Control of Skeletal Muscle Atrophy Associated to Cancer or Corticosteroids by Ceramide Kinase.

Apart from cytokines and chemokines, sphingolipid mediators, particularly sphingosine-1-phosphate (S1P) and ceramide 1-phosphate (C1P), contribute to cancer and inflammation. Cancer, as well as other inflammatory conditions, are associated with skeletal muscle (SkM) atrophy, which is characterized by the unbalance between protein synthesis and degradation. Although the signaling pathways involved in SkM mass wasting are multiple, the regulatory role of simple sphingolipids is limited. Here, we report the impairment of ceramide kinase (CerK), the enzyme responsible for the phosphorylation of ceramide to C1P, associated with the accomplishment of atrophic phenotype in various experimental models of SkM atrophy: in vivo animal model bearing the C26 adenocarcinoma or Lewis lung carcinoma tumors, in human and murine SkM cells treated with the conditioned medium obtained from cancer cells or with the glucocorticoid dexamethasone. Notably, we demonstrate in all the three experimental approaches a drastic decrease of CerK expression. Gene silencing of CerK promotes the up-regulation of atrogin-1/MAFbx expression, which was also observed after cell treatment with C8-ceramide, a biologically active ceramide analogue. Conversely, C1P treatment significantly reduced the corticosteroid's effects. Altogether, these findings provide evidence that CerK, acting as a molecular modulator, may be a new possible target for SkM mass regulation associated with cancer or corticosteroids.

Ceramide/protein phosphatase 2A axis is engaged in gap junction impairment elicited by PCB153 in liver stem-like progenitor cells.

The widespread environmental pollutant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) is a non-dioxin-like toxicant. It is a potential carcinogen compound able to induce gap junction (GJ) intercellular communication impairment, probably the first non-genomic event leading to tumor promotion. Although PCBs have been known for many years, the molecular mode of PCB153 action is still unclear. Recent studies from our research group have shown that the toxicant elicits a transient modulation of connexin (Cx) 43-formed GJs in hepatic stem-like WB-F344 cells involving sphingosine 1-phosphate (S1P) path. Taking into account that other strictly related bioactive sphingolipids, such as ceramide (Cer), may have different effects from S1P, here we aim to clarify the signaling paths engaged by PCB153 in the control of GJs, focusing primarily on the role of Cer. Accordingly, we have achieved a combined biomolecular and electrophysiological analysis of GJs in cultured WB-F344 cells treated with PCB153 at different time points. We have found that the toxicant elicited a time-dependent regulation of GJs formed by different Cx isoforms, through a transient modulation of Cer/Cer kinase (CerK) axis and, in turn, of protein phosphatase 2A (PP2A). Our new findings demonstrate the existence of a specific molecular mechanism downstream to Cer, which distinctly affects the voltage-dependent and -independent GJs in liver stem-like cells, and open new opportunities for the identification of additional potential targets of these environmental toxicants.

Involvement of released sphingosine 1-phosphate/sphingosine 1-phosphate receptor axis in skeletal muscle atrophy.

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known. Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts. Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.

Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation.

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs' ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair.

Sphingosine 1-Phosphate Receptors: Do They Have a Therapeutic Potential in Cardiac Fibrosis?

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is characterized by a peculiar mechanism of action. In fact, S1P, which is produced inside the cell, can act as an intracellular mediator, whereas after its export outside the cell, it can act as ligand of specific G-protein coupled receptors, which were initially named endothelial differentiation gene (Edg) and eventually renamed sphingosine 1-phosphate receptors (S1PRs). Among the five S1PR subtypes, S1PR1, S1PR2 and S1PR3 isoforms show broad tissue gene expression, while S1PR4 is primarily expressed in immune system cells, and S1PR5 is expressed in the central nervous system. There is accumulating evidence for the important role of S1P as a mediator of many processes, such as angiogenesis, carcinogenesis and immunity, and, ultimately, fibrosis. After a tissue injury, the imbalance between the production of extracellular matrix (ECM) and its degradation, which occurs due to chronic inflammatory conditions, leads to an accumulation of ECM and, consequential, organ dysfunction. In these pathological conditions, many factors have been described to act as pro- and anti-fibrotic agents, including S1P. This bioactive lipid exhibits both pro- and anti-fibrotic effects, depending on its site of action. In this review, after a brief description of sphingolipid metabolism and signaling, we emphasize the involvement of the S1P/S1PR axis and the downstream signaling pathways in the development of fibrosis. The current knowledge of the therapeutic potential of S1PR subtype modulators in the treatment of the cardiac functions and fibrinogenesis are also examined.

Vitamin D3 protects against Aß peptide cytotoxicity in differentiated human neuroblastoma SH- SY5Y cells: A role for S1P1/p38MAPK/ATF4 axis.

Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH)2D3. We have found that 1,25(OH)2D3 prevents amyloid-ß peptide (Aß(1-42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo. In differentiated SH-SY5Y cells, Aß(1-42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH)2D3 partially reverts this effect. 1,25(OH)2D3 reverts also the Aß(1-42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH)2D3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH)2D3 prevents the reduction of S1P1 expression promoted by Aß(1-42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH)2D3 protects from aggregated Aß(1-42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH)2D3 and its structural analogue ZK191784 in counteracting the Aß(1-42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells.

Role of sphingosine kinase/S1P axis in ECM remodeling of cardiac cells elicited by relaxin.

The initiation and progression of heart failure is linked to adverse cardiac remodeling of the extracellular matrix (ECM) during disease mainly through the deregulation of myocardial metalloproteinases (MMPs). Relaxin (RLX), a peptide hormone acting as a physiological cardiac effector, is a key regulator of ECM remodeling in reproductive and nonreproductive tissues. Studying primary cultures of mouse cardiac muscle cells and rat H9c2 cardiomyoblasts, we have obtained evidence for a new signaling pathway activated by RLX to induce ECM remodeling that involves the bioactive sphingolipids sphingosine-1-phosphate (S1P) and ceramide. In both cell populations, recombinant human RLX increased sphingosine kinase activity and S1P formation, whereas sphingomyelin and ceramide content were decreased in [(3)H]serine-labeled cells. According to the literature, RLX promoted MMP-2 and MMP-9 expression/release. Pharmacological inhibition of sphingolipid metabolism and silencing of sphingosine kinase 1, the enzyme responsible for S1P formation, were able to prevent MMP expression/release elicited by the hormone and induce the expression of tissue inhibitor of MMPs. In addition, we found that sphingolipid signaling is required for the regulation of connective tissue growth factor, a member of the CCN 1-3 family of genes that are involved in cell proliferation and differentiation. Finally, the induction of cardiomyoblast maturation induced by RLX was also found to be counteracted by inhibition of S1P formation. In conclusion, these findings provide a novel mechanism by which RLX acts on cardiac ECM remodeling and cardiac cell differentiation and offer interesting therapeutic options to prevent heart fibrosis and to favor myocardial regeneration.

Mesenchymal stromal cell secreted sphingosine 1-phosphate (S1P) exerts a stimulatory effect on skeletal myoblast proliferation.

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

Nutraceutical properties of chestnut flours: beneficial effects on skeletal muscle atrophy.

Plants contain a wide range of non-nutritive phytochemicals, many of which have protective or preventive properties for human diseases. The aim of the present work has been to investigate the nutraceutical properties of sweet chestnut flour extracts obtained from fruits collected from 7 geographic areas of Tuscany (Italy), and their ability in modulating skeletal muscle atrophy. We found that the cultivars from different geographic areas are characterized by the composition and quantity of various nutrients and specific bioactive components, such as tocopherols, polyphenols and sphingolipids. The nutraceutical properties of chestnut sweet flours have been evaluated in C2C12 myotubes induced to atrophy by serum deprivation or dexamethasone. We found that the pretreatment with both total extracts of tocopherols and sphingolipids is able to counterbalance cell atrophy, reducing the decrease in myotube size and myonuclei number, and attenuating protein degradation and the increase in expression of MAFbx/atrogin-1 (a muscle-specific atrophy marker). By contrast, polyphenol extracts were not able to prevent atrophy. Since we also found that γ-tocopherol is the major form of tocopherol in sweet flour and its content differs depending on the procedure of sweet flour preparation, the mechanisms by which γ-tocopherol as well as sphingolipids affect skeletal muscle cell atrophy have been also investigated. This is the first evidence that chestnut sweet flour is a natural source of specific bioactive components with a relevant role in the prevention of cell degeneration and maintenance of skeletal muscle mass, opening important implications in designing appropriate nutritional therapeutic approaches to skeletal muscle atrophy.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase.

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)2D3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)2D3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)2D3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)2D3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)2D3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

DNA induced dimerization of a sulfhydryl surfactant in transfection agents studied by ESR spectroscopy.

Synthetic vectors for gene delivery offer a wide variety of functional derivatization, which can be exploited to increase targeting and transfection efficacy. In this field, redox-sensitive agents based on the thiol/disulfide (-SH/-SS-) equilibrium are a class of promising transfectants. Here the thiol group content in lipoplexes formed by a triazine-based sulfhydryl surfactant (SH14) and a plasmid (pGEFP-N1) was probed by Electron Spin Resonance (ESR) of appropriately tailored nitroxides. By modelling the time decay of ESR intensity, details on the process of lipoplex formation were obtained. It was found that the concentration of available -SH groups depended on the contact time between SH14 and DNA, suggesting that lipoplex formation entailed disulfide bridge formation among SH14 molecules. This finding represents the first experimental evidence that the -SH/-SS- equilibrium plays a role in lipoplex formation when DNA is complexed by sulfhydryl-based transfecting agents, which may have profound influence on their mechanism of action.