Ruthenium(II) Polypyridyl Complexes with Benzoxazole Derivatives and Non-Innocent Ligands as Effective Antioxidants in Human Neuroblasts.

Ruthenium(II) polypyridyl complexes continue to raise increasing interest for the encouraging results in several biomedical areas. Considering their vast chemical-physical repertoire, in particular the possibility to switch from the sensitization of reactive oxygen species (ROS) to ROS-scavenging abilities by tuning the nature of their ligands, it is therefore surprising that their potential as antioxidants has not been largely investigated so far. Herein, we explored the antioxidant behaviour of the novel ruthenium compound [Ru(dbpy)(2,3-DAN)Cl]PF6 (Ru1), featuring a benzoxazole derivative (dpby=2,6-bis(4-methyl-2-benzoxazolyl)pyridine) and the non-innocent 2,3-diamminonaftalene (2,3-DAN) ligand, along with the reference tpy-containing analogue [Ru(tpy)(2,3-DAN)Cl]PF6 (Ru2) (tpy=2,2':6',2''-terpyridine). Following the synthesis and the electrochemical characterization, chemical antioxidant assays highlighted the beneficial role of dpby for the ROS-scavenging properties of Ru1. These data have been corroborated by the highest protective effect of Ru1 against the oxidative stress induced in SH-SY5Y human neuroblastoma, which exerts pro-survival and anti-inflammatory actions. The results herein reported highlight the potential of Ru1 as pharmacological tool in neurodegenerative diseases and specially prove that the antioxidant properties of such compounds are likely the result of a non-trivial synergetic action involving the bioactive ligands in their chemical architectures.

Sphingosine-1-phosphate receptor 3 is a non-hormonal target to counteract endometriosis-associated fibrosis.

OBJECTIVE: To study the molecular mechanisms responsible for fibrosis in endometriosis by investigating whether the protein expression levels of sphingosine-1-phosphate receptor 3 (S1PR3), one of the five specific receptors of the bioactive sphingolipid sphingosine-1-phosphate (S1P), correlate with fibrosis extent in endometriotic lesions and which are the cellular mechanisms involved in this process. DESIGN: Case-control laboratory study and cultured endometriotic cells. SETTING: University research institute and university hospital. PATIENT(S): A total of 33 women, with and without endometriosis, were included in the study. INTERVENTIONS(S): Endometriotic lesions were obtained from women with endometriosis (ovarian endometrioma, n = 8; deep infiltrating endometriosis, n = 15; [urological n = 5, gastrointestinal n = 6, and posterior n = 4]) and control endometrium from healthy women, n = 10, by means of laparoscopic and hysteroscopic surgery. The expression of S1PR3 was evaluated using immunohistochemistry and the extent of fibrosis was assessed using Masson's trichrome staining. Human-cultured epithelial endometriotic 12Z cells were used to evaluate the mechanisms involved in the profibrotic effect of S1PR3 activation. MAIN OUTCOME MEASURE(S): The expression of S1PR3 in endometriotic lesions is positively correlated with endometriosis-associated fibrosis. In addition, S1P induced epithelial-mesenchymal transition (EMT) and fibrosis in epithelial endometriotic cells. Using RNA interference and pharmacological approaches, the profibrotic effect of S1P was shown to rely on S1PR3, thus unveiling the molecular mechanism implicated in the profibrotic action of the bioactive sphingolipid. RESULT(S): The protein expression levels of S1PR3 were significantly augmented in the glandular sections of endometrioma and deep infiltrating endometriosis of different localizations with respect to the control endometrium and positively correlated with the extent of fibrosis. Sphingosine-1-phosphate was shown to have a crucial role in the onset of fibrosis in epithelial endometriotic cells, stimulating the expression of EMT and fibrotic markers. Genetic approaches have highlighted that S1PR3 mediates the fibrotic effect of S1P. Downstream of S1PR3, ezrin and extracellular-signal-regulated kinases 1 and 2 signaling were found to be critically implicated in the EMT and fibrosis elicited by S1P. CONCLUSION(S): Sphingosine-1-phosphate receptor 3 may represent a possible innovative pharmacological target for endometriosis.

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target.

RESEARCH QUESTION: Is the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DESIGN: The hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. RESULTS: The hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. CONCLUSIONS: These data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.

Sphingosine 1-phosphate attenuates neuronal dysfunction induced by amyloid-ß oligomers through endocytic internalization of NMDA receptors.

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aß) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aß (Aß42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aß toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aß42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aß42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aß42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aß42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.

Sphingosine 1-phosphate pathway is dysregulated in adenomyosis.

