Phosphatidic Acid Stimulates Myoblast Proliferation through Interaction with LPA1 and LPA2 Receptors.

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.

Regulation of adipogenesis by ceramide 1-phosphate.

We showed previously that ceramide kinase (CerK) expression increases during adipogenesis pointing to a relevant role of intracellular C1P in this process. In the present work we demonstrate that administration of exogenous C1P inhibits the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes through a mechanism involving activation of extracellularly regulated kinases (ERK) 1-2. Exogenous C1P reduced the accumulation of lipid droplets and the content of triacylglycerol in these cells, and potently inhibited the expression of the early and late adipogenic markers C/EBPß and PPARγ, respectively. C1P also reduced the secretion of leptin, which is a crucial regulator of energy balance and appetite in the organism, and is considered to be a late marker of adipogenesis. Interestingly, all of these C1P actions were reversed by pertussis toxin, suggesting the intervention of a Gi protein-coupled receptor previously identified for C1P, in this process. Also, exogenous C1P significantly reduced CerK activity. Altogether, the data presented in this work suggest that exogenous C1P may balance adipogenesis, and that targeting CerK may be a novel way for potential applications in the treatment of obesity or other inflammation-associated diseases.

Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure.

STUDY QUESTION: Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? SUMMARY ANSWER: Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology. WHAT IS KNOWN ALREADY: Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy. STUDY DESIGN, SIZE, DURATION: The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 µl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration. PARTICIPANTS/MATERIALS, SETTING, METHODS: Animals were euthanized by cervical dislocation or cardiac puncture 2 weeks after surgery for collection of blood orovary and uterus samples, which were cleaned of adhering tissue in culture medium and used for subsequent assays. Ovaries were used for Western blotting or immunohistochemical and/or histological analyses or steroid extraction, as required (n = 5-8 per group). A set of mice (n = 3/group) was destined for oocyte recovery and IVF. Finally, another set (n = 5-6/group) was separated to study fertility parameters. MAIN RESULTS AND THE ROLE OF CHANCE: The number of primordial (P < 0.01), primary (P < 0.05) and preantral follicles (P < 0.05) were decreased in Cy-treated mice compared to control animals, while atretic follicles were increased (P < 0.001). In Cy + C1P mice, the ovaries recovered control numbers of these follicular structures, in both C1P doses studied. Cy affected AMH expression, while it was at least partially recovered when C1P is administered as well. Cy caused an increase in serum FSH concentration (P < 0.01), which was prevented by C1P coadministration (P < 0.01). E2 levels in Cy-treated ovaries decreased significantly compared to control ovaries (P < 0.01), whilst C1P restored E2 levels to those of control ovaries (P < 0.01). Cy increased the expression of BAX (P < 0.01) and decreased the expression of BCLX-L compared to control ovaries (P < 0.01). The ovarian BCLX-L:BAX ratio was also lower in Cy-treated mice (P < 0.05). In the Cy + C1P group, the expression levels of BAX, BCLX-L and BCLX-L:BAX ratio were no different than those in control ovaries. In addition, acid sphingomyelinase (A-SMase) expression was higher in Cy-treated ovaries, whilst remaining similar to the control in the Cy + C1P group. Cy increased the apoptotic index (TUNEL-positive follicles/total follicles) in preantral and early antral stages, compared to control ovaries (P < 0.001 and P < 0.01, respectively). C1P protected follicles from this increase. No primordial or primary follicular cells stained for either cleaved caspase-3 or TUNEL when exposed to Cy, therefore, we have found no evidence for follicular reserve depletion in response to Cy being due to apoptosis. Cy caused evident vascular injury, especially in large cortical stromal vessels, and some neovascularization. In the Cy + C1P group, the disruptions in vascular wall continuity were less evident and the number of healthy stromal blood vessels seemed to be restored. In Cy-treated ovaries α-SMA-positive cells showed a less uniform distribution around blood vessels. C1P coadministration partially prevented this Cy-induced effect, with a higher presence of α-SMA-positive cells surrounding vessels. By H&E staining, Cy-treated mice showed endometrial alterations compared to controls, affecting both epithelial and stromal compartments. However, C1P allowed that the stromal tissue to maintain its loose quality and its glandular branches. Cy-treated animals had significantly lower pregnancy rates and smaller litter sizes compared with control mice (P = 0.013 and P < 0.05, respectively), whereas cotreatment with C1P preserved normal fertility. Furthermore, a higher (P < 0.05) proportion of abnormal oocytes was recovered from Cy-treated mice compared to the control, which was prevented by C1P administration. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The results of this study were generated from an in-vivo animal experimental model, already used by several authors. Further studies on C1P functions in female reproduction in pathological conditions such as chemotherapy-induced ovarian failure and on the safety of use of this sphingolipid are required. WIDER IMPLICATIONS OF THE FINDINGS: The present findings showed that C1P administration prior to Cy might be a promising fertility preservation strategy in female patients who undergo chemotherapy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from ANPCyT (PICT 2015-1117), CONICET (PIP 380), Cancer National Institute (INC) and Roemmers Foundation, Argentina. The authors declare no conflicts of interest.

