Effects Of Lysophosphatidic Acid Research Articles

Lysophosphatidic acid protects cervical cancer HeLa cells from apoptosis induced by doxorubicin hydrochloride.

Cervical cancer is one of the most common types of gynecological tumors. Lysophosphatidic acid (LPA), as a bioactive lipid medium, plays an important role in numerous physiological and pathophysiological processes, including the stimulation of cell migration and tumor cell invasion. LPA is increased in the plasma of patients with cervical cancer. Doxorubicin hydrochloride (DOX) is used as a first-line drug in the treatment of cervical cancer in clinics, however, the effect and molecular mechanism of LPA on DOX-induced apoptosis in cervical cancer cells remain unclear. Therefore, the present study aimed to explore the effect and underlying molecular mechanism of LPA on DOX-induced apoptosis in cervical cancer cells. HeLa cells were treated as a control group or with LPA (10 µmol/l), DOX (4 µmol/l) or LPA (10 µmol/l) + DOX (4 µmol/l) for 24 h. Using transmission electron microscopy the results demonstrated that LPA reduced cell death and the degree of chromatin aggregation in DOX-induced HeLa cells. Reverse transcription-quantitative PCR demonstrated that LPA significantly downregulated caspase-3 mRNA expression levels in DOX-induced HeLa cells. Moreover, western blotting demonstrated that LPA significantly reduced caspase-3 and cleaved caspase-3 protein expression levels in DOX-induced HeLa, C33A and SiHa cells. Furthermore, flow cytometry demonstrated that LPA may prevent apoptosis in DOX-induced HeLa cells (P<0.05). Using dichloro-dihydro-fluorescein diacetate assay, it was demonstrated that LPA significantly reduced the intracellular ROS levels induced by DOX. In summary, the present study indicated that LPA may protect HeLa cells from apoptosis induced by DOX. These findings have provided experimental evidence that LPA may be a potential therapeutic target for the treatment of cervical cancer.

Admission Lysophosphatidic Acid Is Related to Impaired Kidney Function in Acute Aortic Dissection: 2-Year Retrospective Follow-Up Study.

BackgroundDelayed treatment of acute aortic dissection (AAD)-related acute kidney injury (AKI) significantly increases the burden of chronic kidney disease (CKD) and mortality. Lysophosphatidic acid (LPA) is a shared mediator of kidney disease and AAD. Here, we evaluated the relationship between LPA and kidney injury in AAD patients.MethodsWe measured the plasma concentration of LPA in a cohort of 80 patients with AAD. Least Absolute Shrinkage and Selection Operator (LASSO) regression and Logistic regression were used to evaluate the effect and interaction of LPA on AKI. Additive generalized model and penalized spline method were used to describe the non-linear association. Multivariable analyses with the Cox proportional-hazards model were used for subgroup analysis and interaction in LPA and subsequent CKD.ResultsThe participant’s average age was 54.27 ± 11.00 years, 68.75% of them were males, and the incidence of AKI was 43.75%. Patients with AKI had higher levels of LPA on admission, and the more significant the increase, the higher the risk of AKI. There was a non-linear positive correlation between admission LPA and AKI, and the premeditated inflection point was 346.33 (μg/dL) through two-piecewise linear regression and recursive algorithm. Subgroup analysis identified a stronger association between admission LPA and AKI in the elder, female and medically treated patients. The incidence of CKD was 22.67% in the 2-year follow-up. Patients with subsequent CKD had higher LPA levels on admission in the follow-up cohort, and a similar interaction trend was also observed through Cox proportional—hazards model.ConclusionAdmission LPA levels show a non-linear positive correlation with AKI and increase the risk of subsequent CKD, which is more pronounced in elderly, female, and medically treated patients.