RESEARCH QUESTION: Is sphingosine 1-phosphate (S1P) pathway involved in the process of fibrosis in adenomyosis? DESIGN: RNA was extracted from paraffin-embedded slices collected from the ectopic endometrium of patients with nodular adenomyosis (n = 27) and eutopic endometrium of healthy controls women (n = 29). Expression of genes involved in the metabolism and signalling of S1P, and actin-alpha-2 smooth muscle, encoded by ACTA2 gene, a gene involved in fibrogenesis, was evaluated by real-time polymerase chain reaction analysis. RESULTS: In adenomyotic samples, the expression of sphingosine kinase 1 (SPHK1), the enzyme responsible for the synthesis of S1P, and of S1P phosphatase 2 (SGPP2), the enzyme responsible for the conversion of S1P back to sphingosine, was lower (P = 0.0006; P = 0.0015), whereas that of calcium and integrin-binding protein 1, responsible for membrane translocation of SPHK1, was higher (P = 0.0001) compared with healthy controls. In S1P signalling, a higher expression of S1P receptor S1P3 (P = 0.001), and a lower expression of S1P2 (P = 0.0019) mRNA levels, were found compared with healthy endometrium. In adenomyotic nodules, a higher expression of ACTA2 mRNA levels were observed (P = 0.0001), which correlated with S1P3 levels (P = 0.0138). CONCLUSION: Present data show a profound dysregulation of the S1P signalling axis in adenomyosis. This study also highlights that the bioactive sphingolipid might be involved in the fibrotic tract of the disease, correlated with the expression of ACTA2, suggesting its role as novel potential biomarker of adenomyosis.

Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes.

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P1-5. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P3 prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect.

OBJECTIVE: To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A. DESIGN: Case-control laboratory study. SETTING: University institute and university hospital. PATIENT(S): Patients with uterine fibroids (n = 26). INTERVENTIONS(S): Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy. MAIN OUTCOME MEASURE(S): Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines. RESULT(S): The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P2, S1P3, and S1P5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P2. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells. CONCLUSION(S): This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.

Sphingosine 1-phosphate receptors are dysregulated in endometriosis: possible implication in transforming growth factor ß-induced fibrosis.

OBJECTIVE: To study the molecular mechanisms involved in the appearance of the fibrotic trait in endometriosis by investigating whether the signaling pathway of the bioactive sphingolipid sphingosine 1-phosphate (S1P) was altered in endometriotic lesions. DESIGN: Case-control laboratory study. SETTING: University research institute and university hospital. PATIENT(S): A total of 75 women, with and without endometriosis, were included in the study. INTERVENTIONS(S): Endometrial samples were obtained from women affected (n = 15 endometrioma [OMA]; n = 30 deep infiltrating endometriosis [DIE]) and not (n = 30) by endometriosis by means of laparoscopic surgery, followed by clinical and imaging investigation and checking for the expression of fibrosis markers and genes implicated in S1P metabolism and signaling by means of real-time polymerase chain reaction. MAIN OUTCOME MEASURE(S): The role of the S1P signaling axis in endometriosis-associated fibrosis was studied in vitro, where RNA interference approaches were used to investigate if S1P synthesis by sphingosine kinases (SKs) and specific S1P receptors (S1PRs) are implicated in the profibrotic effect of the cytokine transforming growth factor (TGF) ß1. RESULT(S): mRNA expression analysis of S1PR demonstrated a deep dysregulation of S1P signaling in endometriosis, characterized by increased expression of fibrosis markers: S1P1 was transcriptionally more expressed in OMA, and S1P3 and S1P5 mRNA levels were significantly augmented in both OMA and DIE. SK1 and its activating protein calcium- and integrin-binding protein 1 (CIB1) were significantly up-regulated in OMA and DIE. A crucial role for the SK/S1PR axis in the profibrotic effect elicited by TGFß1 was highlighted in vitro. CONCLUSION(S): The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis.

Adenosine A2B receptors inhibit K+ currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. No data are available to date about the A2B receptor (A2BR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) are also crucial modulators of OPC development. An interaction between this pathway and the A2BR is reported in peripheral cells. We studied the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC and the effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.

Sphingosine 1-phosphate-mediated activation of ezrin-radixin-moesin proteins contributes to cytoskeletal remodeling and changes of membrane properties in epithelial otic vesicle progenitors.

Hearing loss is among the most prevalent sensory impairments in humans. Cochlear implantable devices represent the current therapies for hearing loss but have various shortcomings. ERM (ezrin- radixin -moesin) are a family of adaptor proteins that link plasma membrane with actin cytoskeleton, playing a crucial role in cell morphology and in the formation of membrane protrusions. Recently, bioactive sphingolipids have emerged as regulators of ERM proteins. Sphingosine 1-phosphate (S1P) is a pleiotropic sphingolipid which regulates fundamental cellular functions such as proliferation, survival, migration as well as processes such as development and inflammation mainly via ligation to its specific receptors S1PR (S1P1-5). Experimental findings demonstrate a key role for S1P signaling axis in the maintenance of auditory function. Preservation of cellular junctions is a fundamental function both for S1P and ERM proteins, crucial for the maintenance of cochlear integrity. In the present work, S1P was found to activate ERM in a S1P2-dependent manner in murine auditory epithelial progenitors US/VOT-E36. S1P-induced ERM activation potently contributed to actin cytoskeletal remodeling and to the appearance of ionic currents and membrane passive properties changes typical of more differentiated cells. Moreover, PKC and Akt activation was found to mediate S1P-induced ERM phosphorylation. The obtained findings contribute to demonstrate the role of S1P signaling pathway in inner ear biology and to disclose potential innovative therapeutical approaches in the field of hearing loss prevention and treatment.

