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Lysophosphatidic acid (LPA) is one of the lipids identified to be involved in stem cell differentiation. It exerts various functions through activation of G protein-coupled lysophosphatidic acid receptors (LPARs). In previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis of human hematopoietic stem cells (HSCs) and zebrafish using molecular and pharmacological approaches. Our results show that treatment with lysophosphatidic acid receptor 2 (LPA2) agonist suppressed erythropoiesis, whereas activation of LPA3 by 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted it, both in vitro and in vivo. Furthermore, we have demonstrated the inhibitory role of LPA3 during megakaryopoiesis. However, the mechanism underlying these observations remains elusive. In the present study, we suggest that the expression pattern of LPARs may be correlated with the transcriptional factors GATA-1 and GATA-2 at different stages of myeloid progenitors. We determined that manipulation of GATA factors affected the expression levels of LPA2 and LPA3 in K562 leukemia cells. Using luciferase assays, we demonstrate that the promoter regions of LPAR2 and LPAR3 genes were regulated by these GATA factors in HEK293T cells. Mutation of GATA-binding sites in these regions abrogated luciferase activity, suggesting that LPA2 and LPA3 are regulated by GATA factors. Moreover, physical interaction between GATA factors and the promoter region of LPAR genes was verified in K562 cells using chromatin immunoprecipitation (ChIP) studies. Taken together, our results suggest that balance between LPA2 and LPA3 expression, which may be determined by GATA factors, is a regulatory switch for lineage commitment in myeloid progenitors. The expression-level balance of LPA receptor subtypes represents a novel mechanism regulating erythropoiesis and megakaryopoiesis.

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression, and resistance. It is produced mainly by autotaxin and acts on six G-protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors may involve any novel, thus far uncharacterized, signaling pathway(s). Here we show that three LPA receptors are involved in tumor progression by activation of both the AKT and ERK signaling pathways. CRISPR-edited LPAR2 and LPAR3 knockouts have opposing effects on ERK activation, whereas LPAR6 is involved in the activation of AKT, affecting cell migration and invasion. Our study identifies specific molecular machinery triggered by LPA and its receptors that modulates tumor cells and can serve as therapeutic target in this malignancy.

The lysophosphatidic acid receptor-3 (LPAR3) is a G protein–coupled receptor that mediates viability among malignant cells and aggressiveness among certain tumors. The study's objective was to determine the interplay between LPAR3 and miRNAs to impact key cellular signaling pathways. Using SK-Mel-2 and SK-Mel-5 melanoma cells, wild-type and mutated receptors were stably expressed to explore molecular mechanisms. LPAR3 signaling induced miR-122-5p intracellularly and subsequently its inclusion into exosomes. This amplification resulted in less abundant Wnt1, maintenance of GSK3 inactivation and to a lesser extent, partial degradation of β-catenin. The surge in miR-122-5p and reduction in Wnt1 originated from signaling at the Src homology 3 (SH3) ligand–binding motif within the third intracellular loop of LPAR3, because mutant receptors did not increase miR-122-5p and had a weakened capacity to reduce Wnt1. In addition, a key mediator of melanoma survival signaling, the peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A/PGC1), was involved in miR-122-5p transcription. In conclusion, this study highlights the powerful role miRNAs have in fine-tuning specific G protein–coupled receptor-mediated signaling events by altering the transcription of signaling transduction pathway components. This study also identifies that LPAR3 increases miR-122-5p expression, which occurs mechanistically through the SH3 domain and helps explain why miR-122-5p increases are detected in cancer patient serum. Implications: LPAR3 is partially responsible for the production and secretion of miR-122-5p, found in the serum of a wide variety of patients with cancer.

Lysophosphatidic acid (LPA) is a small lysophospholipid molecule that activates multiple cellular functions through pathways with G-protein-coupled receptors. So far, six LPA receptors (LPAR1 to LPAR6) have been discovered and each one of them can connect to the downstream cell message-transmitting network. A previous study demonstrated that LPA receptors found in blood-producing stem cells can enhance erythropoietic processes through the activation of LPAR3. In the current study, newly discovered functions of LPAR3 were identified through extensive behavioral tests in lpar3 knockout (KO) zebrafish. It was found that the adult lpar3 KO zebrafish display an abnormal movement orientation and altered exploratory behavior compared to that of the control group in the three-dimensional locomotor and novel tank tests, respectively. Furthermore, consistent with those results, in the circadian rhythm locomotor activity test, the lpar3 KO zebrafish showed a lower level of angular velocity and average speed during the light cycles, indicating an hyperactivity-like behavior. In addition, the mutant fish also exhibited considerably higher locomotor activity during the dark cycle. Supporting those findings, this phenomenon was also displayed in the lpar3 KO zebrafish larvae. Furthermore, several important behavior alterations were also observed in the adult lpar3 KO fish, including a lower degree of aggression, less interest in conspecific social interaction, and looser shoal formation. However, there was no significant difference regarding the predator avoidance behavior between the mutant and the control fish. In addition, lpar3 KO zebrafish displayed memory deficiency in the passive avoidance test. These in vivo results support for the first time that the lpar3 gene plays a novel role in modulating behaviors of anxiety, aggression, social interaction, circadian rhythm locomotor activity, and memory retention in zebrafish.

Background Thyroid cancer (TC) is a member of common malignant tumors in endocrine system. To develop effective treatment, further comprehension of understanding molecular mechanism in TC is necessary. In this research, we attempted to search the underlying molecular mechanism in TC. Methods ZEB1-AS1 expression was analyzed via qRT-PCR analysis. CCK-8, colony formation, flow cytometry and TUNEL assays were used to evaluate TC cell growth. The interaction between miR-133a-3p and LPAR3, EGFR and ZEB1-AS1 was testified through using RNA pull down and luciferase reporter assays. Results LPAR3 and EGFR were expressed at high levels in TC tissues and cell lines. Besides, both LPAR3 and EGFR could promote TC cell growth. Later, miR-133a-3p was searched as an upstream gene of LPAR3 and EGFR, and LPAR3 could partially rescue the suppressive effect of miR-133a-3p overexpression on TC progression, whereas the co-transfection of LPAR3 and EGFR completely restored the inhibition. Next, ZEB1-AS1 was confirmed as a sponge of miR-133a-3p. ZEB1-AS1 has a negative correlation with miR-133a-3p and a positive association with LPAR3 and EGFR through ceRNA analysis. Importantly, ZEB1-AS1 boosted the proliferation and suppressed the apoptosis in TC cells. Through restoration assays, we discovered that ZEB1-AS1 regulated LPAR3 and EGFR expression to mediate TC cell proliferation and apoptosis by sponging miR-133a-3p. Further investigation also indicated the oncogenic role of ZEB1-AS1 by mediating PI3K/AKT/mTOR pathway. Conclusions ZEB1-AS1 could be an underlying biomarker in TC.