Abstract 5810: LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism

Abstract Introduction: Gastric cancer is the second most common cause of cancer-related death worldwide. Altered metabolism is considered a primary hallmark of tumorigenesis, as it can regulate important processes associated with proliferation, migration, and invasion. Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and malignant behavior of the cancer cells through G-protein coupled receptors. Although several signaling pathways have been reported as a critical mediator of gastric cancer, LPA-LPAR2 axis-mediated alteration of energy metabolism in gastric cancer progression has not been established yet. Therefore, our study focused on the molecular mechanism of LPA-LPAR2 axis-mediated alteration of mitochondrial bioenergetics during gastric cancer progression. Method: TCGA database analysis was carried out to check the mRNA levels of the LPA receptors. Expression of LPA receptors was investigated in human gastric cancer samples by immunoblotting, RT-PCR, and immunohistochemical analysis. We elucidated the functional effects of LPA on gastric cancer by performing ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in gastric cancer cells. To confirm the involvement of the specific LPA receptor on gastric cancer progression, we treated the cells with LPA1-3 receptor blocker (Ki16425) and LPA2 receptor-specific antagonist. Following LPA receptor blockade, we analyzed the glycolytic and mitochondrial functions by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a Seahorse XF24 analyzer. Result: Our TCGA dataset analysis, LPA ELISA, and the Western blotting result showed the presence of LPA receptors in human gastric cancer tissues, specifically LPAR2 was robustly increased (P&amp;lt;0.001) in human gastric cancer tissue samples, and LPA level was significantly higher (P&amp;lt;0.001), suggesting a possible role of LPA in gastric cancer. LPA treatment increased proliferation, migration, and invasion activity in AGS and NCI-N87 gastric cancer cell lines. In contrast, LPA receptor antagonist Ki16425 abrogated the LPA-induced effect. Our seahorse analysis results showed that LPA treatment increased the OCR (P&amp;lt;0.001) and the ECAR (P&amp;lt;0.001), whereas LPAR2 specific receptor antagonists abrogated this effect (P&amp;lt;0.001). Conclusion: Together, our results suggested that the LPA-LPAR2 axis mediates gastric cancer initiation and progression by increasing energy metabolism via oxidative phosphorylation and glycolysis. Thus, targeting the LPAR2 receptor may give us a novel therapeutic approach to treat gastric cancer. Citation Format: Hosne Ara, Susmita Bhattarai, Sudha Sharma, Utsab Subedi, Xiuping Yu, Md. Shenuarin Bhuiyan, Sumitra Miriyala, Manikandan Panchatcharam. LPAR2 depletion suppresses gastric cancer via alteration of mitochondrial energy metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5810.

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1

Lysophosphatidic acid (LPA) is a bioactive compound known to regulate various vascular functions. However, despite the fact that many vascular functions are regulated by peri-vascular cells such as pericytes, the effect of LPA on brain pericytes has not been fully evaluated. Thus, we designed this study to evaluate the effects of LPA on brain pericytes. These experiments revealed that while LPA receptors (LPARs) are expressed in cultured pericytes from mouse brains, LPA treatment does not influence the proliferation of these cells but does have a profound impact on their migration, which is regulated via the expression of LPAR1. LPAR1 expression was also detected in human pericyte culture and LPA treatment of these cells also induced migration. Taken together these findings imply that LPA-LPAR1 signaling is one of the key mechanisms modulating pericyte migration, which may help to control vascular function during development and repair processes.

AgRP neurons control feeding behaviour at cortical synapses via peripherally derived lysophospholipids.

