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.

Ceramide Metabolism Enzymes-Therapeutic Targets against Cancer.

Sphingolipids are both structural molecules that are essential for cell architecture and second messengers that are involved in numerous cell functions. Ceramide is the central hub of sphingolipid metabolism. In addition to being the precursor of complex sphingolipids, ceramides induce cell cycle arrest and promote cell death and inflammation. At least some of the enzymes involved in the regulation of sphingolipid metabolism are altered in carcinogenesis, and some are targets for anticancer drugs. A number of scientific reports have shown how alterations in sphingolipid pools can affect cell proliferation, survival and migration. Determination of sphingolipid levels and the regulation of the enzymes that are implicated in their metabolism is a key factor for developing novel therapeutic strategies or improving conventional therapies. The present review highlights the importance of bioactive sphingolipids and their regulatory enzymes as targets for therapeutic interventions with especial emphasis in carcinogenesis and cancer dissemination.

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.

Prevalence and mortality of patients with palliative needs in an acute respiratory setting / Prevalencia y mortalidad de pacientes con necesidades paliativas en una planta de Neumología. Estudio prospectivo

Introduction: NECPAL is a tool for identification of patients with advanced chronic disease in need of palliative care. The main objective of the study is to know the prevalence of patients with palliative needs in an acute respiratory ward in a Spanish tertiary hospital using NECPAL. A second objective of the study is to know the annual mortality rate of these patients.Materials and methods: Cross sectional study and prospective monitoring of a cohort identified as palliative patients with the NECPAL tool for 12 months. Patient identification was performed in patients admitted to the respiratory ward of our hospital for longer than 3 days. We have assessed the annual vital status (deceased or not deceased) of patients and have recorded demographics, clinical and functional data, as well as the use of healthcare resources.Results: We monitored a cohort of 363 patients. Of them, 87 patients (24.3%) (IC 95% 19–30) were identified as NECPAL positive. 60% of patients (n = 64) died within 12 months of their admission. There was no significant difference in the mortality ratio of oncologic versus non oncologic patients. In a multivariable analysis, mortality was associated with demand by patients or relatives for palliative care and with the presence of specific disease progression markers or indicators.Conclusions: prevalence of patients with palliative needs in acute respiratory wards is high (one out of four patients). 60% of the patients identified as NECPAL positive in our cohort died in the first 12 months. Training of healthcare professionals as well as availability of appropriate resources are indispensable factors to improve care of this population. (AU)

Introducción: El instrumento NECPAL es una herramienta para la identificación de personas en situación de enfermedad crónica avanzada con necesidades paliativas. El objetivo del estudio es conocer la prevalencia de pacientes con necesidades paliativas en un Servicio de Neumología de un hospital terciario mediante la herramienta NECPAL. Como objetivo secundario se plantea conocer la mortalidad al año de estos pacientes.Material y métodos: Estudio trasversal y seguimiento prospectivo de la cohorte de los pacientes identificados como paliativos con la herramienta NECPAL durante 12 meses. La identificación se realiza en pacientes ingresados > 3 días en una planta de Neumología. Hemos evaluado el estatus vital (fallecido o no fallecido) al año, así como datos demográficos clínicos, funcionales y uso de recursos sanitarios.Resultados: Se analizaron 363 pacientes. De ellos, 87 (24,3%) (IC 95%, 19–30) fueron identificados como NECPAL positivos. En el seguimiento durante un año fallecieron 54 pacientes, un 60%. No hubo diferencias en mortalidad entre diagnósticos oncológicos y no oncológicos. En el análisis multivariante, la mortalidad se asoció a la demanda de paciente o familiares de atención paliativa y a la presencia de indicadores específicos de progresión de la enfermedad.Conclusiones: La prevalencia de pacientes con necesidades paliativas en una planta de hospitalización de agudos de Neumología es alta (uno de cada cuatro pacientes). El 60% de los identificados como NECPAL positivos en nuestra serie fallece en los primeros 12 meses. Son necesarios formación y recursos para atender a estos pacientes. (AU)

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.

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.

Prevalence and Mortality of Patients with Palliative Needs in an Acute Respiratory Setting. / Prevalencia y mortalidad de pacientes con necesidades paliativas en una planta de Neumología. Estudio prospectivo.