C1P Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Preventing NF-κB Activation in Neutrophils.

Recently, ceramide-1-phosphate (C1P) has been shown to modulate acute inflammatory events. Acute lung injury (Arnalich et al. 2000. Infect. Immun. 68: 1942-1945) is characterized by rapid alveolar injury, lung inflammation, induced cytokine production, neutrophil accumulation, and vascular leakage leading to lung edema. The aim of this study was to investigate the role of C1P during LPS-induced acute lung injury in mice. To evaluate the effect of C1P, we used a prophylactic and therapeutic LPS-induced ALI model in C57BL/6 male mice. Our studies revealed that intrapulmonary application of C1P before (prophylactic) or 24 h after (therapeutic) LPS instillation decreased neutrophil trafficking to the lung, proinflammatory cytokine levels in bronchoalveolar lavage, and alveolar capillary leakage. Mechanistically, C1P inhibited the LPS-triggered NF-κB levels in lung tissue in vivo. In addition, ex vivo experiments revealed that C1P also attenuates LPS-induced NF-κB phosphorylation and IL-8 production in human neutrophils. These results indicate C1P playing a role in dampening LPS-induced acute lung inflammation and suggest that C1P could be a valuable candidate for treatment of ALI.

Vascular endothelial growth factor mediates ceramide 1-phosphate-stimulated macrophage proliferation.

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates cell division in a variety of cell types including macrophages. However, the mechanisms involved in this action are not completely understood. In the present work we show that C1P stimulates the release of vascular endothelial growth factor (VEGF) in RAW264.7 macrophages, and that this growth factor is essential for stimulation of cell proliferation by C1P. The stimulation of VEGF release was dependent upon activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB-1 also known as Akt-1), and mitogen-activated protein kinase-kinase (MEK)/extracellularly regulated kinase-2 (ERK-2) pathways, as inhibition of these kinases with selective pharmacological inhibitors or with specific gene silencing siRNA, abrogated VEGF release. A key observation was that sequestration of VEGF with a neutralizing antibody, or treatment with VEGF siRNA abolished C1P-stimulated macrophage growth. Also, inhibition of the pathways involved in C1P-stimulated VEGF release inhibited the stimulation of macrophage growth by C1P. Moreover, blockade of VEGF receptor-2 (VEGFR-2), which is the primary receptor for VEGF, with the pharmacological inhibitor DMH4, or with specific VEGFR-2 siRNA, substantially inhibited C1P-stimulated cell growth. It can be concluded that stimulation of VEGF release is a key factor in the promotion of macrophage proliferation by C1P.

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.