Phospholipid levels are influenced by peripheral metabolism. Within the central nervous system, synaptic phospholipids regulate glutamatergic transmission and cortical excitability. Whether changes in peripheral metabolism affect brain lipid levels and cortical excitability remains unknown. Here, we show that levels of lysophosphatidic acid (LPA) species in the blood and cerebrospinal fluid are elevated after overnight fasting and lead to higher cortical excitability. LPA-related cortical excitability increases fasting-induced hyperphagia, and is decreased following inhibition of LPA synthesis. Mice expressing a human mutation (Prg-1R346T) leading to higher synaptic lipid-mediated cortical excitability display increased fasting-induced hyperphagia. Accordingly, human subjects with this mutation have higher body mass index and prevalence of type 2 diabetes. We further show that the effects of LPA following fasting are under the control of hypothalamic agouti-related peptide (AgRP) neurons. Depletion of AgRP-expressing cells in adult mice decreases fasting-induced elevation of circulating LPAs, as well as cortical excitability, while blunting hyperphagia. These findings reveal a direct influence of circulating LPAs under the control of hypothalamic AgRP neurons on cortical excitability, unmasking an alternative non-neuronal route by which the hypothalamus can exert a robust impact on the cortex and thereby affect food intake.

Lysophosphatidic acid supports the development of vitrified ovarian follicles by decreasing the incidence of cell death: An experimental study

BackgroundLysophosphatidic acid (LPA) contributes to follicular activation, oocyte maturation, in vitro fertilization, and embryo implantation.ObjectiveThis study was designed to evaluate the effects of LPA to improve the development of isolated follicles derived from whole mouse cultured vitrified ovaries.Materials and Methods In this experimental study, first, the 1-wk-old mouse ovaries in the non-vitrified and vitrified groups were cultured in the presence of 20 µM of LPA for 1 wk. Then, their isolated preantral follicles were cultured individually for 12 days in the presence or absence of 40 µM of LPA. The following evaluations were done for the cultured follicles: a viability test using Calcein AM staining, flow cytometry using annexin V/Pi, and analysis of the expression of genes by real-time reverse transcription polymerase chain reaction. The maturation rates of the oocytes were compared among groups and some of the released metaphase II oocytes were subjected to in vitro fertilization.ResultsIn all LPA treated groups, the rates of survival and follicular development were higher, and the incidence of cell death and expression of pro-apoptotic genes were lower, than in the non-LPA supplemented groups (p = 0.035). There was no significant difference between the vitrified and non-vitrified groups regarding follicular or oocyte development, but the expression of Bad and LPA receptors genes was significantly altered in the vitrified LPA supplemented group in comparison with the non-vitrified LPA supplemented group (p = 0.028).ConclusionLPA improved the survival and developmental potential of the isolated follicles. Despite some alterations in the expression of apoptosis-related genes in the vitrified ovaries, LPA had positive effects on the survival and development of these follicles.

Autotaxin signaling facilitates β cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency

Objectiveβ cell dedifferentiation may underlie the reversible reduction in pancreatic β cell mass and function in type 2 diabetes (T2D). We previously reported that β cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic β cell dedifferentiation in Sirt3-deficient β cells. MethodsWe applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on β cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the β cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. ResultsIn Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by β cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced β cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. ConclusionsSirt3 prevents β cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse β cell dysfunction associated with glucolipotoxicity.

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

Autotaxin/Lysophosphatidic Acid Axis: From Bone Biology to Bone Disorders.

Lysophosphatidic acid (LPA) is a natural bioactive phospholipid with pleiotropic activities affecting multiple tissues, including bone. LPA exerts its biological functions by binding to G-protein coupled LPA receptors (LPA1-6) to stimulate cell migration, proliferation, and survival. It is largely produced by autotaxin (ATX), a secreted enzyme with lysophospholipase D activity that converts lysophosphatidylcholine (LPC) into active LPA. Beyond its enzymatic activity, ATX serves as a docking molecule facilitating the efficient delivery of LPA to its specific cell surface receptors. Thus, LPA effects are the result of local production by ATX in a given tissue or cell type. As a consequence, the ATX/LPA axis should be considered as an entity to better understand their roles in physiology and pathophysiology and to propose novel therapeutic strategies. Herein, we provide not only an extensive overview of the relevance of the ATX/LPA axis in bone cell commitment and differentiation, skeletal development, and bone disorders, but also discuss new working hypotheses emerging from the interplay of ATX/LPA with well-established signaling pathways regulating bone mass.