Cancer cell death induced by ferritins and the peculiar role of their labile iron pool.

Cellular uptake of human H-ferritin loaded with 50 or 350 iron ions results in significant cytotoxicity on HeLa cells at submicromolar concentrations. Conversely, Horse Spleen Ferritin, that can be considered a model of L-cages, as it contains only about 10% of H subunits, even when loaded with 1000 iron ions, is toxic only at >1 order of magnitude higher protein concentrations. We propose here that the different cytotoxicity of the two ferritin cages originates from the presence in H-ferritin of a pool of non-biomineralized iron ions bound at the ferroxidase catalytic sites of H-ferritin subunits. This iron pool is readily released during the endosomal-mediated H-ferritin internalization.

S1P promotes migration, differentiation and immune regulatory activity in amniotic-fluid-derived stem cells.

Stem cells have high potential for cell therapy in regenerative medicine. We previously isolated stem cell types from human amniotic fluid, derived from prenatal amniocentesis. One type, characterized by a fast doubling time, was designated as fast human amniotic stem cells (fHASCs). These cells exhibited high differentiation potential and immunoregulatory properties. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that influences stem-cell pluripotency, differentiation, mobility, and regulates immune functions. In this study, we investigated the influence of S1P on fHASC migration, proliferation, differentiation and immune regulatory functions. We found that fHASC stimulation with S1P potentiated their migratory and proliferative activity in vitro. Notably, short fHASC exposure to S1P enhanced their differentiation towards multiple lineages, including adipocytes, osteocytes and endothelial cells, an effect that was associated with downregulation of the main transcription factors involved in the maintenance of a stem-cell undifferentiated state. A specific crosstalk between S1P and tumor growth factor ß1 (TGF-ß1) has recently been demonstrated. We found that fHASC exposure to S1P in combination with TGF-ß1 promoted the expression of the immune regulatory pathway of indoleamine 2,3-dioxygenase 1 (IDO1). In addition, human peripheral blood mononuclear cells, co-cultured with fHASCs treated with S1P and TGF-ß1, expanded regulatory T-cells, via a mechanism requiring IDO1. Overall, this study demonstrates that S1P potentiates several properties in fHASCs, an effect that may be critical for exploiting the therapeutic potential of fHASCs and might explain the specific effects of S1P on stem cells during pregnancy.

Lysophosphatidic Acid Signaling Axis Mediates Ceramide 1-Phosphate-Induced Proliferation of C2C12 Myoblasts.

Sphingolipids are not only crucial for membrane architecture but act as critical regulators of cell functions. The bioactive sphingolipid ceramide 1-phosphate (C1P), generated by the action of ceramide kinase, has been reported to stimulate cell proliferation, cell migration and to regulate inflammatory responses via activation of different signaling pathways. We have previously shown that skeletal muscle is a tissue target for C1P since the phosphosphingolipid plays a positive role in myoblast proliferation implying a role in muscle regeneration. Skeletal muscle displays strong capacity of regeneration thanks to the presence of quiescent adult stem cells called satellite cells that upon trauma enter into the cell cycle and start proliferating. However, at present, the exact molecular mechanism by which C1P triggers its mitogenic effect in myoblasts is lacking. Here, we report for the first time that C1P stimulates C2C12 myoblast proliferation via lysophosphatidic acid (LPA) signaling axis. Indeed, C1P subsequently to phospholipase A2 activation leads to LPA1 and LPA3 engagement, which in turn drive Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinases 1/2) activation, thus stimulating DNA synthesis. The present findings shed new light on the key role of bioactive sphingolipids in skeletal muscle and provide further support to the notion that these pleiotropic molecules might be useful therapeutic targets for skeletal muscle regeneration.

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

NMR metabolomics highlights sphingosine kinase-1 as a new molecular switch in the orchestration of aberrant metabolic phenotype in cancer cells.

Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR-based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO2 production. Additionally, SK1-expressing cells displayed a significant increase in glucose uptake paralleled by GLUT3 transporter upregulation. The role of SK1 is not limited to the induction of aerobic glycolysis, affecting metabolic pathways that appear to support the biosynthesis of macromolecules. These findings highlight the role of SK1 signaling axis in cancer metabolic reprogramming, pointing out innovative strategies for cancer therapies.

Ferroxidase Activity in Eukaryotic Ferritin is Controlled by Accessory-Iron-Binding Sites in the Catalytic Cavity.

Ferritins are iron-storage nanocage proteins that catalyze the oxidation of Fe2+ to Fe3+ at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe2+ substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild-type cages.

Iron binding to human heavy-chain ferritin.

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein from Rana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with its R. catesbeiana counterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.

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.

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-3ß 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 G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) 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 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.