Unique uterine localization and regulation may differentiate LPA3 from other lysophospholipid receptors for its role in embryo implantation.
ABSTRACT: To determine factors differentiating LPA3 from other lysophospholipid (LP) receptors for its role in embryo implantation.Experimental mouse models.Institute/university research laboratories.Wild-type, Lpar3(-/-), Lpar1(-/-)Lpar2(-/-), and S1pr2(-/-)S1pr3(-/-) mice.Ovariectomy.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).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.LE localization and up-regulation by P differentiate LPA3 from the other nine LP receptors and may underlie its essential role in embryo implantation.
Project description:Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3(-/-) uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3(-/-) uterus. An observed delay of COL III and COL VI clearance in the Lpar3(-/-) uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3(-/-) uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3(-/-) uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3(-/-) uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.
Project description: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.
Project description:Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1-6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1(-/-) mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1(-/-) mice but not in Lpar2(-/-) and Lpar3(-/-) animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1(-/-) osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP(+) osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.
Project description:A bioactive lipid, sphingosine 1-phosphate (S1P), acts extracellularly as a potent mediator, and is implicated in the progression of various cancers including hepatocellular carcinoma (HCC). S1P exerts its functions by binding to five types of specific receptors, S1P receptor 1 (S1PR1), S1PR2, S1PR3, S1PR4 and S1PR5 on the plasma membrane. However, the exact roles of S1P and each S1PR in HCC cells remain to be clarified. In the present study, we investigated the effect of S1P on the hepatocyte growth factor (HGF)-induced migration of human HCC-derived HuH7 cells, and the involvement of each S1PR. S1P dose-dependently reduced the HGF-induced migration of HuH7 cells. We found that all S1PRs exist in the HuH7 cells. Among each selective agonist for five S1PRs, CYM5520, a selective S1PR2 agonist, significantly suppressed the HGF-induced HuH7 cell migration whereas selective agonists for S1PR1, S1PR3, S1PR4 or S1PR5 failed to affect the migration. The reduction of the HGF-induced migration by S1P was markedly reversed by treatment of JTE013, a selective antagonist for S1PR2, and S1PR2- siRNA. These results strongly suggest that S1P reduces the HGF-induced HCC cell migration via S1PR2. Our findings may provide a novel potential of S1PR2 to therapeutic strategy for metastasis of HCC.
Project description:Sphingosine-1-phosphate (S1P) has been reported to enhance the function of islet β-cells, providing a potential therapeutic target for diabetes mellitus. In the present study, the effects of S1P on the proliferation and apoptosis of β-cells in type 2 diabetic mice were investigated. The mice were administered intraperitoneal S1P solution daily at a dose of 20 µg/kg for three weeks. The intraperitoneal glucose tolerance test (IPGTT) and homeostatic model assessment of insulin resistance (HOMA-IR) index determination were carried out. Immunohistochemical staining was used to detect the protein expression of insulin, antigen Ki-67 and S1P receptor isoforms (S1PR1/S1PR2/S1PR3) in pancreatic islets. Compared with the diabetic control (DC) group, the IPGTT results and HOMA-IR index in the S1P treatment group were decreased. The islets in the S1P group exhibited higher insulin immunostaining intensity than the DC group, as well as higher proliferation (P<0.05) and lower apoptosis rates (P<0.05). Positive staining for the S1P receptors S1PR1, S1PR2 and S1PR3 was observed in the cytoplasm and membrane of the islet cells. S1PR1 and S1PR2 proteins showed increased expression in the S1P and DC groups compared with the normal control group (P<0.01 and P<0.05, respectively), whereas no significant difference was observed in the expression of S1PR3 among these groups. In conclusion, extracellular S1P can induce islet β-cell proliferation and decrease cell apoptosis in diabetic mice. S1P function may be mediated via S1PR1 and S1PR2; therefore, targeting S1P/S1PR signalling pathways may be a novel therapeutic strategy for diabetes mellitus.
Project description:Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.