INTRODUCTION: NECPAL is a tool for identification of patients with advanced chronic disease in need of palliative care. The main objective of the study is to know the prevalence of patients with palliative needs in an acute respiratory ward in a Spanish tertiary hospital using NECPAL. A second objective of the study is to know the annual mortality rate of these patients. MATERIALS AND METHODS: Cross sectional study and prospective monitoring of a cohort identified as palliative patients with the NECPAL tool for 12 months. Patient identification was performed in patients admitted to the respiratory ward of our hospital for longer than 3 days. We have assessed the annual vital status (deceased or not deceased) of patients and have recorded demographics, clinical and functional data, as well as the use of healthcare resources. RESULTS: We monitored a cohort of 363 patients. Of them, 87 patients (24.3%) (IC 95% 19-30) were identified as NECPAL positive. 60% of patients (n = 64) died within 12 months of their admission. There was no significant difference in the mortality ratio of oncologic versus non oncologic patients. In a multivariable analysis, mortality was associated with demand by patients or relatives for palliative care and with the presence of specific disease progression markers or indicators. CONCLUSIONS: prevalence of patients with palliative needs in acute respiratory wards is high (one out of four patients). 60% of the patients identified as NECPAL positive in our cohort died in the first 12 months. Training of healthcare professionals as well as availability of appropriate resources are indispensable factors to improve care of this population.

Role of bioactive sphingolipids in physiology and pathology.

Sphingolipids are a class of complex lipids containing a backbone of sphingoid bases, namely the organic aliphatic amino alcohol sphingosine (Sph), that are essential constituents of eukaryotic cells. They were first described as major components of cell membrane architecture, but it is now well established that some sphingolipids are bioactive and can regulate key biological functions. These include cell growth and survival, cell differentiation, angiogenesis, autophagy, cell migration, or organogenesis. Furthermore, some bioactive sphingolipids are implicated in pathological processes including inflammation-associated illnesses such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease (namely Crohn's disease and ulcerative colitis), type II diabetes, obesity, and cancer. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism and can act as second messenger, especially when it is produced at the plasma membrane of cells. Ceramides promote cell cycle arrest and apoptosis. However, ceramide 1-phosphate (C1P), the product of ceramide kinase (CerK), and Sph 1-phosphate (S1P), which is generated by the action of Sph kinases (SphK), stimulate cell proliferation and inhibit apoptosis. Recently, C1P has been implicated in the spontaneous migration of cells from some types of cancer, and can enhance cell migration/invasion of malignant cells through interaction with a Gi protein-coupled receptor. In addition, CerK and SphK are implicated in inflammatory responses, some of which are associated with cancer progression and metastasis. Hence, targeting these sphingolipid kinases to inhibit C1P or S1P production, or blockade of their receptors might contribute to the development of novel therapeutic strategies to reduce metabolic alterations and disease.

Novel signaling aspects of ceramide 1-phosphate.

The bioactive sphingolipid ceramide 1-phosphate (C1P) regulates key physiologic cell functions and is implicated in a number of metabolic alterations and pathological processes. Initial studies using different types of fibroblasts and monocytes/macrophages revealed that C1P was mitogenic and that it promoted cell survival through inhibition of apoptosis. Subsequent studies implicated C1P in inflammatory responses with a specific role as pro-inflammatory agent. Specifically, C1P potently stimulated cytosolic phospholipase A2 (cPLA2) resulting in elevation of arachidonic acid and pro-inflammatory eicosanoid levels. However, increasing experimental evidence suggests that C1P can also exert anti-inflammatory actions in some cell types and tissues. Specifically, it has been demonstrated that C1P inhibits the release of pro-inflammatory cytokines and blocks activation of the pro-inflammatory transcription factor NF-κB in some cell types. Moreover, C1P was shown to increase the release of anti-inflammatory interleukin-10 in macrophages, and to overcome airway inflammation and reduce lung emphysema in vivo. Noteworthy, C1P stimulated cell migration, an action that is associated with diverse physiological cell functions, as well as with inflammatory responses and tumor dissemination. More recently, ceramide kinase (CerK), the enzyme that produces C1P in mammalian cells, has been shown to be upregulated during differentiation of pre-adipocytes into mature adipocytes, and that exogenous C1P, acting through a putative Gi protein-coupled receptor, negatively regulates adipogenesis. Although the latter actions seem to be contradictory, it is plausible that exogenous C1P may balance the adipogenic effects of intracellularly generated (CerK-derived) C1P in adipose tissue. The present review highlights novel signaling aspects of C1P and its impact in the regulation of cell growth and survival, inflammation and tumor dissemination.

Achalasia with massive oesophageal dilation causing tracheomalacia and asthma symptoms.

Achalasia is an uncommon oesophageal motor disorder characterized by failure of relaxation of the lower oesophageal sphincter and muscle hypertrophy, resulting in a loss of peristalsis and a dilated oesophagus. Gastrointestinal symptoms are invariably present in all cases of achalasia observed in adults. We report a case of a 34 year-old female patient with long standing history of asthma-like symptoms, labelled as uncontrolled and steroid resistant asthma with no gastrointestinal manifestations. Thoracic CT scan revealed a massive oesophagus due to achalasia, which caused severe tracheomalacia as a result of tracheal compression. Her symptoms regressed completely after a laparoscopic Heller myotomy surgery intervention.