Extracellular lysophosphatidic acid (LPA) and the G-protein-coupled LPA receptors (LPAR) are involved in cell migration and invasion and found in the human endometrium. However, underlying mechanisms resulting in cellular invasion have been rarely investigated. We used stromal endometrial T-HESC, epithelial endometriotic 12Z, 49Z and Ishikawa cells. Interestingly, proliferation of T-HESC cells was strongly increased after LPA treatment, whereas the epithelial cell lines only showed a moderate increase. LPA increased invasion of 12Z and 49Z strongly and significantly. The LPAR inhibitor Ki16425 (LPAR1/3) attenuated significantly LPA-induced invasiveness of 12Z, which was confirmed by LPAR1 and LPAR3 siRNAs, showing that both LPA receptors contribute to invasiveness of 12Z cells. Investigation of cell invasion with an antibody-based protease array revealed mainly differences in cathepsins and especially cathepsin B between 12Z compared to the less invasive Ishikawa. Stimulation with LPA showed a time- and dose-dependent increased secretion of cathepsin B which was inhibited by the Gq inhibitor YM-254890 and Gi/o inhibitor pertussis toxin in the 12Z cells, again highlighting the importance of LPAR1/3. The activity of intracellular and secreted cathepsin B was significantly upregulated in LPA-treated samples. Inhibition of cathepsin B with the specific inhibitor CA074 significantly reduced LPA-increased invasion of 12Z. Our results reveal a novel role of LPA-mediated secretion of cathepsin B which stimulated invasion of endometriotic epithelial cells mainly via LPAR1 and LPAR3. These findings may deepen our understanding how endometriotic cells invade into ectopic sites, and provide new insights into the role of LPA and cathepsin B in cellular invasion.

Lysophosphatidic acid (LPA) is a small ubiquitous lipid exerting diverse biological functions. Its role in reproduction in different species has created great interest in recent times. In the present study, we aimed to elucidate LPA signaling in nonpregnant buffalo uterus by in vitro studies. Standard techniques like real-time PCR (for mRNA expression of LPARs and COX-2 and iNOS), Western blot (for PPARγ protein expression), sandwich ELISA (for PGE2 and PGF2α assay) and histopathology (for assessment of endometrial architecture in culture) were used in this study. The buffalo uterine tissues were collected from the local slaughterhouse and were selected for the study on the basis of the presence of corpus luteum on the ovary (n = 5). The LPAR3 receptor was the highest expressed receptor as compared to LPAR1 and LPAR6 in non-pregnant uterine tissues after 6 h incubation in Dulbecco's Modified Eagle Medium (DMEM). 50 μM LPA increased the mRNA expressions of COX-2 and iNOS enzymes which were attenuated by the treatment of LPAR1/3 antagonist Ki16425. PPARγ antagonist GW9662 prevented the LPA-induced increase in iNOS mRNA expression but did not alter the COX-2 expression. LPA also enhanced the PGE2 to PGF2α ratio in uterine tissue homogenates which was inhibited by all the receptor antagonists as well as by the inhibitors of COX-2 and iNOS. LPA also increased the total nitrite level in tissue homogenates in LPAR1/3- and iNOS-dependent manner. Additionally, we demonstrate PPARγ mRNA and protein expressions in nonpregnant buffalo endometrium. In conclusion, the results of the present study suggest that LPA acts as a luteotropic factor during the estrus cycle in nonpregnant buffalo uterus by enhancing PGE2 to PGF2α ratio and NO level through multiple receptors.

Lysophosphatidic acid (LPA) is a simple biological lipid and mediates several biological functions with LPA receptors (LPA1 to LPA6). In the present study, to assess whether LPA receptors promote cell-invasive activity of pancreatic cancer cells, highly invasion PANC-R9 cells were established from PANC-1 cells, using Matrigel-coated Cell Culture Insert. The cell-invasive activity of PANC-R9 cells was shown to be approximately 15 times higher than that of PANC-1 cells. LPAR1 expression level was markedly elevated in PANC-R9 cells in comparison with PANC-1 cells, while LPAR3 expression level was reduced. The cell-invasive activity of PANC-R9 cells was enhanced by LPA, but LPA had no impact on PANC-1 cell invasion. Before initiation of the cell invasion assay, PANC-R9 cells were pretreated with dioctanoylglycerol pyrophosphate (DGPP), an antagonist of LPA1/LPA3. The invasive activity of PANC-R9 cells was markedly suppressed by DGPP. Autotaxin (ATX) is a key enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to LPA. ATX expression level was elevated in PANC-R9 cells compared with PANC-1 cells. In the presence of LPC, the cell motile activity of PANC-R9 cells was markedly stimulated. In contrast, LPC did not affect the cell motile activity of PANC-1 cells. PANC-R9 cell motility was inhibited by an ATX inhibitor, PF-8380. These results suggest that LPA signaling via LPA1 is a potent molecular target for the regulation of tumor progression in PANC-1 cells.

During pregnancy, up‐regulation of heparin‐binding (HB‐) EGF and cyclooxygenase‐2 (COX‐2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G‐protein‐coupled receptor LPA 3 in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA‐producing enzyme autotaxin (ATX) in pregnant mice leads to HB‐EGF and COX‐2 down‐regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA 3 induces decidualization via up‐regulation of HB‐EGF and COX‐2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre‐implantation‐stage embryos, respectively. Our results indicate that ATX–LPA–LPA 3 signaling at the embryo‐epithelial boundary induces decidualization via the canonical HB‐EGF and COX‐2 pathways.

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA1 and LPA3 in cellular functions during tumor progression in pancreatic cancer cells. LPA1 and LPA3 knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA1 and LPA3 knockdown. In gelatin zymography, LPA1 and LPA3 knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA1 and LPA3 regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPAR1 and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA1 and LPA3 knockdown as well as colony formation. These results suggest that LPA signaling via LPA1 and LPA3 play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells.

