Autotaxin-lysophosphatidic acid-LPA3 signaling at the embryo-epithelial boundary controls decidualization pathways.
ABSTRACT: 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 LPA3 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 LPA3 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-LPA3 signaling at the embryo-epithelial boundary induces decidualization via the canonical HB-EGF and COX-2 pathways.
Project description:We previously reported that intrathecal injection of lysophosphatidylcholine (LPC) induced neuropathic pain through activation of the lysophosphatidic acid (LPA)-1 receptor, possibly via conversion to LPA by autotaxin (ATX).We examined in vivo LPA-induced LPA production using a biological titration assay with B103 cells expressing LPA1 receptors. Intrathecal administration of LPC caused time-related production of LPA in the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve or sciatic nerve. LPC-induced LPA production was markedly diminished in ATX heterozygotes, and was abolished in mice that were deficient in LPA3, but not LPA1 or LPA2 receptors. Similar time-related and LPA3 receptor-mediated production of LPA was observed following intrathecal administration of LPA. In an in vitro study using spinal cord slices, LPA-induced LPA production was also mediated by ATX and the LPA3 receptor. Intrathecal administration of LPA, in contrast, induced neuropathic pain, which was abolished in mice deficient in LPA1 or LPA3 receptors.These findings suggest that feed-forward LPA production is involved in LPA-induced neuropathic pain.
Project description:Background: Lysophosphatidic acid (LPA) is a small glycerophospholipid that acts as a potent extracellular signal in various biological processes and diseases. Our previous work demonstrated that the expression of the LPA receptors LPA1 and LPA3 is elevated in the early postnatal heart. However, the role of this stage-specific expression of LPA1 and LPA3 in the heart is unknown. Methods and Results: By using LPA3 and LPA1 knockout mice, and neonatal SD rats treated with Ki16425 (LPA1/LPA3 inhibitor), we found that the number of proliferating cardiomyocytes, detected by coimmunostaining pH3, Ki67 or BrdU with cardiac troponin T, was significantly decreased in the LPA3 knockout mice and the Ki16425-treated rats but not in the LPA1 knockout mice during the first week of postnatal life. Using a myocardial infarction (MI) model, we found that cardiac function and the number of proliferating cardiomyocytes were decreased in the neonatal LPA3 KO mice and increased in the AAV9-mediated cardiac-specific LPA3 overexpression mice. By using lineage tracing and AAV9-LPA3, we further found that LPA3 overexpression in adult mice enhances cardiac function and heart regeneration as assessed by pH3-, Ki67-, and Aurora B-positive cardiomyocytes and clonal cardiomyocytes after MI. Genome-wide transcriptional profiling and additional mechanistic studies showed that LPA induces cardiomyocyte proliferation through the PI3K/AKT, BMP-Smad1/5, Hippo/YAP and MAPK/ERK pathways in vitro, whereas only ERK was confirmed to be activated by LPA-LPA3 signaling in vivo. Conclusion: Our study reports that LPA3-mediated LPA signaling is a crucial factor for cardiomyocyte proliferation in the early postnatal heart. Cardiac-specific LPA3 overexpression improved cardiac function and promoted cardiac regeneration after myocardial injury induced by MI. This finding suggested that activation of LPA3 potentially through AAV-mediated gene therapy might be a therapeutic strategy to improve the outcome after MI.
Project description:Despite wide margins and high dose irradiation, unresectable malignant glioma (MG) is less responsive to radiation and is uniformly fatal. We previously found that cytosolic phospholipase A2 (cPLA(2)) is a molecular target for radiosensitizing cancer through the vascular endothelium. Autotaxin (ATX) and lysophosphatidic acid (LPA) receptors are downstream from cPLA(2) and highly expressed in MG. Using the ATX and LPA receptor inhibitor, ?-bromomethylene phosphonate LPA (BrP-LPA), we studied ATX and LPA receptors as potential molecular targets for the radiosensitization of tumor vasculature in MG. Treatment of Human Umbilical Endothelial cells (HUVEC) and mouse brain microvascular cells bEND.3 with 5 µmol/L BrP-LPA and 3 Gy irradiation showed decreased clonogenic survival, tubule formation, and migration. Exogenous addition of LPA showed radioprotection that was abrogated in the presence of BrP-LPA. In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells. Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation. However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect. Using heterotopic tumor models of GL-261, mice treated with BrP-LPA and irradiation showed a tumor growth delay of 6.8 days compared to mice treated with irradiation alone indicating that inhibition of ATX and LPA receptors may significantly improve malignant glioma response to radiation therapy. These findings identify ATX and LPA receptors as molecular targets for the development of radiosensitizers for MG.
Project description:This research aimed to evaluate the expression and clinical implication of autotaxin (ATX)-lysophosphatidate (LPA) signaling-related proteins in breast cancer with adipose stroma. To this end, a tissue microarray (TMA) was constructed from 137 breast cancer tissues with adipose stroma and 329 breast cancer tissues with non-adipose stroma (inflammatory stroma: n = 81, 24.6%; fibrous stroma: n = 246, 75.4%). Immunohistochemical staining for ATX-LPA signaling-related proteins (ATX, LPA1, LPA2, and LPA3) was performed on the TMA. The results showed that LPA2 in tumor cells and LPA3 in stromal cells were highly expressed in breast cancer with adipose stroma and breast cancer with adipose and inflammatory stroma, respectively. Stromal LPA1 positivity (p = 0.017) and stromal LPA3 positivity (p = 0.004) were higher in breast cancer with adipose stroma containing CD68-positive crown-like structures (CLS). Stromal ATX positivity (p = 0.010) and stromal LPA3 positivity (p = 0.009) were higher in breast cancer with adipose tissue containing CD163-positive CLS. In breast cancer with adipose stroma, the number of CD163-positive macrophages was greater with stromal ATX positivity (p = 0.003), and the number of CD68-positive and CD163-positive macrophages were greater in cases with stromal LPA3 positivity. In conclusion, ATX-LPA signaling-related proteins are highly expressed in breast cancer with adipose stroma, with associated macrophage infiltration.