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.

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.

Ceramide 1-phosphate regulates cell migration and invasion of human pancreatic cancer cells.

Pancreatic cancer is an aggressive and devastating disease characterized by invasiveness, rapid progression and profound resistance to treatment. Despite years of intense investigation, the prognosis of this type of cancer is poor and there is no efficacious treatment to overcome the disease. Using human PANC-1 and MIA PaCa-2 cells, we demonstrate that the bioactive sphingolipid ceramide 1-phosphate (C1P) increases pancreatic cancer cell migration and invasion. Treatment of these cells with selective inhibitors of phosphatidylinositol 3-kinase (PI3K), Akt1, or mammalian target of rapamycin 1 (mTOR1), or with specific siRNAs to silence the genes encoding these kinases, resulted in potent inhibition of C1P-induced cell migration and invasion. Likewise, the extracellularly regulated kinases 1 and 2 (ERK1-2), and the small GTPase RhoA, which regulates cytoskeleton reorganization, were also found to be implicated in C1P-stimulated ROCK1-dependent cancer cell migration and invasion. In addition, pre-treatment of the cancer cells with pertussis toxin abrogated C1P-induced cell migration, suggesting the intervention of a Gi protein-coupled receptor in this process. Pancreatic cancer cells engineered to overexpress ceramide kinase (CerK), the enzyme responsible for C1P biosynthesis in mammalian cells, showed enhanced spontaneous cell migration that was potently blocked by treatment with the selective CerK inhibitor NVP-231, or by treatment with specific CerK siRNA. Moreover, overexpression of CerK with concomitant elevations in C1P enhanced migration of pancreatic cancer cells. Collectively, these data demonstrate that C1P is a key regulator of pancreatic cancer cell motility, and suggest that targeting CerK expression/activity and C1P may be relevant factors for controlling pancreatic cancer cell dissemination.

Control of inflammatory responses by ceramide, sphingosine 1-phosphate and ceramide 1-phosphate.

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.

Caged ceramide 1-phosphate (C1P) analogs: Novel tools for studying C1P biology.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid metabolite that is produced in cells by the action of ceramide kinase (CerK) acting upon ceramide, and is also found in the circulation. C1P was first demonstrated to be mitogenic and antiapoptotic in different cell types, and was later shown to induce cell migration. Understanding the precise mechanisms by which C1P exerts its biological effects has been possible using specific photosensitive caged C1P analogues synthesized by Robert Bittman's group. These compounds are cell permeable, bypass cell plasma membrane receptors, and can be released into the cytosol upon light irradiation, thereby allowing precise determination of the intracellular mechanisms of actions of C1P. Two derivatives of N-palmitoyl-ceramide 1-phosphate have been used in most studies. In one C1P derivative the cage was 7-(N,N-diethylamino)coumarin (DECM-C1P) while in the other it was a 4-bromo-5-hydroxy-2-nitrobenzhydryl moiety (BHNB-C1P). The uncaging process released C1P in the cytosol, and this was accompanied by stimulation of cell proliferation, inhibition of apoptosis, and production of low levels of reactive oxygen species. However, intracellular accumulation of C1P did not affect chemotaxis. The caged C1P analogues allowed distinction between the extracellular events evoked by C1P, as for example through interaction with a putative cell-surface receptor, from its intracellular effects.

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.

Phosphatidic acid inhibits ceramide 1-phosphate-stimulated macrophage migration.

Ceramide 1-phosphate (C1P) was recently demonstrated to potently induce cell migration. This action could only be observed when C1P was applied exogenously to cells in culture, and was inhibited by pertussis toxin. However, the mechanisms involved in this process are poorly understood. In this work, we found that phosphatidic acid (PA), which is structurally related to C1P, displaced radiolabeled C1P from its membrane-binding site and inhibited C1P-stimulated macrophage migration. This effect was independent of the saturated fatty acid chain length or the presence of a double bond in each of the fatty acyl chains of PA. Treatment of RAW264.7 macrophages with exogenous phospholipase D (PLD), an enzyme that produces PA from membrane phospholipids, also inhibited C1P-stimulated cell migration. Likewise, PA or exogenous PLD inhibited C1P-stimulated extracellularly regulated kinases (ERK) 1 and 2 phosphorylation, leading to inhibition of cell migration. However, PA did not inhibit C1P-stimulated Akt phosphorylation. It is concluded that PA is a physiological regulator of C1P-stimulated macrophage migration. These actions of PA may have important implications in the control of pathophysiological functions that are regulated by C1P, including inflammation and various cellular processes associated with cell migration such as organogenesis or tumor metastasis.