post-infarcted patients.

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 S1P(2) 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 S1P(2).

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43⌬130 -136 reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.