Problem Recent advances in lipid research have revealed that impairments in lipid mediator signaling can be involved in the pathoetiology of a variety of diseases. We previously reported aberrant expression of autotaxin, a key enzyme for lysophosphatidic acid (LPA) production, in placentas from women with preeclampsia. The present study aimed to further explore the involvement of LPA signaling in the pathoetiology of preeclampsia. Method of study Term placentas were obtained from deliveries after uncomplicated pregnancy (n = 18) and those complicated by preeclampsia (n = 24). First-trimester placental tissues were collected after elective terminations of pregnancy (n = 20). Placental expression of the six identified LPARs (LPAR1-6) was analyzed at protein and mRNA levels. Results In normal pregnancy, the mRNA expression levels of all LPARs except LPAR4 were significantly higher in term. Levels of mRNA encoding LPAR2-5 were significantly increased in preeclampsia placentas compared with those in the normal term placentas. Using Western immunoblotting, only LPAR3 was noted to be increased at the protein level in placentas from preeclamptic pregnancies. This was validated by immunohistochemistry. Conclusion In summary, the placental expression of LPARs, particularly LPAR3, is enhanced in preeclampsia, suggesting that disturbances in placental LPA signaling may be involved in the pathogenesis of preeclampsia.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of biological responses. In tumor microenvironment, endothelial cells promote cancer cell functions. In this study, we investigated the roles of endothelial cells in the regulation of cell motile activity via LPA2 and LPA3 in human osteosarcoma MG-63 cells. In cell motility assay, the cell motile activity of MG-63 cells was markedly increased by the supernatants of endothelial F2 cells. MG-63 cell motility elevated by the supernatants was enhanced by GRI-977143 (LPA2 agonist) and reduced by (2S)-OMPT (LPA3 agonist). LPAR2 and LPAR3 expressions were increased in highly migratory MG63-CR7(F2) cells, which were generated from MG-63 cells by co-culture with F2 cell supernatants. MG63-CR7(F2) cell motility was stimulated by LPA treatment. In the presence of F2 cell supernatants, MG63-CR7(F2) cell motility was markedly enhanced by GRI-977143 and suppressed by (2S)-OMPT. Autotaxin (ATX) enzymatically converts lysophosphatidylcholine (LPC) to LPA. ATX expression was higher in MG63-CR(F2) cells than in MG-63 cells. MG63-CR7(F2) cell motility was markedly increased by LPC in comparison with MG-63 cells. In addition, MG63-CR(F2) cell motility was significantly stimulated by the supernatants of LPC treated F2 cells. The present results suggest that the activation of LPA signaling via LPA2 and LPA3 by endothelial cells is involved in the modulation of cell motile activity of MG-63 cells.

Lysophosphatidic acid (LPA) mediates a variety of biological functions via the binding of G protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). This study aimed to investigate the roles of LPA2 and LPA3 in the modulation of chemoresistance to anticancer drug in lung cancer A549 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate to CDDP of A549 cells was significantly elevated by an LPA2 agonist, GRI-977143. To evaluate the roles of LPA2-mediated signaling in cell survival during tumor progression, highly migratory (A549-R10) cells were generated from A549 cells. In the presence of GRI-977143, the cell survival rate to CDDP of A549-R10 cells were markedly higher than that of A549 cells, correlating with LPAR2 expression level. Moreover, to assess the effects of long-term anticancer drug treatment on cell survival, the long-term CDDP treated (A549-CDDP) cells were established from A549 cells. The cell survival rate to CDDP of A549-CDDP cells was elevated by GRI-977143. Since LPAR3 expression level was significantly higher in A549-CDDP cells than in A549 cells, we investigated the roles of LPA3 in the cell survival to CDDP of A549 cells, using an LPA3 agonist, 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate ((2S)-OMPT). The cell survival rate to CDDP of A549 cells was significantly reduced by (2S)-OMPT treatment. In the presence of (2S)-OMPT, the cell survival rate to CDDP of A549 cells was elevated by LPA3 knockdown. These results suggest that LPA signaling via LPA2 and LPA3 is involved in the regulation of chemoresistance in A549 cells treated with CDDP.

Diabetic nephropathy (DN) is one of the major long-term complications of diabetes. Lysophosphatidic acid (LPA) signaling has been implicated in renal fibrosis. In our previous study, we found that the LPA receptor 1/3 (LPAR1/3) antagonist, ki16425, protected against DN in diabetic db/db mice. Here, we investigated the effects of a specific pharmacological inhibitor of LPA receptor 1 (LPA1), AM095, on DN in streptozotocin (STZ)-induced diabetic mice to exclude a possible contribution of LPAR3 inhibition. AM095 treatment significantly reduced albuminuria and the albumin to creatinine ratio and significantly decreased the glomerular volume and tuft area in the treated group compared with the STZ-vehicle group. In the kidney of STZ-induced diabetic mice, the expression of LPAR1 mRNA and protein was positively correlated with oxidative stress. AM095 treatment inhibited LPA-induced reactive oxygen species production and NADPH oxidase expression as well as LPA-induced toll like receptor 4 (TLR4) expression in mesangial cells and in the kidney of STZ-induced diabetic mice. In addition, AM095 treatment suppressed LPA-induced pro-inflammatory cytokines and fibrotic factors expression through downregulation of phosphorylated NFκBp65 and c-Jun N-terminal kinases (JNK) in vitro and in the kidney of STZ-induced diabetic mice. Pharmacological or siRNA inhibition of TLR4 and NADPH oxidase mimicked the effects of AM095 in vitro. In conclusion, AM095 is effective in preventing the pathogenesis of DN by inhibiting TLR4/NF-κB and the NADPH oxidase system, consequently inhibiting the inflammatory signaling cascade in renal tissue of diabetic mice, suggesting that LPAR1 antagonism might provide a potential therapeutic target for DN.

The aim of this study was to analyze the expression, biological role and clinical relevance of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA), and LPA receptors (LPAR) in high-grade serous carcinoma (HGSC). mRNA expression by qRT-PCR of LPAR1-6 was analyzed in 155 HGSC specimens (88 effusions, 67 solid lesions). ATX mRNA expression was analyzed in 97 specimens. ATX, ERK, and AKT protein expression was studied by Western blotting. LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein. Higher LPAR1 levels were significantly related to longer overall survival (OS) in pre-chemotherapy effusions (p = 0.027). Conversely, higher expression of LPAR1, LPAR2, and LPAR5 in post-chemotherapy effusions was significantly associated with shorter OS (p = 0.037, p = 0.025 and p = 0.021, respectively) and progression-free survival (PFS) (p

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors mediates various biological effects in cancer cells. This study aimed to investigate the roles of LPA receptors in the regulation of cellular functions during tumor progression in osteosarcoma cells. Long-term cisplatin (CDDP)-treated MG63-C and MG63-R7-C cells were generated from osteosarcoma MG-63 and highly-migratory MG63-R7 cells, respectively. LPAR2 and LPAR3 expression levels were significantly higher in MG63-C cells than in MG-63 cells, while LPAR1 expression was reduced. MG63-C cells were highly motile, compared with MG-63 cells. MG63-C cell motility was suppressed by LPA2 knockdown and enhanced by the LPA1/LPA3 antagonist, dioctanoylglycerol pyrophosphate. LPAR2 and LPAR3 expression levels were significantly elevated in MG63-R7-C cells in comparison with MG63-R7 cells. MG63-R7-C cells were found to be highly invasive, correlating with metalloproteinase-2 activation. MG63-R7-C cells formed large colonies, whereas colony formation was absent from MG63-R7 cells. Notably, MG63-R7-C cell activities were inhibited by LPA2 knockdown. These results suggest that LPA signaling via LPA2 plays an important role in the acquisition of malignant properties during tumor progression in MG-63 cells.

