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Human ferritin platforms containing Ru(ii)-polypyridyl-based photosensitizers effectively target cancer cells and provide cytotoxic effects upon light-activation.

Ovarian cancer recurrence is frequent and associated with chemoresistance, leading to extremely poor prognosis. Herein, we explored the potential anti-cancer effect of a series of highly charged Ru(II)-polypyridyl complexes as photosensitizers in photodynamic therapy (PDT), which were able to efficiently sensitize the formation of singlet oxygen upon irradiation (Ru12+ and Ru22+) and to produce reactive oxygen species (ROS) in their corresponding dinuclear metal complexes with the Fenton active Cu(II) ion/s ([CuRu1]4+ and [Cu2Ru2]6+). Their cytotoxic and anti-tumor effects were evaluated on human ovarian cancer A2780 cells both in the absence or presence of photoirradiation, respectively. All the compounds tested were well tolerated under dark conditions, whereas they switched to exert anti-tumor activity following photoirradiation. The specific effect was mediated by the onset of programed cell death, but only in the case of Ru12+ and Ru22+ was preceded by the loss of mitochondrial membrane potential soon after photoactivation and ROS production, thus supporting the occurrence of apoptosis via type II photochemical reactions. Thus, Ru(II)-polypyridyl-based photosensitizers represent challenging tools to be further investigated in the identification of new therapeutic approaches to overcome the innate chemoresistance to platinum derivatives of some ovarian epithelial cancers and to find innovative drugs for recurrent ovarian cancer.

Endometriosis is a chronic inflammatory condition affecting women of reproductive age. A relevant feature of endometriosis is the presence of fibrotic tissue inside and around the lesions, thus contributing to the classic endometriosis-related symptoms, pain, and infertility. The molecular mechanisms responsible for the development of fibrosis in endometriosis are not yet defined. The present review aimed to examine the biological mechanisms and signalling pathways involved in fibrogenesis of endometriotic lesions, highlighting the difference between deep infiltrating and ovarian endometriosis. The main cell types involved in the development of fibrosis are platelets, myofibroblasts, macrophages, and sensory nerve fibers. Members of the transforming growth factor (TGF) -β family, as well as the receptor Notch, or the bioactive sphingolipid sphingosine 1-phosphate (S1P), play a role in the development of tissue fibrosis, resulting in their metabolism and/or their signalling pathways altered in endometriotic lesions. It is relevant the knowledge of the molecular mechanisms that guide and support fibrosis in endometriosis, to identify new drug targets and provide new therapeutic approaches to patients.

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aβ (Aβ

Urinary tract infections (UTIs) are among the most frequent bacterial diseases in infants and children. Physician adherence to recommendations is notoriously often poor, but no data are available on UTIs management in the emergency setting. In this multicenter national study, we investigated the policies regarding UTIs management in children aged 2 months to 3 years in Italian emergency units. Between April and June 2021, directors of the emergency units were invited to answer an online survey on the following items: diagnostic approach to children with fever without an apparent source, therapeutic approach to UTIs, the use of kidney and urinary tract ultrasound, and the criteria for hospitalization. A total of 121 (89%) out of 139 of invited units participated in the study. Overall, units manage children with a suspected or confirmed UTI according to available recommendations for most of the items. However, in almost 80% (n = 94) of units, a sterile perineal bag is used to collect urine for culture. When urine is collected by cathether, heterogeneity exists on the threshold of bacterial load considered for UTI diagnosis. Conclusions: Available recommendations on UTIs in children are followed by Italian emergency units for most of the items. However, the methods to collect urine specimens for culture, one of the crucial steps of the diagnostic work-up, often do not align with current recommendations and CFU thresholds considered for diagnosis largely vary among centers. Efforts should be addressed to validate and implement new child and family friendly urine collection techniques. What is Known:• Several guidelines are published on the management of children with suspected or confirmed urinary tract infection.• No data are available on the management of pediatric urinary tract infections in the emergency setting. What is New:• Almost 80% of the Italian emergency units employ a sterile perineal bag to collect urine for culture.• Diagnostic CFU thresholds largely vary among centers.

The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

Is sphingosine 1-phosphate (S1P) pathway involved in the process of fibrosis in adenomyosis?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.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 S1PPresent 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.

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [3H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation. This research was funded by ‘Departamento de Educación del Gobierno Vasco (Gasteiz-Vitoria, Basque Country, Spain) grant number IT-1106-16 and ‘Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain) grant number SAF2016-79695-R.

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 (TNF&alpha, ), 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&ndash, 5. Here, we provide experimental evidence that TNF&alpha, 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 TNF&alpha, challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNF&alpha, 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 TNF&alpha, induced atrophy in skeletal muscle.