Regulation of cell migration and inflammation by ceramide 1-phosphate.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid metabolite first shown to regulate cell growth and death. Subsequent studies revealed that C1P was a potent stimulator of cytosolic phospholipase A2 (cPLA2) with ensuing release of arachidonic acid and prostaglandin biosynthesis. The latter findings placed C1P on the list of pro-inflammatory metabolites. More recently, C1P was found to potently stimulate cell migration, an action that is associated to diverse physiological effects, as well as to inflammatory responses and tumor dissemination. The implication of C1P in inflammation has gained further interest in the last few years due to the discovery that it can exert anti-inflammatory actions in some cell types and tissues. In particular, C1P has been demonstrated to inhibit pro-inflammatory cytokine release and blockade of the pro-inflammatory transcription factor NF-κB in some cell types, as well as to reduce airway inflammation and lung emphysema. The present review is focused on novel aspects of C1P regulation of cell migration and the impact of C1P as novel anti-inflammatory agent. GLOSS: Ceramide 1-phosphate (C1P) is a phosphosphingolipid with potent biological activities. It promotes cell growth and survival, and is a key regulator of cell migration. Both C1P and the enzyme that catalyzes its biosynthesis, ceramide kinase, are implicated in inflammatory responses. Although C1P has pro-inflammatory properties, it reduces pulmonary emphysema and exerts anti-inflammatory actions in the lung. Synthetic C1P analogs may be promising tools to treat lung inflammation.

Implication of Ceramide Kinase in Adipogenesis.

Ceramide kinase (CerK) plays a critical role in the regulation of cell growth and survival and has been implicated in proinflammatory responses. In this work, we demonstrate that CerK regulates adipocyte differentiation, a process associated with obesity, which causes chronic low-grade inflammation. CerK was upregulated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Noteworthy, knockdown of CerK using specific siRNA to silence the gene encoding this kinase resulted in substantial decrease of lipid droplet formation and potent depletion in the content of triacylglycerols in the adipocytes. Additionally, CerK knockdown caused blockade of leptin secretion, an adipokine that is crucial for regulation of energy balance in the organism and that is increased in the obese state. Moreover, CerK gene silencing decreased the expression of peroxisome proliferator-activated receptor gamma (PPARγ), which is considered the master regulator of adipogenesis. It can be concluded that CerK is a novel regulator of adipogenesis, an action that may have potential implications in the development of obesity, and that targeting this kinase may be beneficial for treatment of obesity-associated diseases.

Regulation of ceramide generation during macrophage apoptosis by ASMase and de novo synthesis.

The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.

Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-methyltransferase-deficient mice.

Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt(-/-) mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt(-/-) mice. We fed Pemt(-/-) and Pemt(+/+) mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt(-/-) mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt(+/+) mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPß were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt(-/-) mice. The in vivo conversion of [¹4C]acetate to [¹4C]TG in WAT was also lower in Pemt(-/-) mice. The release of glycerol from WAT explants was comparable between Pemt(+/+) and Pemt(-/-) mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt(-/-) mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt(+/+) mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt(-/-) mice.

Vagus nerve contributes to the development of steatohepatitis and obesity in phosphatidylethanolamine N-methyltransferase deficient mice.

BACKGROUND & AIMS: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine biosynthesis. When fed a high-fat diet (HFD), Pemt(-/-) mice are protected from HF-induced obesity; however, they develop steatohepatitis. The vagus nerve relays signals between liver and brain that regulate peripheral adiposity and pancreas function. Here we explore a possible role of the hepatic branch of the vagus nerve in the development of diet induced obesity and steatohepatitis in Pemt(-/-) mice. METHODS: 8-week old Pemt(-/-) and Pemt(+/+) mice were subjected to hepatic vagotomy (HV) or capsaicin treatment, which selectively disrupts afferent nerves, and were compared to sham-operated or vehicle-treatment, respectively. After surgery, mice were fed a HFD for 10 weeks. RESULTS: HV abolished the protection against the HFD-induced obesity and glucose intolerance in Pemt(-/-) mice. HV normalized phospholipid content and prevented steatohepatitis in Pemt(-/-) mice. Moreover, HV increased the hepatic anti-inflammatory cytokine interleukin-10, reduced chemokine monocyte chemotactic protein-1 and the ER stress marker C/EBP homologous protein. Furthermore, HV normalized the expression of mitochondrial electron transport chain proteins and of proteins involved in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase in Pemt(-/-) mice. However, disruption of the hepatic afferent vagus nerve by capsaicin failed to reverse either the protection against the HFD-induced obesity or the development of HF-induced steatohepatitis in Pemt(-/-) mice. CONCLUSIONS: Neuronal signals via the hepatic vagus nerve contribute to the development of steatohepatitis and protection against obesity in HFD fed Pemt(-/-) mice.