Ferroptosis is an iron- and oxidative-dependent form of regulated cell death and may play important roles in maintaining myocardium homeostasis and pathology of cardiovascular diseases. Currently, the regulatory roles of lipid signals in regulating cardiomyocytes ferroptosis has not been explored. In this study, we show that ENPP2, as a lipid kinase involved in lipid metabolism, protects against erastin-induced ferroptosis in cardiomyocytes. The classical ferroptosis inducer erastin remarkably inhibits the growth which could be rescued by the small molecule Fer-1 in H9c2 cells. Adenovirus mediated ENPP2 overexpression modestly promotes migration and proliferation and significantly inhibits erastin-induced ferroptosis of H9c2 cells. ENPP2 overexpression leads to increase the LPA level in supernatant of H9c2 cells. H9c2 cells express the LPAR1, LPAR3, LPAR4 and LPAR5 receptors. The supernatant of ENPP2 transduced cardiomyocytes could protects the cells from erastin-induced ferroptosis of H9c2 cells. Furthermore, we observed that ENPP2 overexpression regulates ferroptosis-associated gene GPX4, ACSL4 and NRF2 expression and modulates MAPK and AKT signal in H9c2 cells. Collectively, these findings demonstrated that ENPP2/LPA protects cardiomyocytes from erastin-induced ferroptosis through modulating GPX4, ACSL4 and NRF2 expression and enhancing AKT survival signal.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and is characterized by rapid tumor expansion and metastasis. Lysophosphatidic acid (LPA) signaling, via LPA receptors 1–6 (LPARs1–6), regulates diverse cell functions including motility, migration, and proliferation, yet the role of LPARs in hepatic tumor pathology is poorly understood. We sought to determine the expression and function of endothelial differentiation gene (EDG) LPARs (LPAR1–3) in human HCC and complimentary in vitro models. Human HCC were characterized by significantly elevated LPAR1/LPAR3 expression in the microenvironment between the tumor and non-tumor liver (NTL), a finding mirrored in human SKHep1 cells. Analysis of human tissue and human hepatic tumor cells in vitro revealed cells that express LPAR3 (HCC-NTL margin in vivo and SKHep1 in vitro) also express cancer stem cell markers in the absence of hepatocyte markers. Treatment of SKHep1 cells with exogenous LPA led to significantly increased cell motility but not proliferation. Using pharmacological agents and cells transfected to knock-down LPAR1 or LPAR3 demonstrated LPA-dependent cell migration occurs via an LPAR3-Gi-ERK-pathway independent of LPAR1. These data suggest cells that stain positive for both LPAR3 and cancer stem cell markers are distinct from the tumor mass per se, and may mediate tumor invasiveness/expansion via LPA-LPAR3 signaling.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6 ) mediates a variety of cellular functions, including cell motility. In the present study, we investigated the effects of LPA receptors on cell motile activity during multi-stage hepatocarcinogenesis in rat liver epithelial WB-F344 cells treated with chemical liver carcinogens. Cells were treated with a initiator (N-nitrosodiethylamine (DEN)) and three promoters (phenobarbital (PB), okadaic acid (OA) and clofibrate) every 24 h for 2 days. Cell motile activity was elevated by DEN, correlating with Lpar3 expression. PB, OA, and clofibrate elevated Lpar1 expression and inhibited cell motile activity. To evaluate the effects of long-term treatment on cell motility, cells were treated with DEN and/or PB for at least 6 months. Lpar3 expression and cell motile activity were significantly elevated by the long-term DEN treatment with or without further PB treatment. In contrast, long-term PB treatment with or without further DEN elevated Lpar1 expression and inhibited cell motility. When the synthesis of extracellular LPA was blocked by a potent ATX inhibitor S32826 before cell motility assay, the cell motility induced by DEN and PB was markedly suppressed. These results suggest that activation of the different LPA receptors may regulate the biological functions of cells treated with chemical carcinogens. © 2015 Wiley Periodicals, Inc.

Essential hypertension is a complex disease affected by genetic and environmental factors and serves as a major risk factor for cardiovascular diseases. Serum lysophosphatidic acid correlates with an elevated blood pressure in rats, and lysophosphatidic acid interacts with 6 subtypes of receptors. In this study, we assessed the genetic association of lysophosphatidic acid receptors with essential hypertension by genotyping 28 single-nucleotide polymorphisms from genes encoding for lysophosphatidic acid receptors, LPAR1 , LPAR2 , LPAR3 , LPAR4 , LPAR5 , and LPAR6 and their flanking sequences, in 3 Han Chinese cohorts consisting of 2630 patients and 3171 controls in total. We identified a single-nucleotide polymorphism, rs531003 in the 3′-flanking genomic region of LPAR1 , associated with hypertension (the Bonferroni corrected P =1.09×10 –5 , odds ratio [95% confidence interval]=1.23 [1.13–1.33]). The risk allele C of rs531003 is associated with the increased expression of LPAR1 and the susceptibility of hypertension, particularly in those with a shortage of sleep ( P =4.73×10 –5 , odds ratio [95% confidence interval]=1.75 [1.34–2.28]). We further demonstrated that blood pressure elevation caused by sleep deprivation and phenylephrine-induced vasoconstriction was both diminished in LPAR1 -deficient mice. Together, we show that LPAR1 is a novel susceptibility gene for human essential hypertension and that stress, such as shortage of sleep, increases the susceptibility of patients with risk allele to essential hypertension.

In order to study lysophosphatidic acid (LPA) signaling associated with type 1 endometrial carcinoma (EC), we evaluated the LPA receptors (LPARs), autotaxin (ATX) and phospholipase A2 (PLA2) expression in EC and normal endometrium with correlation to clinicopathological features. We investigated LPAR1, LPAR2, LPAR3, LPAR4, ATX and PLA2 expression at mRNA and protein levels using quantitative real-time PCR and western blot analyses in 37 ECs and 10 normal endometria. All the examined LPARs (except for LPAR3 protein), ATX and PLA2 were overexpressed in cancerous compared to healthy endometrium. The studied ECs showed the highest LPAR2 and LPAR1 expression. Statistically positive correlations were found between depth of myoinvasion and levels of LPAR1, LPAR2 and PLA2 transcripts and proteins. We also found positive correlations between LPAR1, LPAR2, LPAR4 and PLA2 expression with the International Federation of Gynecology and Obstetrics (FIGO) stage. The expression of LPAR1, LPAR2 and PLA2 was positively associated with the age of patients. Positive correlations were found between the expression of LPAR1 mRNA, LPAR2 mRNA and protein and LPAR3 mRNA and body mass index (BMI) of the examined patients. We found no association between the expression levels of the studied factors and diabetes or hypertension among the examined patients. Owing to the highest LPAR2 and LPAR1 expression in EC and positive correlations of these two receptors with the depth of myoinvasion and the FIGO stage, we believe that LPAR2 and LPAR1 show promise as predictors of the EC progression as well as the main receptors responsible for LPA action in the EC tissue.

