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.This work was supported by Grant IT1720-22 from “Departamento de Educación, Viceconsejería de Universidades e Investigación del Gobierno Vasco” (GV/EJ, Basque Country, Spain). N. Presa is the recipient of a fellowship from “Departamento de Educación, Universidades e Investigación del Gobierno Vasco” (GV/EJ, Basque Country, Spain)

Identification of substrates for human deubiquitinating enzymes (DUBs): An up-to-date review and a case study for neurodevelopmental disorders

Similar to the reversal of kinase-mediated protein phosphorylation by phosphatases, deubiquitinating enzymes (DUBs) oppose the action of E3 ubiquitin ligases and reverse the ubiquitination of proteins. A total of 99 human DUBs, classified in 7 families, allow in this way for a precise control of cellular function and homeostasis. Ubiquitination regulates a myriad of cellular processes, and is altered in many pathological conditions. Thus, ubiquitination-regulating enzymes are increasingly regarded as potential candidates for therapeutic intervention. In this context, given the predicted easier pharmacological control of DUBs relative to E3 ligases, a significant effort is now being directed to better understand the processes and substrates regulated by each DUB. Classical studies have identified specific DUB substrate candidates by traditional molecular biology techniques in a case-by-case manner. Lately, single experiments can identify thousands of ubiquitinated proteins at a specific cellular context and narrow down which of those are regulated by a given DUB, thanks to the development of new strategies to isolate and enrich ubiquitinated material and to improvements in mass spectrometry detection capabilities. Here we present an overview of both types of studies, discussing the criteria that, in our view, need to be fulfilled for a protein to be considered as a high-confidence substrate of a given DUB. Applying these criteria, we have manually reviewed the relevant literature currently available in a systematic manner, and identified 650 high-confidence substrates of human DUBs. We make this information easily accessible to the research community through an updated version of the DUBase website (https://ehubio.ehu.eus/dubase/). Finally, in order to illustrate how this information can contribute to a better understanding of the physiopathological role of DUBs, we place a special emphasis on a subset of these enzymes that have been associated with neurodevelopmental disorders.This work was supported by Spanish MINECO grant SAF2016-76898-P to UM and by the University of the Basque Country (UPV/EHU), grant numbers US19/05 and COLAB19/18

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 inflammatory processes by ceramide kinase and phosphatidylethanolamine N-methyltransferase in lung cells, adipocytes and liver tissue.

188 p.Nowadays, around 60% of the world's population dies due to chronic inflammatory conditions, such as chronic respiratory diseases, obesity, fatty liver disease and cancer. All these pathologies are associated to alterations in sphingolipid metabolism. Thus, understanding the mechanisms responsible for the establishment and evolution of those diseases may be useful for developing novel strategies to control their development and progression. In this thesis, we demonstrate that ROCK1 is a key regulatory enzyme necessary for cigarette smoke extract (CSE)-induced monocyte chemoattractant protein-1 (MCP-1) release in lung epithelial cells, and that AKT2 and the Ceramide Kinase (CerK)/ ceramide 1-phosphate (C1P) axis elicit the opposite effects, pointing to an anti-inflammatory role of C1P in the lungs. We also provide evidence suggesting that adipogenesis is associated with an increase in phosphatidylethanolamine N-methyltransferase-2 (PEMT-2) protein expression, whose depletion leads to impaired adipocyte differentiation, as seen by a reduced expression of adipogenic markers, as well as reduced lipid droplet formation, triglyceride (TG) content and leptin release. Moreover, we demonstrate that the inhibition of adipocyte differentiation by exogenous C1P occurs by modulating PEMT expression. Besides, we havealso found an abnormal sphingolipid metabolism in Pemt-/- mice fed a HFD (a well-known non-alcoholic fatty liver disease mouse model), with elevation of ceramides, sphingomyelin, sphinganine, sphingosine, 1-deoxyceramides, and C26:1 C1P as well as higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (CerK). Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis, and restored Asah1 and CerK mRNA and sphingolipid levels, showing that vitamin E treatment efficiently prevents the progression from simple steatosis to steatohepatitis in mice lacking PEMT, and that sphingolipid metabolites and Asah1 and CerK may be important factors in this action

Regulation of inflammatory processes by ceramide kinase and phosphatidylethanolamine N-methyltransferase in lung cells, adipocytes and liver tissue.

188 p.Nowadays, around 60% of the world's population dies due to chronic inflammatory conditions, such as chronic respiratory diseases, obesity, fatty liver disease and cancer. All these pathologies are associated to alterations in sphingolipid metabolism. Thus, understanding the mechanisms responsible for the establishment and evolution of those diseases may be useful for developing novel strategies to control their development and progression. In this thesis, we demonstrate that ROCK1 is a key regulatory enzyme necessary for cigarette smoke extract (CSE)-induced monocyte chemoattractant protein-1 (MCP-1) release in lung epithelial cells, and that AKT2 and the Ceramide Kinase (CerK)/ ceramide 1-phosphate (C1P) axis elicit the opposite effects, pointing to an anti-inflammatory role of C1P in the lungs. We also provide evidence suggesting that adipogenesis is associated with an increase in phosphatidylethanolamine N-methyltransferase-2 (PEMT-2) protein expression, whose depletion leads to impaired adipocyte differentiation, as seen by a reduced expression of adipogenic markers, as well as reduced lipid droplet formation, triglyceride (TG) content and leptin release. Moreover, we demonstrate that the inhibition of adipocyte differentiation by exogenous C1P occurs by modulating PEMT expression. Besides, we havealso found an abnormal sphingolipid metabolism in Pemt-/- mice fed a HFD (a well-known non-alcoholic fatty liver disease mouse model), with elevation of ceramides, sphingomyelin, sphinganine, sphingosine, 1-deoxyceramides, and C26:1 C1P as well as higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (CerK). Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis, and restored Asah1 and CerK mRNA and sphingolipid levels, showing that vitamin E treatment efficiently prevents the progression from simple steatosis to steatohepatitis in mice lacking PEMT, and that sphingolipid metabolites and Asah1 and CerK may be important factors in this action

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)