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

LPA effect on integrin recruitment and actin remodeling in aged vascular smooth muscle cells

Arterial aging is associated with decreased arterial contractility and altered mechanosensitive response to external stimuli in vascular smooth muscle cells. This study investigates the effect of lysophosphatidic acid (LPA) on integrin-mediated cell adhesion and actin fiber formation in aged cells. Vascular smooth muscle cells were isolated from soleus feed arteries from young (4 months) and old (24 months) male Fischer 344 rats. To test the effect of LPA on integrin recruitment at cell-matrix adhesions and actin stress fiber formation, cells were plated on cell culture dishes functionalized with fibronectin and collagen-I.

The Effect of Bioactive Phospholipids on Normal and Malignant Hematopoiesis—Modulation of Their Effects By Downregulating the Activities of Heme Oxygenase 1 (HO-1) and Inducible Nitric Oxygenase Synthase (iNOS)

Background. Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), haveemerged as important mediators regulating the trafficking of several types of normal and malignant cells. As we have demonstrated, these factors are upregulated in tissues, including in the bone marrow microenvironment after radio/chemotherapy [Leukemia 2010; 24: 976-985] . While the role of S1P in regulating the trafficking of normal and malignant hematopoietic cells is well established, in this work we focused on the biological effects of C1P, LPC, and LPA. Hypothesis. We hypothesized that not only S1P but also other bioactive phospholipids are involved in regulating hematopoietic cell trafficking and expansion within hematopoietic tissues. Moreover, based on our results showing negative effects of heme oxygenase 1 (HO-1) [ Stem Cell Rev . 2015, 11, 110-118 ] and inducible nitric oxygenase synthase (iNOS) [ Stem Cell Rev . 2017, 13, 92-103 ] on the trafficking of normal hematopoietic cells, we hypothesized that this process could be inhibited by upregulating HO-1 and iNOS in malignant hematopoietic cells.Materials and Methods. We employed eight human myeloid and lymphoid cell lines as well as cells from patients diagnosed with acute myeloid leukemia. We phenotyped these cells for expression of bioactive lipid receptors and tested the functionality of these receptors by assaying for activation of p42/44 MAPK and AKT after stimulation with S1P, C1P, LPC, and LPA. We also tested the effect of bioactive phospholipids on cell migration, adhesion, and proliferation. In parallel experiments, we evaluated the effect of bioactive phospholipids on the expression of HO-1 and iNOS in human leukemic cells and the involvement of p38 MAPK in these phenomena. Furthermore, in in vivo experiments the expression of HO-1 and iNOS was upregulated by administration of the p38 MAPK inhibitor SB203580. We also performed in vivo seeding efficiency studies in immunodeficient mice with cells stimulated by bioactive phospholipids in the presence or absence of SB203580. In parallel, we also tested the chemotactic responsiveness of normal human CD34+ cells and clonogenic blasts from AML patients to bioactive phospholipids in chemotaxis and adhesion experiments. Results. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express functional receptors for other bioactive lipids and that C1P, LPC, and LPA are, as has been reported for S1P, important chemoattractants and pro-adhesive factors for human leukemic cells. In contrast only S1P and C1P, but not LPC or LPA stimulated migration and adhesion of normal human clonogeneic CD34+ cells. At the same time, we report that all of the tested phospholipids did not stimulate proliferation of human clonogeneic CD34+ or leukemic cells. We also demonstrate that these pro-migratory effects of bioactive phospholipids can be explained, at least partially, by downregulation HO-1 and iNOS activities in a p38 MAPK-dependent manner. Stimulation of leukemic cells before injection into immunodeficient mice with bioactive phospholipids increased their seeding efficiency to BM, liver, and lungs. This effect was significantly inhibited after exposure of these cells to SB203580. Conclusions. We demonstrate for the first time that, besides S1P receptors, human leukemic cells express other functional receptors that enable chemotaxis in response to, as well as adhesion to, several bioactive phospholipids. Since this effect is mediated, at least partially, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, inhibitors of p38 MAPK may find application in inhibiting the spread of leukemic cells in situations in which S1P, C1P, LPC, and LPA become upregulated in the tissues. This may occur, for example, after radio- and chemotherapy, leading to the unwanted spread of leukemic cells that have escaped therapy. DisclosuresNo relevant conflicts of interest to declare.