Ceramide-1-phosphate inhibits cigarette smoke-induced airway inflammation.

Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.

New insights on the role of ceramide 1-phosphate in inflammation.

Inflammation is a complex biological process involving a variety of locally produced molecules, as well as different types of white blood cells. Some of the so-called inflammatory mediators include cytokines, chemokines, interleukins, prostaglandins, or bioactive lipids, all of which provide protection from infection and foreign substances, such as bacteria, yeast, viruses or some chemicals. Under some circumstances, however, the organism inappropriately activates the immune system triggering an inflammatory response in the absence of foreign insults thereby leading to the establishment of autoimmune diseases. Therefore, inflammation must be tightly regulated in order to ensure sufficient protection to the organism in the absence of unwanted, and at times dangerous, side effects. Increasing experimental evidence implicates sphingolipids as major inducers of inflammatory responses and regulators of immune cell functions. In particular, ceramides and sphingosine 1-phosphate have been extensively implicated in inflammation, and ceramide 1-phosphate has also been shown to participate in these processes. The present review highlights novel aspects on the regulation of inflammation by sphingolipids, with special emphasis to the role played by ceramide 1-phosphate and ceramide kinase, the enzyme responsible for its biosynthesis, in inflammatory responses.

Ceramide 1-phosphate induces macrophage chemoattractant protein-1 release: involvement in ceramide 1-phosphate-stimulated cell migration.

The bioactive sphingolipid ceramide 1-phosphate (C1P) is implicated in inflammatory responses and was recently shown to promote cell migration. However, the mechanisms involved in these actions are poorly described. Using J774A.1 macrophages, we have now discovered a new biological activity of C1P: stimulation of monocyte chemoattractant protein-1 (MCP-1) release. This novel effect of C1P was pertussis toxin (PTX) sensitive, suggesting the intervention of Gi protein-coupled receptors. Treatment of the macrophages with C1P caused activation of the phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase kinase (MEK)/extracellularly regulated kinases (ERK), and p38 pathways. Inhibition of these kinases using selective inhibitors or specific siRNA blocked the stimulation of MCP-1 release by C1P. C1P stimulated nuclear factor-κB activity, and blockade of this transcription factor also resulted in complete inhibition of MCP-1 release. Also, C1P stimulated MCP-1 release and cell migration in human THP-1 monocytes and 3T3-L1 preadipocytes. A key observation was that sequestration of MCP-1 with a neutralizing antibody or treatment with MCP-1 siRNA abolished C1P-stimulated cell migration. Also, inhibition of the pathways involved in C1P-stimulated MCP-1 release completely blocked the stimulation of cell migration by C1P. It can be concluded that C1P promotes MCP-1 release in different cell types and that this chemokine is a major mediator of C1P-stimulated cell migration. The PI3K/Akt, MEK/ERK, and p38 pathways are important downstream effectors in this action.

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate.

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A(2) and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA(2)-α in this pathway.

Phosphatidic Acid Stimulates Lung Cancer Cell Migration through Interaction with the LPA1 Receptor and Subsequent Activation of MAP Kinases and STAT3.