Project description:Increased levels of circulating sphingosine-1-phosphate (S1P) have been reported in ulcerative colitis. The objective of this study was to examine the effect of S1P on colonic smooth muscle contractility and how is it affected by colitis.Colonic inflammation was induced by intrarectal administration of trinitrobenzene sulfonic acid. Five days later colon segments were isolated and used for contractility experiments and immunoblotting.S1P contracted control and inflamed colon segments and the contraction was significantly greater in inflamed colon segments. S1P-induced contraction was mediated by S1PR1 and S1PR2 in control and S1PR2 in inflamed colon segments. S1PR3 did not play a significant role in S1P-induced contractions in control or inflamed colon. S1PR1, S1PR2 and S1PR3 proteins were expressed in colon segments from both groups. The expression of S1PR1 and S1PR2 was significantly enhanced in control and inflamed colon segments, respectively. S1PR3 levels however were not significantly different between the two groups. Nifedipine significantly reduced S1P-induced contraction in control but not inflamed colon segments. Thapsigargin significantly reduced S1P-induced contraction of the inflamed colon. GF 109203X and Y-27632, alone abolished S1P-induced contraction of the control but not inflamed colon segments. Combination of GF 109203X, Y-27632 and thapsigargin abolished S1P-induced contraction of inflamed colon segments.S1P contracted control colon via S1PR1 and S1PR2 and inflamed colon exclusively via S1PR2. Calcium influx (control) or release (inflamed) and calcium sensitization are involved in S1P-induced contraction. Exacerbated response to S1P in colitic colon segments may explain altered colonic motility reported in patients and experimental models of inflammatory bowel disease.
Project description:Lysophosphatidic acid (LPA) is a bioactive lipid molecule produced by the plasma lysophospholipase D enzyme autotaxin that is present at ?100 nmol/L in plasma. Local administration of LPA promotes systemic arterial remodeling in rodents. To determine whether LPA contributes to remodeling of the pulmonary vasculature, we examined responses in mice with alterations in LPA signaling and metabolism.Enpp2(+/-) mice, which are heterozygous for the autotaxin-encoding gene and which have reduced expression of autotaxin/lysophospholipase D and approximately half normal plasma LPA, were hyperresponsive to hypoxia-induced vasoconstriction and remodeling, as evidenced by the development of higher right ventricular (RV) systolic pressure, greater decline in peak flow velocity across the pulmonary valve, and a higher percentage of muscularized arterioles. Mice lacking LPA(1) and LPA(2), 2 LPA receptors abundantly expressed in the vasculature, also had enhanced hypoxia-induced pulmonary remodeling. With age, Lpar1(-/-)2(-/-) mice spontaneously developed elevated RV systolic pressure and RV hypertrophy that was not observed in Lpar1(-/-) mice or Lpar2(-/-) mice. Expression of endothelin-1, a potent vasoconstrictor, was elevated in lungs of Lpar1(-/-)2(-/-) mice, and expression of endothelin(B) receptor, which promotes vasodilation and clears endothelin, was reduced in Enpp2(+/-) and Lpar1(-/-)2(-/-) mice.Our findings indicate that LPA may negatively regulate pulmonary vascular pressure through LPA(1) and LPA(2) receptors and that in the absence of LPA signaling, upregulation in the endothelin system favors remodeling.
Project description:Mesenchymal stem cells (MSCs) are a multipotent cell population acquired most prominently from bone marrow with the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, and others. MSCs demonstrate the capacity to home to sites of injury and contribute to tissue repair. Sphingosine 1-phosphate (S1P) is a biologically active sphingolipid impacting proliferation, apoptosis, inflammation, and angiogenesis with changes in S1P concentration providing significant implications for various disease conditions including cancer, diabetes, and cardiac disease. These functions are primarily mediated by interactions with 5 G-protein coupled S1P receptors (S1PR1-5). In this paper, we demonstrate that inhibition of S1PR2 results in increased MSC clonogenicity, migration, and proliferation; features dependent on Erk phosphorylation. Furthermore, decreased S1PR2 expression decreases the differentiation of MSCs into adipocytes and mature osteoblasts that may be the result of increased expression of MSC pluripotency factors including Nanog, Sox-9, and Oct-4. Inhibition of S1PR1 and S1PR3 in contrast does not impact MSC migration or Erk activation although increased proliferation is observed. In the study, we describe the essential role of S1PR2 in MSC differentiation pathways through modification of pluripotency factors. We propose a MAPK dependent mechanism through S1PR2 inhibition that promotes equally multipotent MSC proliferation.
Project description: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.