Lysophosphatidic acid activates nociceptors and causes pain or itch depending on the application mode in human skin.

Lysophosphatidic acid (LPA) is involved in the pathophysiology of cholestatic pruritus and neuropathic pain. Slowly conducting peripheral afferent C-nerve fibers are crucial in the sensations of itch and pain. In animal studies, specialized neurons ("pruriceptors") have been described, expressing specific receptors, eg, from the Mas-related G-protein-coupled receptor family. Human nerve fibers involved in pain signaling ("nociceptors") can elicit itch if activated by focalized stimuli such as cowhage spicules. In this study, we scrutinized the effects of LPA in humans by 2 different application modes on the level of psychophysics and single nerve fiber recordings (microneurography). In healthy human subjects, intracutaneous LPA microinjections elicited burning pain, whereas LPA application through inactivated cowhage spicules evoked a moderate itch sensation. Lysophosphatidic acid microinjections induced heat hyperalgesia and hypersensitivity to higher electrical stimulus frequencies. Pharmacological blockade of transient receptor potential channel A1 or transient receptor potential channel vanilloid 1 reduced heat hyperalgesia, but not acute chemical pain. Microneurography revealed an application mode-dependent differential activation of mechanosensitive (CM) and mechanoinsensitive C (CMi) fibers. Lysophosphatidic acid microinjections activated a greater proportion of CMi fibers and more strongly than CM fibers; spicule application of LPA activated CM and CMi fibers to a similar extent but excited CM fibers more and CMi fibers less intensely than microinjections. In conclusion, we show for the first time in humans that LPA can cause pain as well as itch dependent on the mode of application and activates afferent human C fibers. Itch may arise from focal activation of few nerve fibers with distinct spatial contrast to unexcited surrounding afferents and a specific combination of activated fiber subclasses might contribute.

Lysophosphatidic acid improves oocyte quality during IVM by activating the ERK1/2 pathway in cumulus cells and oocytes.

Oocyte IVM technology is an option for fertility preservation in some groups of patients, such as those with polycystic ovary syndrome, patients with ovarian hyperstimulation syndrome, and for patients with cancer. However, the developmental potential of oocytes from IVM still needs to improve. Several previous studies have reported that lysophosphatidic acid (LPA) promotes glucose metabolism, cumulus cell (CC) expansion, and oocyte nuclear maturation. However, the effect of LPA on oocyte cytoplasmic maturation, particularly mitochondrial function, has rarely been studied and the underlying mechanism is largely unknown, which impedes (pre)clinical applications of LPA. In this study, cumulus-oocyte complexes (COCs) and cumulus-denuded germinal vesicle oocytes (DOs) were treated with various concentrations of LPA during IVM, in the presence or absence of the oxidative stressor cyclophosphamide (CTX). In both normal and CTX-damaged COCs, the 25 μM LPA group exhibited improved CC expansion capacity, a higher nuclear maturation rate, and superior mitochondrial function, compared to no LPA treatment. When the concentration of LPA was over 40 μM, detrimental effects of LPA on oocyte maturation occurred. Compared with COCs, the addition of LPA slightly improved oocyte nuclear and cytoplasmic maturation of DOs, but this was not statistically significant. We observed that LPA promotes the activation of extracellular signal-regulated kinase (ERK)1/2, although this was not statistically significant in DOs. Furthermore, LPA could not reverse the negative effect of CC expansion and mitochondrial function after inactivation of ERK1/2 by U0126. RNA-sequencing and RT-PCR results showed that LPA upregulated several ERK1/2 downstream genes related to CC expansion, such as Areg, Cited4, and Ptgs2. This study demonstrates that LPA improves oocyte quality during IVM through the activation of ERK1/2 pathway CCs and oocytes, which provides evidence for the potential addition of LPA to IVM medium.