Phosphatidic acid (PA) is a key bioactive glycerophospholipid that is implicated in the regulation of vital cell functions such as cell growth, differentiation, and migration, and is involved in a variety of pathologic processes. However, the molecular mechanisms by which PA exerts its pathophysiological actions are incompletely understood. In the present work, we demonstrate that PA stimulates the migration of the human non-small cell lung cancer (NSCLC) A549 adenocarcinoma cells, as determined by the transwell migration assay. PA induced the rapid phosphorylation of mitogen-activated protein kinases (MAPKs) ERK1-2, p38, and JNK, and the pretreatment of cells with selective inhibitors of these kinases blocked the PA-stimulated migration of cancer cells. In addition, the chemotactic effect of PA was inhibited by preincubating the cells with pertussis toxin (PTX), a Gi protein inhibitor, suggesting the implication of a Gi protein-coupled receptor in this action. Noteworthy, a blockade of LPA receptor 1 (LPA1) with the specific LPA1 antagonist AM966, or with the selective LPA1 inhibitors Ki1645 or VPC32193, abolished PA-stimulated cell migration. Moreover, PA stimulated the phosphorylation of the transcription factor STAT3 downstream of JAK2, and inhibitors of either JAK2 or STAT3 blocked PA-stimulated cell migration. It can be concluded that PA stimulates lung adenocarcinoma cell migration through an interaction with the LPA1 receptor and subsequent activation of the MAPKs ERK1-2, p38, and JNK, and that the JAK2/STAT3 pathway is also important in this process. These findings suggest that targeting PA formation and/or the LPA1 receptor may provide new strategies to reduce malignancy in lung cancer.

Implication of Ceramide Kinase/C1P in Cancer Development and Progression.

Cancer cells rewire their metabolic programs to favor biological processes that promote cell survival, proliferation, and dissemination. Among this relevant reprogramming, sphingolipid metabolism provides metabolites that can favor or oppose these hallmarks of cancer. The sphingolipid ceramide 1-phosphate (C1P) and the enzyme responsible for its biosynthesis, ceramide kinase (CERK), are well established regulators of cell growth and survival in normal, as well as malignant cells through stress-regulated signaling pathways. This metabolite also promotes cell survival, which has been associated with the feedback regulation of other antitumoral sphingolipids or second messengers. C1P also regulates cancer cell invasion and migration of different types of cancer, including lung, breast, pancreas, prostate, or leukemia cells. More recently, CERK and C1P have been implicated in the control of inflammatory responses. The present review provides an updated view on the important role of CERK/C1P in the regulation of cancer cell growth, survival, and dissemination.

Regulation of cell growth, survival and migration by ceramide 1-phosphate - implications in lung cancer progression and inflammation.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of vital cellular functions and plays key roles in a number of inflammation-associated pathologies. C1P was first described as mitogenic for fibroblasts and macrophages and was later found to promote cell survival in different cell types. The mechanisms involved in the mitogenic actions of C1P include activation of MEK/ERK1-2, PI3K/Akt/mTOR, or PKC-α, whereas promotion of cell survival required a substantial reduction of ceramide levels through inhibition of serine palmitoyl transferase or sphingomyelinase activities. C1P and ceramide kinase (CerK), the enzyme responsible for its biosynthesis in mammalian cells, play key roles in tumor promotion and dissemination. CerK-derived C1P can be secreted to the extracellular milieu by different cell types and is also present in extracellular vesicles. In this context, whilst cell proliferation is regulated by intracellularly generated C1P, stimulation of cell migration/invasion requires the intervention of exogenous C1P. Regarding inflammation, C1P was first described as pro-inflammatory in a variety of cell types. However, cigarette smoke- or lipopolysaccharide-induced lung inflammation in mouse or human cells was overcome by pretreatment with natural or synthetic C1P analogs. Both acute and chronic lung inflammation, and the development of lung emphysema were substantially reduced by exogenous C1P applications, pointing to an anti-inflammatory action of C1P in the lungs. The molecular mechanisms involved in the regulation of cell growth, survival and migration with especial emphasis in the control of lung cancer biology are discussed.

Ceramide Metabolism and Parkinson's Disease-Therapeutic Targets.

Ceramide is a bioactive sphingolipid involved in numerous cellular processes. In addition to being the precursor of complex sphingolipids, ceramides can act as second messengers, especially when they are generated at the plasma membrane of cells. Its metabolic dysfunction may lead to or be a consequence of an underlying disease. Recent reports on transcriptomics and electrospray ionization mass spectrometry analysis have demonstrated the variation of specific levels of sphingolipids and enzymes involved in their metabolism in different neurodegenerative diseases. In the present review, we highlight the most relevant discoveries related to ceramide and neurodegeneration, with a special focus on Parkinson's disease.

Identification of Androgen Receptor Metabolic Correlome Reveals the Repression of Ceramide Kinase by Androgens.

Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.