Lysophosphatidic acid (LPA) functions through activation of LPA receptors (LPARs). LPA-LPAR signaling has been implicated in development of fibrosis. However, the role of LPA-LPAR signaling in development of diabetic nephropathy (DN) has not been studied. We examined whether BMS002, a novel dual LPAR1 and LPAR3 antagonist, affects development of DN in endothelial nitric oxide synthase-knockout db/db mice. Treatment of these mice with BMS002 from 8 to 20 weeks of age led to a significant reduction in albuminuria, similar to that observed with renin-angiotensin system inhibition (losartan plus enalapril). LPAR inhibition also prevented the decline in GFR observed in vehicle-treated mice, such that GFR at week 20 differed significantly between vehicle and LPAR inhibitor groups (P

Lysophosphatidic acid (LPA) is a small ubiquitous lipid found in vertebrate and nonvertebrate organisms that mediates diverse biological actions and demonstrates medicinal relevance. LPA's functional roles are driven by extracellular signaling through at least six 7-transmembrane G protein-coupled receptors. These receptors are named LPA1–6 and signal through numerous effector pathways activated by heterotrimeric G proteins, including Gi/o, G12/13, Gq, and Gs. LPA receptor-mediated effects have been described in numerous cell types and model systems, both in vitro and in vivo, through gain- and loss-of-function studies. These studies have revealed physiological and pathophysiological influences on virtually every organ system and developmental stage of an organism. These include the nervous, cardiovascular, reproductive, and pulmonary systems. Disturbances in normal LPA signaling may contribute to a range of diseases, including neurodevelopmental and neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, fibrosis, cancer, infertility, and obesity. These studies underscore the potential of LPA receptor subtypes and related signaling mechanisms to provide novel therapeutic targets.

Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA1 to LPA6) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.

Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) indicate a variety of cellular responses, such as cell proliferation, migration, differentiation, and morphogenesis. However, the role of each LPA receptor is not functionally equivalent. Ethionine, an ethyl analog of methionine, is well known to be one of the potent liver carcinogens in rats. In this study, to assess whether ethionine may regulate cell motile activity through LPA receptors, rat liver epithelial (WB-F344) cells were treated with ethionine for 48 h. In cell motility assay with a cell culture insert, the treatment of ethionine at 1.0 and 10 μM enhanced significantly high cell motile activity, compared with untreated cells. The expression levels of LPA receptor genes in cells treated with ethionine were measured by quantitative real time RT-PCR analysis. The expression of the Lpar3 gene in ethionine-treated cells was significantly higher than that in untreated cells. Furthermore, to confirm an involvement of LPA3 on cell motility increased by ethionine, the Lpar3 knockdown cells were also used. The cell motile activity by ethionine was completely suppressed in the Lpar3 knockdown cells. These results suggest that LPA signaling through LPA3 may be involved in cell motile activity stimulated by ethionine in WB-F344 cells.

Lysophosphatidic acid (LPA), a natural phospholipid, is able to modulate diverse cellular responses through LPA receptors (LPARs). Several studies have reported that LPAR2 gene expression is increased in a variety of cancer cells, suggesting that LPAR2 is involved in gastric cancer. The present study investigated the expression profiles of the LPAR and involvement of the receptor subtypes in the LPA-induced migration of gastric cancer cells using cell migration assays, RNA interference, quantitative real-time PCR and western blotting. LPAR2 was observed to be highly expressed in SGC-7901 cells, a human gastric cancer cell line, while LPAR1 and LPAR3 were not. Transient transfection with LPAR2 siRNA was observed to reduce LPAR2 mRNA in SGC-7901 cells and eliminate the LPA-induced cell migration. It was also observed that LPA-induced SGC-7901 cell migration was inhibited by the inhibitor for Gq/11 protein and p38. The results suggest that the LPAR2/Gq/11/p38 pathway regulates LPA-induced SGC-7901 cell migration. The present findings suggest that LPAR2 may be a potential target for the clinical treatment of gastric cancer.

Lysophosphatidic acid (LPA) is a phospholipid with a variety of fatty acyl groups that mediates diverse biological effects on various types of cells through specific G protein-coupled receptors. LPA appears to play a significant role in many reproductive processes, including luteolysis, implantation, and placentation. Our previous study in pigs demonstrated that LPA and the LPA receptor system are present at the maternal-conceptus interface and that LPA increases uterine endometrial expression of prostaglandin-endoperoxide synthase 2 (PTGS2) through LPA receptor 3 (LPAR3). However, the role of LPA in conceptuses during early pregnancy has not been determined. Therefore, this study examined the effects of LPA in cell proliferation, migration, and activation of the intracellular signaling pathway in porcine conceptuses by using an established porcine trophectoderm (pTr) cell line isolated from Day 12 conceptuses. All examined LPA species with various fatty acid lengths increased proliferation and migration of pTr cells as the dosage increased. Immunoblot analyses found that LPA activated intracellular signaling molecules, extracellular signal-regulated kinase 1/2 (ERK1/2), ribosomal protein S6 kinase 90 kDa (P90RSK), ribosomal protein S6 (RPS6), and P38 in pTr cells. Furthermore, LPA increased expression of PTGS2 and urokinase-type plasminogen activator (PLAU), and the LPA-induced increases in PTGS2 and PLAU expression were inhibited by LPAR3 siRNA. Collectively, these results showed that LPA promotes proliferation, migration, and differentiation of pTr cells by activating the ERK1/2-P90RSK-RPS6 and P38 pathways, indicating that the LPA-LPAR3 system may be involved in the development of trophoblast during early pregnancy in pigs.

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of β-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). Recently, we have reported that LPA(3) indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA(3) on cell migration of sarcoma cells, we generated LPA receptor-3 (LPAR3)-knockdown (HT1080-sh3 and HOS-sh3, respectively) cells from fibrosarcoma HT1080 and osteosarcoma HOS cells, and measured their cell migration abilities. In cell motility assay with a Cell Culture Insert, both LPAR3-knockdown cells showed significantly lower cell motile activities than control cells. Next, to investigate the effect of LPAR3-knockdown on invasion activity, which degraded the extracellular matrices, the Matrigel-coated filter was used. HT1080-sh3 cells showed significantly low invasive activity compared with control cells, while no invasive activity was found in HOS-sh3 cells. In gelatin zymography, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 activities were detected in all cells. The results indicated that LPA(3) acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA(3) may be involved in the progression of sarcoma cells.