Project description:HB-EGF is essential for uterine decidualization, but its antioxidant function remains largely unclear. Here, we found that HB-EGF promoted the proliferation of stromal cells followed by the accelerated transition of the cell cycle from G1 to S phase and enhanced the expression or activity of Prl8a2, Prl3c1, and ALP which were well-established markers for uterine stromal cell differentiation during decidualization. Under oxidative stress, stromal cell differentiation was impaired, but this impairment was abrogated by rHB-EGF accompanied with the reduced levels of ROS and MDA which were regarded as the biomarkers for oxidative stress, indicating an antioxidant role of HB-EGF. Further analysis revealed that HB-EGF enhanced the activities of antioxidant enzymes SOD, CAT, and GPX, where addition of GPX inhibitor MS attenuated the induction of rHB-EGF on Prl8a2, Prl3c1, and ALP. Meanwhile, HB-EGF rescued the content of GSH and restored the ratio of GSH/GSSG after exposure to H2O2 but did not alter NOX activity. Along with a decline for mitochondrial superoxide, exogenous rHB-EGF improved the damage of oxidative stress on mtDNA copy number, ATP level, mitochondrial membrane potential, and activities of mitochondrial respiratory chain complex I and III whose blockage by ROT and AA led to a failure of rHB-EGF in protecting stromal cell differentiation against injury. Moreover, HB-EGF prevented stromal cell apoptosis by inhibiting Caspase-3 activity and Bax expression and recovering the level of Bcl-2 mRNA. Collectively, HB-EGF might ameliorate oxidative stress-mediated uterine decidualization damage.
Project description:BACKGROUND:We aimed to investigate the expression of proteins related with autotaxin (ATX)-lysophosphatidate (LPA) signaling and the clinical implications in primary and metastatic thyroid tumors. METHODS:We constructed tissue microarrays with 545 primary thyroid tumors [338 papillary thyroid carcinoma (PTC), 111 follicular carcinoma (FC), 69 medullary carcinoma (MC), 23 poorly differentiated carcinoma (PDC), and four anaplastic carcinoma (AC)]. Immunohistochemical stains for proteins related to ATX-LPA signaling (e.g., ATX, LPA1, LPA2, and LPA3) were performed. RESULTS:The expression of ATX was highest in MC, while the LPA1 expression was higher in PDC and AC, and the expression of LPA2 and LPA3 was highest in PTC (p < 0.001). Additionally, the expression of ATX, LPA1, and LPA2 was higher in conventional-type PTC than in follicular-variant PTC (p < 0.05). PTC with BRAF V600E mutation showed higher expression of ATX, LPA1, LPA2, and LPA3 than PTC without BRAF V600E mutation (p < 0.001). In univariate analysis, ATX positivity (p = 0.005) and LPA1 positivity (p = 0.014) were correlated with shorter overall survival in PTC. CONCLUSION:Proteins related to the ATX-LPA axis showed different levels of expression in primary thyroid tumors according to subtype.
Project description:Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2-4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.
Project description:Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective.Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor.Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G protein-coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish.
Project description:<h4>Objective</h4>To determine factors differentiating LPA3 from other lysophospholipid (LP) receptors for its role in embryo implantation.<h4>Design</h4>Experimental mouse models.<h4>Setting</h4>Institute/university research laboratories.<h4>Animal(s)</h4>Wild-type, Lpar3(-/-), Lpar1(-/-)Lpar2(-/-), and S1pr2(-/-)S1pr3(-/-) mice.<h4>Intervention(s)</h4>Ovariectomy.<h4>Main outcome measure(s)</h4>Blue dye injection for determining implantation sites on gestation day 4.5. Real-time polymerase chain reaction for measuring gene expression in whole uterus and separated uterine layers. In situ hybridization for detecting progesterone (P)-induced Lpar3 expression in the uterine luminal epithelium (LE).<h4>Result(s)</h4>Normal implantation was observed in Lpar1(-/-)Lpar2(-/-) and S1pr2(-/-)S1pr3(-/-) females. Temporal expression showed peak expression of Lpar3 in the preimplantation uterus and constitutive expression of the other nine LP receptors in the periimplantation uterus. Spatial localization revealed main expression of Lpar3 in the LE and broad expression of the remaining LP receptors in all three main uterine layers: LE, stromal, and myometrial layers. Hormonal regulation in ovariectomized uterus indicated up-regulation of Lpar3 but down-regulation or no effect of the remaining nine LP receptors by P, and down-regulation of most LP receptors, including Lpar3, by 17?-estradiol.<h4>Conclusion(s)</h4>LE localization and up-regulation by P differentiate LPA3 from the other nine LP receptors and may underlie its essential role in embryo implantation.