High Expression of Lysophosphatidic Acid Induces Nerve Injury in LSS Patients via AKT Mediated NF-κB p65 Pathway.

Lumbar spinal stenosis (LSS) is a spinal degenerative disease, complicated with nerve injury. Lysophosphatidic acid (LPA), a kind of glycerophospholipid molecule is elevated in the initial stages of neural injury. This research aimed to investigate the patho-mechanism of nerve injury caused by LPA in LSS patients. Twenty-five LSS patients and fifteen idiopathic scoliosis patients (without neurological symptoms) were recruited from Xianyang Central Hospital of Shanxi Province. We measured the concentration of LPA in cerebrospinal fluid samples of all subjects. Different concentrations (0.1, 1, and 10 mol/L) of LPA were used to stimulate Rat Neurons-spinal cord (RN-SC) cells. The effects of LPA on cell injury was detected by MTT and LDH (lactate dehydrogenase) assay. Cell apoptosis was determined by FCM (flow cytometry) and TUNEL staining. The changes in the expression of key proteins involved in Akt mediated NF-κB p65 pathway intervened by LPA were determined by western blot. RN-SC cells were pretreated with JSH-23 (NF-κB inhibitor) before LPA exposure, followed by cell apoptosis measurement. The concentration of LPA in LSS patients was notably higher than that in control patients (p < 0.01). The level of LPA was positively correlated with the severity of LSS. LPA treatment induced RN-SC cells displaying oval or rounded cell body with degenerated protrusion dose dependently. In addition, LPA decreased RN-SC cell viability and promoted cell apoptosis in a dose-dependent manner. LPA initiated Akt phosphorylation, IKB phosphorylation, and NF-κB nuclear translocation in a dose-dependent manner. However, JSH-23 (NF-κB inhibitor) pre-treatment prevented effects of LPA. The high levels of LPA induced nerve injury by reducing the viability of RN-SC cells and promoted cell apoptosis through Akt mediated NF-κB p65 signaling pathway. LPA might be a new therapeutic target for relieving nerve injury in LSS patients.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Lysophosphatidic acid induces thrombospondin-1 production in primary cultured rat cortical astrocytes.

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.

Deregulation of lysophosphatidic acid metabolism in oral cancer promotes cell migration via the up-regulation of COX-2.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide and accounts for 300,000 new cases yearly. The five-year survival rate is approximately 50% and the major challenges to improving patient prognosis include late presentation, treatment resistance, second primary tumours and the lack of targeted therapies. Therefore, there is a compelling need to develop novel therapeutic strategies. In this study, we have examined the effect of lysophosphatidic acid (LPA) on OSCC cell migration, invasion and response to radiation, and investigated the contribution of cyclooxygenase-2 (COX-2) in mediating the tumour promoting effects of LPA. Using the TCGA data set, we show that the expression of the lipid phosphate phosphatases (LPP), LPP1 and LPP3, was significantly down-regulated in OSCC tissues. There was no significant difference in the expression of the ENPP2 gene, which encodes for the enzyme autotaxin (ATX) that produces LPA, between OSCCs and control tissues but ENPP2 levels were elevated in a subgroup of OSCCs. To explore the phenotypic effects of LPA, we treated OSCC cell lines with LPA and showed that the lipid enhanced migration and invasion as well as suppressed the response of the cells to irradiation. We also show that LPA increased COX-2 mRNA and protein levels in OSCC cell lines and inhibition of COX-2 activity with the COX-2 inhibitor, NS398, attenuated LPA-induced OSCC cell migration. Collectively, our data show for the first time that COX-2 mediates some of the pro-tumorigenic effects of LPA in OSCC and identifies the ATX-LPP-LPA-COX-2 pathway as a potential therapeutic target for this disease.