In the present study, we examined the lysophosphatidic acid (LPA) pathway in the ovine uterus during the estrous cycle and early pregnancy. With the use of quantitative reverse transcription PCR, expression of LPAR1 and LPAR3 was analyzed. Both receptors were present in the ovine uterus. Immunolocalization showed that LPAR1 was mainly present in the stroma of the ovine endometrium, whereas LPAR3 was mostly restricted to epithelial compartments. In luminal and glandular epithelia, LPAR1 and LPAR3 levels were affected by pregnancy status, day, or the day-by-status interaction, whereas in stroma the receptors were not modified. Analysis of the whole endometrium from ovariectomized ewes showed that the expression of LPAR3 but not LPAR1 was regulated by the administration of progesterone. However, the examination of receptors at cellular levels showed that progesterone increases LPAR1 and LPAR3 in glandular epithelium and, in a minor extent, in endometrial stroma. Emerging evidence suggests that LPA is an essential component in the estrous cycle and early pregnancy regulation. We demonstrated that LPA induced stress fiber formation in ovine uterine epithelial cells, suggesting that LPA may be involved in cytoskeleton reorganization occurring cyclically in ovine uterus.

Lung fibrosis is a common and serious complication of radiation therapy for lung cancer, for which there are no efficient treatments. Emerging evidence indicates that lysophosphatidic acid (LPA) and its receptors (LPARs) are involved in the pathogenesis of fibrosis. Here, we reported that thoracic radiation with 16Gy in mice induced development of radiation lung fibrosis (RLF) accompanied by obvious increases in LPA release and LPAR1 and LPAR3 (LPAR1/3) transcripts. RLF was significantly alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. VPC12249 administration effectively prolonged animal survival, restored lung structure, inhibited fibroblast accumulation and reduced collagen deposition. Moreover, profibrotic cytokines in radiation-challenged lungs obviously decreased following administration of VPC12249, including transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF). In vitro, LPA induced both fibroblast proliferation and CTGF expression in a dose-dependent manner, and both were suppressed by blockade of LPAR1/3. The pro-proliferative activity of LPA on fibroblasts was inhibited by siRNA directed against CTGF. Together, our data suggest that the LPA-LPAR1/3 signaling system is involved in the development of RLF through promoting fibroblast proliferation in a CTGF-dependent manner. The LPA-LPAR1/3-CTGF pathway may be a potential target for RLF therapy.

The tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates cell migration of several tumor cells. Recently, we reported that loss of lysophosphatidic acid (LPA) receptor-3 (LPA(3)) enhanced cell migration of murine lung tumor LL/2 cells. In the present study, we investigated whether LPA(3) is involved in cell migration of mouse lung tumor cells stimulated by TPA. Exogenous LPA(3) gene (Lpar3)-expressing (LL/2-a3) cells and LL/2-AB cells as a vector control generated from LL/2 cells were used. In a cell migration assay, TPA treatment significantly stimulated cell migration of LL/2-AB and LL/2-a3 cells, while the cell migration abilities of LL/2-a3 were markedly lower than those of LL/2-AB cells. Using quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis, no effect of TPA treatment on the expression levels of LPA(1), LPA(2) and LPA(3) genes was detected in either type of cells. These results suggest that the LPA(3) may not be involved in the enhanced migration ability by TPA in mouse lung tumor cells.

Lysophosphatidic acid (LPA), a water-soluble phospholipid, has gained significant attention in recent years since the discovery that it acts as a potent signaling molecule with wide-ranging effects on many different target tissues. There are currently five identified G protein-coupled receptors for LPA and more are undergoing validation. The complexity of the expression pattern and signaling properties of LPA receptors results in multiple influences on developmental, physiological, and pathological processes. This review provides a summary of LPA receptor signaling and current views on the potential involvement of this pathway in human diseases that include cardiovascular, cancer, neuropathic pain, neuropsychiatric disorders, reproductive disorders, and fibrosis. The involvement of LPA signaling in these processes implicates multiple, potential drug targets including LPA receptor subtypes and LPA metabolizing enzymes. Modulation of LPA signaling may thus provide therapeutic inroads for the treatment of human disease.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA1 gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N -nitrosodiethylamine (DEN) and a choline-deficient l -amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCs induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) – polymerase chain reaction (PCR) – single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%) induced by DEN. Five out of 12 HCCs (41.7%) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved in rat liver carcinogenesis.

Lysophosphatidic acid (LPA) modulates prostaglandin (PG) synthesis via LPA receptor 3 (LPAR3) in the murine endometrium. The lack of functional LPAR3 in mice may lead to embryo mortality. In the present study, we examined the role of LPA in the bovine uterus. We confirmed that LPA is locally produced and released from the bovine endometrium. Moreover, there are enzymes involved in LPA synthesis (phospholipase(PL)D2and PLA2G1B) in the bovine endometrium during estrous cycle and early pregnancy. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression ofPGE2synthase(PGES) and negatively correlated with the expression ofPGF2αsynthase(aldose reductase with 20 α-hydroxysteroid dehydrogenase activity –PGFS) during early pregnancy.In vivoLPA induced P4 and PGE2secretion was inhibited by LPAR1 antagonist (Ki16425). The overall results indicate that LPA is locally produced and released from the bovine endometrium. Moreover,LPAR1gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus.LPAR1gene expression is positively correlated with the expression of the enzyme responsible for luteotropic PGE2production (PGES) in endometrium. In cow, LPA stimulates P4 and PGE2secretion. Thus, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support corpus luteum action via the increase of P4 synthesis and the increase of PGE2/PGF2αratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.

We investigated the lysophosphatidic acid (LPA) pathway during early pregnancy in sheep. LPA was detected in the uteri of early-stage pregnant ewes. Using quantitative RT-PCR, the expression of autotaxin, the LPA-generating enzyme, was found in the endometrium and conceptus. In the latter autotaxin, transcript levels were low on d 12–14 and increased on d 15–16, in parallel with the level of LPA. Autotaxin was localized in the luminal epithelium and superficial glands of the endometrium and in trophectoderm cells of the conceptus. The expression of G protein-coupled receptors for LPA was also examined in the ovine conceptus. LPA receptor LPAR1 and LPAR3 transcripts were expressed during early pregnancy and displayed a peak on d 14, whereas the highest level of protein for both receptors was observed at d 17. LPAR1 was localized in cellular membranes and nuclear compartments of the trophectoderm cells, whereas LPAR3 was revealed only in membranes. LPA activated phosphorylation of the MAPK ERK1/2 in ovine trophectoderm-derived cells. Moreover, the bioactive lipid increased the proliferation of trophectoderm cells in culture, as shown by thymidine and bromodeoxyuridine incorporation. Furthermore, LPA induced changes to the organization of β-actin and α-tubulin, suggesting a role for it in rearrangement of trophectoderm cells cytoskeleton. Because a link had previously been established between prostaglandin and LPA pathways, we analyzed the effect of LPA on prostaglandin synthesis. LPA induced an increase in the release of prostaglandin F2α and prostaglandin E2, with no significant modifications to cytosolic phospholipase A2α and prostaglandin synthase-2 expression. Taken together, our results suggest a new role for LPA-mediated signaling in the ovine conceptus at the time of implantation.Lysophosphatidic acid (LPA) receptor 1 (R1) and LPAR3 mediate signaling of lysophosphatidic acid produced by autotaxin and induce prostaglandin biosynthesis and cytoskeleton changes in ovine trophectoderm cells at implantation time.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) is a simple phospholipid but displays an intriguing cell biology that is mediated via interactions with G protein-coupled seven transmembrane receptors (GPCRs). So far, five GPCRs, designated LPA1-5, and, more recently, two additional GPCRs, GPR87 and P2Y5, have been identified as receptors for LPA. These LPA receptors can be classified into two families, the EDG and P2Y families, depending on their primary structures. Recent studies on gene targeting mice and family diseases of these receptors revealed that LPA is involved in both pathological and physiological states including brain development (LPA1), neuropathy pain (LPA1), lung fibrosis (LPA1), renal fibrosis (LPA1) protection against radiation-induced intestinal injury (LPA2), implantation (LPA3) and hair growth (P2Y5). LPA is produced both in cells and biological fluids, where multiple synthetic reactions occur. There are at least two pathways for LPA production. In serum or plasma, LPA is predominantly produced by a plasma enzyme called autotaxin (ATX). ATX is a multifunctional ectoenzyme and is involved in many patho-physiological conditions such as cancer, neuropathy pain, lymphocyte tracking in lymph nodes, obesity, diabetes and embryonic blood vessel formation. LPA is also produced from phosphatidic acid (PA) by its deacylation catalyzed by phospholipase A (PLA)-type enzymes. However, the physiological roles of this pathway as well as the enzymes involved remained to be solved. A number of phospholipase A1 and A2 isozymes could be involved in this pathway. One PA-selective PLA1 called mPA-PLA1alpha/LIPH is specifically expressed in hair follicles, where it has a critical role in hair growth by producing LPA through a novel LPA receptor called P2Y5.