Fibrosis is characterized by the excessive accumulation of extracellular matrix components, leading to loss of tissue function in affected organs. Although the majority of fibrotic diseases have different origins, they have in common a persistent inflammatory stimulus and lymphocyte-monocyte interactions that determine the production of numerous fibrogenic cytokines. Treatment to contrast fibrosis is urgently needed, since some fibrotic diseases lead to systemic fibrosis and represent a major cause of death. In this article, the role of the bioactive sphingolipid sphingosine 1-phosphate (S1P) and its signalling pathway in the fibrosis of different tissue contexts is extensively reviewed, highlighting that it may represent an innovative and promising pharmacological therapeutic target for treating this devastating multifaceted disease. In multiple tissues S1P influences different aspects of fibrosis modulating the recruitment of inflammatory cells, as well as cell proliferation, migration and transdifferentiation into myofibroblasts, the cell type mainly involved in fibrosis development. Moreover, at the level of fibrotic lesions, S1P metabolism is profoundly influenced by multiple cross-talk with profibrotic mediators, such as transforming growth factor β, thus finely regulating the development of fibrosis. This article is part of a Special Issue entitled "Physiological and pathological roles of bioactive sphingolipids".

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.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P(2) receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.

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) β1. 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 TGFβ1 was highlighted in vitro. Conclusion(s) The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis.

We investigated the effects of S1P3deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (−28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.

Hearing loss affects millions of people in the world. In mammals the auditory system comprises diverse cell types which are terminally differentiated and with no regenerative potential. There is a tremendous research interest aimed at identifying cell therapy based solutions or pharmacological approaches that could be applied therapeutically alongside auditory devices to prevent hair cell and neuron loss. Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid that plays key role in the regulation of many physiological and pathological functions. S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P1-5. In this study, murine auditory neuroblasts named US/VOT-N33 have been used as progenitors of neurons of the spiral ganglion. We demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action was dependent on SK1, SK2 as well as S1P1 and S1P2. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. Additionally, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively. Taken together, these findings demonstrate a crucial role for S1P signaling axis in the proliferation and the survival of otic vesicle neuroprogenitors, highlighting the identification of possible novel therapeutical approaches to prevent neuronal degeneration during hearing loss.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P

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 A

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.

Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

[Image: see text] Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator associated with diverse homeostatic and signaling roles. Enhanced biosynthesis of S1P, mediated by the sphingosine kinase isozymes (SK1 and SK2), is implicated in several pathophysiological conditions and diseases, including skeletal muscle fibrosis, inflammation, multiple sclerosis, and cancer. Therefore, therapeutic approaches that control S1P production have focused on the development of SK1/2 inhibitors. In this framework, we designed a series of natural monosaccharide-based compounds to enhance anchoring of the known SK1 inhibitor PF-543 at the polar head of the J-shaped substrate-binding channel. Herein, we describe the structure-based design and synthesis of new glycan-containing PF-543 analogues and we demonstrate their efficiency in a TGFβ1-induced pro-fibrotic assay.

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G

We report a case of urinary schistosomiasis in an adolescent refugee from Gambia (arrived to Italy illegally), who was brought to the Emergency Department of our hospital. The patient complained of gross hematuria and, in the absence of clinical evidence of bacterial urinary infection, was admitted to the pediatric ward, considering his provenience and social setting. An appropriate collection and microscopic analysis of urine samples led to the detection of bilharzia. Much attention should be paid to this emerging disease in Europe by physicians in order to recognize and treat it timely, which could prevent future and higher costs for public health systems and could reduce the potential risk of environmental spreading. In fact, there are some areas in Italy where the parasite can find its intermediate host to complete its lifecycle.

Background In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. Methods We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. Results SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by 1 H– 15 N heteronuclear NMR. Conclusions and general significance We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-β and the thromboxane A2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gαq and PLCβ. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.

Neuroblastoma (NB) is the most frequently observed among extracranial pediatric solid tumors. It displays an extreme clinical heterogeneity, in particular for the presentation at diagnosis and response to treatment, often depending on cancer cell differentiation/stemness. The frequent presence of elevated hematic and urinary levels of catecholamines in patients affected by NB suggests that the dissection of adrenergic system is crucial for a better understanding of this cancer. β3-adrenoreceptor (β3-AR) is the last identified member of adrenergic receptors, involved in different tumor conditions, such as melanoma. Multiple studies have shown that the dysregulation of the bioactive lipid sphingosine 1-phosphate (S1P) metabolism and signaling is involved in many pathological diseases including cancer. However, whether S1P is crucial for NB progression and aggressiveness is still under investigation. Here we provide experimental evidence that β3-AR is expressed in NB, both human specimens and cell lines, where it is critically involved in the activation of proliferation and the regulation between stemness/differentiation, via its functional cross-talk with sphingosine kinase 2 (SK2)/S1P receptor 2 (S1P2) axis. The specific antagonism of β3-AR by SR59230A inhibits NB growth and tumor progression, by switching from stemness to cell differentiation both in vivo and in vitro through the specific blockade of SK2/S1P2 signaling.