Activation of Macrophages by Lysophosphatidic Acid through the Lysophosphatidic Acid Receptor 1 as a Novel Mechanism in Multiple Sclerosis Pathogenesis.

Multiple sclerosis (MS) is a neuroinflammatory disease whose pathogenesis remains unclear. Lysophosphatidic acid (LPA) is an endogenous phospholipid involved in multiple immune cell functions and dysregulated in MS. Its receptor LPA1 is expressed in macrophages and regulates their activation, which is of interest due to the role of macrophage activation in MS in both destruction and repair. In this study, we studied the genetic deletion and pharmaceutical inhibition of LPA1 in the mouse MS model, experimental autoimmune encephalomyelitis (EAE). LPA1 expression was analyzed in EAE mice and MS patient immune cells. The effect of LPA and LPA1 on macrophage activation was studied in human monocyte-derived macrophages. We show that lack of LPA1 activity induces milder clinical EAE course and that Lpar1 expression in peripheral blood mononuclear cells (PBMC) correlates with onset of relapses and severity in EAE. We see the same over-expression in PBMC from MS patients during relapse compared with progressive forms of the disease and in stimulated monocyte-derived macrophages. LPA induced a proinflammatory-like response in macrophages through LPA1, providing a plausible way in which LPA and LPA1 dysregulation can lead to the inflammation in MS. These data show a new mechanism of LPA signaling in the MS pathogenesis, prompting further research into its use as a therapeutic target biomarker.

Lysophosphatidic acid as a regulator of endometrial connective tissue growth factor and prostaglandin secretion during estrous cycle and endometrosis in the mare

BackgroundEquine endometrosis is a chronic degenerative condition, described as endometrial fibrosis that forms in the stroma, under the basement membrane and around the endometrial glands. The role of lysophosphatidic acid (LPA) in the development of tissue fibrosis varies depending on the organ, and its profibrotic role in mare endometrosis remains unclear. The study aimed to establish the endometrial presence of LPA and its receptors (LPAR1–4), together with its effects on connective tissue growth factor (CTGF) and prostaglandins (PG) secretion from equine endometrium under physiological (estrous cycle), or pathological conditions (endometrosis). Mare endometria in the mid-luteal phase (n = 5 for each category I, IIA, IIB, III of Kenney and Doig) and in the follicular phase (n = 5 for each category I, IIA, III and n = 4 for IIB) were used. In experiment 1, the levels of LPA, LPAR1–4 mRNA level and protein abundance were investigated in endometria at different stages of endometrosis. In experiment 2, the in vitro effect of LPA (10− 9 M) on the secretion of CTGF and PGs from endometrial tissue explants at different stages of endometrosis were determined.ResultsEndometrial LPA concentration was higher in the mid-luteal phase compared to the follicular phase in category I endometrium (P < 0.01). There was an alteration in endometrial concentrations of LPA and LPAR1–4 protein abundance in the follicular phase at different stages of endometrosis (P < 0.05). Additionally, LPA increased the secretion of PGE2 from category I endometrium in both phases of the estrous cycle (P < 0.05). The effect of LPA on the secretion of CTGF and PGF2α from endometrial tissue was altered depending on different stages of endometrosis (P < 0.05).ConclusionOur data indicate that endometrosis disturbs proper endometrial function and is associated with altered endometrial LPA concentration, its receptor expression and protein abundance, PGE2/PGF2α ratio, and CTGF secretion in response to LPA. These changes could influence several physiological events occurring in endometrium in mare during estrous cycle and early pregnancy.