Lpar3 encodes LPA3, the third G protein-coupled receptor for lysophosphatidic acid (LPA). Lpar3(-/-) female mice had delayed embryo implantation. Their serum progesterone and estrogen levels were comparable with control on Gestation Day 3.5 (D3.5) at 1100 h. There was reduced cell proliferation in D3.5 and D4.5 Lpar3(-/-) stroma. Progesterone receptor (PGR) disappeared from D4.5 Lpar3(+/+) uterine luminal epithelium (LE) but remained highly expressed in D4.5 Lpar3(-/-) LE. Pgr and PGR- target genes but not estrogen receptor alpha (ERalpha [Esr1]) or ESR target genes, were upregulated in D4.5 Lpar3(-/-) LE. It was hypothesized that suppression of PGR activity in LE could restore on-time uterine receptivity in Lpar3(-/-) mice. A low dose of RU486 (5 μg/mouse) given on D3.5 at 900 h rescued delayed implantation in all pregnant Lpar3(-/-) females and significantly increased number of implantation sites compared to vehicle-treated pregnant Lpar3(-/-) females detected on D4.5. E2 (25 ng/mouse) had a similar effect as 5 μg RU486 on embryo implantation in Lpar3(-/-) females. However, when the ovaries were removed on late D2.5 to create an experimentally induced delayed implantation model, 25 ng E2 activated implantation in Lpar3(+/+) but not Lpar3(-/-) females detected on D4.5. These results demonstrate that deletion of Lpar3 leads to an increased ratio of progesterone signaling/estrogen signaling that can be optimized by low doses of RU486 or E2 to restore on-time implantation in Lpar3(-/-) females.

We sought to evaluate lysophosphatidic acid (LPA) signaling improvement in lung development by assessing the expression of autotaxin and LPA receptor 1 and 3 (LPAR1 and LPAR3) in the neonatal rat lung during normal perinatal development and in response to hyperoxia. In the developmental study, rats were sacrificed on days 17, 19, and 21 of gestation; on postnatal days 1, 4, and 7; and at adulthood (postnatal 9 weeks). In the hyperoxia study, 42 postnatal 4-day-old rat pups were divided into seven groups and exposed to either 85% O2 for 24, 72, or 120 h or room air for 0, 24, 72, or 120 h. The rats were then euthanized after 0, 24, 72, and 120 h of exposure. Immunofluorescence demonstrated that autotaxin, LPAR1, and LPAR3 proteins were broadly colocalized in airway epithelial cells, but mainly distributed in vascular endothelial and mesenchymal cells during the first postnatal week. The expression of autotaxin, LPAR1, and LPAR3 were increased during late gestation and then decreased after birth. Autotaxin expression and enzymatic activity were significantly increased at 72 and 120 h after exposure to hyperoxia. LPAR1 and LPAR3 expression was also increased after 120 h of hyperoxic exposure. These findings suggest that LPA-associated molecules were upregulated at birth and induced by hyperoxia in the developing rat lung. Therefore, the LPA pathway may be involved in normal lung development, including vascular development, as well as wound-healing processes of injured neonatal lung tissue, which is at risk of neonatal hyperoxic acute lung injury.

Lysophosphatidic acid (LPA) is a simple bioactive phospholipid with diverse effects on various cells, that interacts with three G protein-coupled transmembrane receptors, LPA1, LPA2, and LPA3. The expression pattern and functions of these LPA receptors in various tumors have not been fully examined, except in ovarian cancer. To evaluate the LPA receptor expression profile in human colorectal cancer and in normal mucosa, we used real-time reverse transcription-polymerase chain reaction (RT-PCR) and measured the expression levels of LPA1, LPA2, and LPA3 messenger RNA (mRNA) in 26 colorectal cancers and 16 corresponding normal tissue samples. Normal epithelium expressed both LPA1 and LPA2 mRNA at similar levels. In comparison, colorectal cancers expressed LPA1 mRNA at a significantly lower level (0.3-fold; P

Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa(1)/lp(A1)/Edg-2/Gpcr26, lpa(2)/lp(A2)/Edg-4, and lpa(3)/lp(A3)/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa(1) in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa(2) in mice. Homozygous knockout (lpa(2)((-/-))) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa(1)((-/-)) lpa(2)((-/-)) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa(1)((-/-)) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa(1)((-/-)) lpa(2)((-/-)) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca(2+) mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa(1)((-/-)) lpa(2)((-/-)) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa(1)((-/-)) fibroblasts], these responses were only partially affected in lpa(1)((-/-)) and lpa(2)((-/-)) fibroblasts. Thus, although LPA(2) is not essential for normal mouse development, it does act redundantly with LPA(1) to mediate most LPA responses in fibroblasts.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including G(i), G(q) and G(12/13) to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.

