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.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

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.Work in AGM lab is supported by the ‘Departamento de Educación del Gobierno Vasco (Gasteiz-Vitoria, Basque Country, Spain)’ [grant number IT-1106-16]

Ceramide 1-phosphate (C1P) induces macrophage chemoattractant protein-1 release: involvement in C1P-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 (also known as protein kinase B), 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-kappa B 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.This work was supported by grants BFU2009-13314/BFI from Ministerio de Ciencia e Innovación (MICINN) (Madrid, Spain), IT-705-13 from Departamento de Educación, Universidades e Investigación del Gobierno Vasco (GV/EJ, Spain), S-PE11UN017, and S-PE12UN040 from Departamento de Industria, Comercio y Turismo del Gobierno Vasco (Basque Government, GV/EJ, Spain). LA and AO are the recipients of fellowships from the Basque Government. I-G.R is the recipient of a fellowship from Ministerio de Ciencia e Innovación (MICINN) (Madrid, Spain), and MO is the recipient of a fellowship from the University of the Basque Country (GV/EJ, Spain)

Sphingomyelinase D/Ceramide 1-Phosphate in Cell Survival and Inflammation

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.Work in AGM lab is supported by Departamento de Educacion, Universidades e Investigacion del Gobierno Vasco (Gazteiz-Vitoria, Basque Country), and Ministerio de Economia y Competitividad (Madrid, Spain)

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 A2 and protein kinase C-, 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- and cPLA2- in this pathway.This work was supported by grants BFU2009-13314/BFI from Ministerio de Ciencia e Innovación (MICINN) (Madrid, Spain), IT-353-10 from Departamento de Educación, Universidades e Investigación del Gobierno Vasco (GV/EJ), SA-2010/00013 from Departamento de Industria, Comercio y Turismo del Gobierno Vasco (Basque Government, GV/EJ) to AGM, and grant HL083187 from the National Institutes of Health to RB. LA and AO are the recipients of fellowships from the Basque Government