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.

Volume 20, no. 24, p. 9236–9246, 2000, [https://doi.org/10.1128/MCB.20.24.9236-9246.2000][1]. The publisher hereby retracts this article. Questions have been raised by concerned readers about the integrity of the data. The American Society for Microbiology has reviewed the figures and confirmed

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P2 receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P

Regulation of the motility of skeletal muscle precursor cells, such as satellite cells, is critically important for their proper recruitment at the site of tissue damage, and ultimately for its correct repair. Here we show that lysophosphatidic acid (LPA), which is well-recognized as a powerful bioactive agent, strongly stimulates cell migration of activated murine satellite cells. The biological effect exerted by LPA was found to be induced via activation of LPA1 and LPA3 , being abolished by cell treatment with the antagonist Ki16425, and severely impaired by siRNA-mediated down-regulation of the two receptor isoforms. In contrast, silencing of LPA2 potentiated the stimulation of cell motility by LPA, suggesting that it is negatively coupled to cell migration. Pharmacological inhibition of both sphingosine kinase (SK) isoforms using VPC96047, or the selective blocking of SK1 using VPC96091, abolished cell responsiveness to LPA; in agreement, gene silencing of SK1 or SK2 significantly reduced the biological effect of LPA. Moreover, the LPA-dependent stimulation of cell chemotaxis was found to be impaired by down-regulation of the sphingosine 1-phosphate (S1P) receptors S1P1 or S1P4 by specific siRNAs. In summary, the results obtained support the notion that the sphingosine kinase/sphingosine 1-phosphate (SK/S1P) axis is critically involved in the mechanism by which LPA elicits its pro-migratory action. This study provides compelling new information on the regulatory mechanisms of satellite cell motility, and reinforces the view that the SK/S1P signaling pathway plays a crucial role in the control of skeletal muscle precursor cell biology.

Evidences in the central nervous system are in favor that adenosine under basal conditions is released by a direct excitation-secretion modality. However, till now, there is no direct evidence that adenosine is contained in synaptic vesicles. Eight synaptic vesicle fractions were recovered on a discontinuous sucrose gradient after ultracentrifugation of the crude synaptosomal fraction (pellet P2) of rat brain. The adenosine content in each fraction was measured by high-performance liquid chromatography coupled to a fluorescence detector (minimum sensitivity 10 femtomoles). The immunoblot analysis, to detect synaptophysin, a molecular marker for the vesicle membrane, showed that fractions from 3 to 8 were rich in synaptophysin. The sum of adenosine found in fractions 3-8 was (mean ± SEM, n = 4) 3325.6 ± 94.6 pmol/mg of tissue protein. We proved that adenosine measured in synaptic vesicle fractions was not contaminated by cytosolic adenosine, as adenosine exogenously added to the P2 preferentially distributed in fractions 1 and 2 that are synaptophysin-free and did not contaminate the vesicle pellet P3. Data provide direct demonstration that adenosine is present in rat brain synaptic vesicle fractions. This information is consistent with the notion that adenosine is stored in synaptic vesicles and is released under normoxic physiological conditions by an excitation-secretion mechanism typical of neuronal cells.

In view of its multiple detrimental effects, transforming growth factor β1 (TGFβ1) is recognized as critical negative regulator of skeletal muscle repair. Apoptosis of skeletal muscle precursor cells driven by TGFβ1 contributes to the negative role exerted by the cytokine in tissue repair, although the underlying molecular mechanisms are still elusive. Herein we report the identification of a new signaling pathway, relying on Rho kinase-2 stimulation, subsequent to SMAD-dependent S1P4 up-regulation and transactivation via sphingosine kinase (SK)-2, that accounts for TGFβ1-induced apoptosis in cultured myoblasts. S1P4-specific gene silencing reduced by almost 50% activation of caspase-3 and poly-ADP ribosyl transferase cleavage elicited by TGFβ1. Moreover, the selective S1P4 antagonist CYM50358 also reduced the TGFβ1 proapoptotic effects. By employing pharmacological and molecular biological approaches, the involvement of SK2 and ROCK2 in the transmission of the TGFβ1 apoptotic action was also demonstrated. These results reinforce the notion that the SK/S1P axis plays a fundamental role in TGFβ1 mode of action in skeletal muscle cells and, by disclosing a novel mechanism by which TGFβ1 exerts its harmful action, pinpoint new molecular targets that in principle could be beneficial in the treatment of several skeletal muscle disorders or aging-dependent muscle atrophy.

We investigated the effects of S1P