Introduction: Changes in lipid biosynthesis has attracted significant attention in the study of cancer. Its value in our understanding of pancreatic cancer has not yet been realized. This study examined lysophosphatidic acid (LPA) variant biosynthesis in patients with stage II-III pancreatic ductal adenocarcinoma (PDAC), evaluating serum, bile, and tissue levels of LPA, its receptors, and downstream metabolites. LPA, a product of phospholipase D activity of the enzyme autotaxin (ATX) is a potent mitogenic agent. Data suggests that changes in lipid biosynthesis contribute to the tumor microenvironment in PDAC. Methods: PDAC, matched adjacent non-malignant tissues, gall bladder bile and serum samples were collected from patients with Stage II-III pancreatic cancer at the time of surgery. Control bile and serum were collected from patients undergoing cholecystectomy for benign disease and living renal donors respectively. Results: Serum and biliary LPA levels were increased in patients with PDAC when compared to controls. ATX activity, and LPA receptors, LPAR1, LPAR2, and LPAR5 were greater in PDAC adjacent pancreatic tissue when compared to PDAC tissue. In contrast, TNFα receptors TNFα1 and TNFα2 and LPA receptor LPAR3 receptor expression was greater in PDAC lesions when compared to adjacent tissue. Conclusion: PDAC patients display a modified LPA biosynthesis profile, when compared to non-PDAC patients, potentially providing a paracrine-signaling cascade to promote pancreatic tumorigenesis through increased expression of mitogenic cytokines and growth factors. Citation Format: Nicholas J. Skill, Mary A. Maluccio. Lysophosphatidic acid receptor signaling and pancreatic adenocarcinoma tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4941. doi:10.1158/1538-7445.AM2017-4941

In vascular smooth muscle cells (VSMC) increased integrin adhesion to extracellular matrix (ECM) proteins, as well as the production of reactive oxygen species (ROS) are strongly stimulated by lysophosphatidic acid (LPA). We hypothesized that LPA-induced generation of ROS increases integrin adhesion to the ECM. Using atomic force microscopy (AFM) we determined the effects of LPA on integrin adhesion to fibronectin (FN) in VSMC isolated from rat (Sprague–Dawley) skeletal muscle arterioles. In VSMC, exposure to LPA (2 μM) doubled integrin-FN adhesion compared to control cells (P < 0.05). LPA-induced integrin-FN adhesion was reduced by pre-incubation with antibodies against β1 and β3 integrins (50 μg/ml) by 66% (P < 0.05). Inhibition of LPA signaling via blockade of the LPA G-protein coupled receptors LPAR1 and LPAR3 with 10 μM Ki16425 reduced the LPA-enhanced adhesion of VSCM to FN by 40% (P < 0.05). Suppression of ROS with tempol (250 μM) or apocynin (300 μM) also reduced the LPA-induced FN adhesion by 47% (P < 0.05) and 59% (P < 0.05), respectively. Using confocal microscopy, we observed that blockade of LPA signaling, with Ki16425, reduced ROS by 45% (P < 0.05), to levels similar to control VSMC unexposed to LPA. In intact isolated arterioles, LPA (2 μM) exposure augmented the myogenic constriction response to step increases in intraluminal pressure (between 40 and 100 mm Hg) by 71% (P < 0.05). The blockade of LPA signaling, with Ki16425, decreased the LPA-enhanced myogenic constriction by 58% (P < 0.05). Similarly, blockade of LPA-induced ROS release with tempol or gp91 ds-tat decreased the LPA-enhanced myogenic constriction by 56% (P < 0.05) and 55% (P < 0.05), respectively. These results indicate that, in VSMC, LPA-induced integrin activation involves the G-protein coupled receptors LPAR1 and LPAR3, and the production of ROS, and that LPA may play an important role in the control of myogenic behavior in resistance vessels through ROS modulation of integrin activity.

Lysophosphatidic acid (LPA) is a lipid-derived signaling molecule that plays key roles in diverse biological processes including inflammation and uterine remodeling. Although the function of LPA and its receptors has been extensively studied using knock-out mice, the temporal-spatial expression of LPA receptors is less well-characterized. To gain further insight into the dynamic regulation of LPA receptor 3 (Lpar3) expression in vivo by bioluminescence imaging, we generated and characterized mice transgenic for a putative Lpar3 promoter fragment. A non-coding region of the Lpar3 gene immediately upstream of the start site was subcloned adjacent to the luciferase gene. Promoter activity was determined by in vitro luciferase assays, in vivo bioluminescent imaging or by semi-quantitative real-time PCR. The air-pouch model was used to investigate Lpar3 promoter activity in the context of inflammation. The putative Lpar3 promoter fragment behaved similarly to the endogenous promoter in vitro and in vivo. In male mice, elevated levels of Lpar3-induced luciferase activity were observed in the testis. In female mice, the basal level of luciferase activity in the uterus significantly increased during pseudopregnancy. Moreover, luciferase activity was upregulated by TNF-α in the air-pouch model. We report the identification of a functional Lpar3 promoter fragment and the generation of a transgenic mouse model to investigate the regulation of Lpar3 promoter activity non-invasively in vivo by bioluminescence imaging. This mouse model is a valuable tool for reproductive biology and inflammation research as well as other biological processes in which this receptor is involved.

Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, lp A1 / vzg-1 / edg-2 , was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of lp A1 results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among lp A1 (−/−) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and/or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an lp receptor gene and indicate that LPA signaling through the LP A1 receptor is required for normal development of an inborn, neonatal behavior.

Two G protein-coupled receptors (Edg-2) and (Edg-4) for the lysolipid phosphoric acid mediator lysophosphatidic acid have been described by molecular cloning. However, the calcium-mobilizing receptor Edg-4 is not expressed in some cell lines that exhibit robust calcium responses to this ligand, thus predicting the existence of additional receptor subtypes. We report here on the characterization of a third human lysophosphatidic acid receptor subtype, Edg-7, which mediates lysophosphatidic acid-evoked calcium mobilization. In a rat hepatoma Rh7777 cell line that lacks endogenous responses to lysophosphatidic acid, this lipid mediator, but not others, evokes calcium transients when the cells have been transfected with Edg-7 or Edg-4 DNAs. Furthermore, frog oocytes exhibit a calcium-mediated chloride conductance in response to mammalian-selective lysophosphatidic acid mimetics after injection of Edg-7 mRNA. Edg-7-expressing Rh7777 cells do not show inhibition of forskolin-driven rises in cAMP in response to lysophosphatidic acid. However, membranes from HEK293T cells cotransfected with Edg-7 and Gi2α protein DNAs show lysophosphatidic acid dose-dependent increases in [γ-35S]GTP binding with an EC50 value of 195 nM. When we used this assay to compare various synthetic LPA analogs at Edg-2, Edg-4, and Edg-7 receptors, we found that ethanolamine-based compounds, which are full LPA mimetics at Edg-2 and Edg-4, exhibit little activity at the Edg-7 receptor. Edg-7 RNA was detected in extracts of several rat and human tissues including prostate. Together, our data indicate that Edg-7 is a third lysophosphatidic acid receptor that couples predominantly to Gq/11α proteins.

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl-L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 ( vzg-1 ) (also referred to as lp A1 / edg-2 ) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [ 3 H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1 , can activate multiple LPA-dependent responses in cells from distinct tissue lineages.