Loss of lysophosphatidic acid receptor-3 suppresses cell migration activity of human sarcoma cells

Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors (LPA₁-LPA₆). Recently, we have reported that LPA₃ indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA₃ 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 LPA₃ acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA₃ may be involved in the progression of sarcoma cells.     

A novel function of hepatocyte growth factor in the activation of checkpoint kinase 1 phosphorylation in colon cancer cells

Lysophosphatidic acid (LPA) is a simple biophysical lipid which interacts with at least six subtypes of G protein-coupled LPA receptors (LPA₁–LPA₆). In cancer cells, LPA signaling via LPA receptors is involved in the regulation of malignant properties, such as cell growth, motility, and invasion. The aim of this study was to assess whether LPA receptors regulate cellular functions of fibrosarcoma cells treated with anticancer drug. HT1080 cells were maintained by the stepwise treatment of cisplatin (CDDP) at a range of 0.01 to 1.0 µM for approximately 6 months. The cell motile and invasive activities of long-term CDDP-treated (HT-CDDP) cells were significantly stimulated by LPA treatment, while HT-CDDP cells in the static state showed the low cell motile and invasive activities in comparison with HT1080 cells. Since the expression level of LPAR2 gene was markedly elevated in HT-CDDP cells, LPA₂ knockdown cells were generated from HT-CDDP cells. The cell motile and invasive activities of HT-CDDP cells were reduced by LPA₂ knockdown. In colony assay, large-sized colonies formed by long-term CDDP treatment were suppressed by LPA₂ knockdown. In addition, LPA₂ knockdown cells reduced LPA production by autotaxin (ATX), correlating with ATX expression level. These results suggest that LPA signaling via LPA₂ may play an important role in the regulation of cellular functions in HT1080 cells treated with CDDP.     

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA₁–LPA₆) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA₁ inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA₅ in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA₁ and LPA₅ on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA₅ may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA₁.     

Inhibitory effects of LPA1 on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells

Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA₃) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA₁ in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA₁ on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA₁ inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.     

Promotion of cell-invasive activity through the induction of LPA receptor-1 in pancreatic cancer cells

Abstract

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.     

Ethionine regulates cell motile activity through LPA receptor-3 in liver epithelial 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.     

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

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

     

Effects of bisphenol A and 4-nonylphenol on cellular responses through the different induction of LPA receptors in liver epithelial WB-F344 cells

Abstract

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.     

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility

Lysophosphatidic acid (LPA) is a ligand of multiple G protein–coupled receptors. The LPA_1–3 receptors are members of the endothelial cell differentiation gene (Edg) family. LPA₄/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA₅/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa₄ in mice. Although LPA₄-deficient mice displayed no apparent abnormalities, LPA₄-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA₄, LPA₄ deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA₄ converted LPA₄-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA₄ strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA₁ in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA₄ attenuated LPA₁-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA₄ is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.     

Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade

Introduction

Lysophosphatidic acid (LPA) is a bioactive lipid that binds to G protein–coupled receptors (LPA_1–6). Recently, we reported that abrogation of LPA receptor 1 (LPA₁) ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis. In this study, we examined the importance of the LPA–LPA₁ axis in cell proliferation, cytokine/chemokine production and lymphocyte transmigration in fibroblast-like synoviocytes (FLSs) obtained from the synovial tissues of rheumatoid arthritis (RA) patients.

Methods

FLSs were prepared from synovial tissues of RA patients. Expression of LPA_1–6 was examined by quantitative real-time RT-PCR. Cell surface LPA₁ expression was analyzed by flow cytometry. Cell proliferation was analyzed using a cell-counting kit. Production of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), chemokine (C-C motif) ligand 2 (CCL2), metalloproteinase 3 (MMP-3) and chemokine (C-X-C motif) ligand 12 (CXCL12) was measured by enzyme-linked immunosorbent assay. Pseudoemperipolesis was evaluated using a coculture of RA FLSs and T or B cells. Cell motility was examined by scrape motility assay. Expression of adhesion molecules was determined by flow cytometry.

Results

The expression of LPA₁ mRNA and cell surface LPA₁ was higher in RA FLSs than in FLSs from osteoarthritis tissue. Stimulation with LPA enhanced the proliferation of RA FLSs and the production of IL-6, VEGF, CCL2 and MMP-3 by FLSs, which were suppressed by an LPA₁ inhibitor (LA-01). Ki16425, another LPA₁ antagonist, also suppressed IL-6 production by LPA-stimulated RA FLSs. However, the production of CXCL12 was not altered by stimulation with LPA. LPA induced the pseudoemperipolesis of T and B cells cocultured with RA FLSs, which was suppressed by LPA₁ inhibition. In addition, LPA enhanced the migration of RA FLSs and expression of vascular cell adhesion molecule and intercellular adhesion molecule on RA FLSs, which were also inhibited by an LPA₁ antagonist.

Conclusions

Collectively, these results indicate that LPA–LPA₁ signaling contributes to the activation of RA FLSs.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

Different effects on cell proliferation and migration abilities of endothelial cells by LPA1 and LPA3 in mammary tumor FM3A cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA₁ and LPA₃ may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.     

The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell’s Response to LPA

Lysophosphatidic acid (LPA) mediates diverse cellular responses through the activation of at least six LPA receptors – LPA_1–6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA₁ contains a PDZ binding motif (SVV) identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA₁ but not that of other LPA receptors. LPA₁ colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA₁ to EEA1 early endosomes and promoted LPA₁ mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA₁ and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.     

Effects of lysophosphatidic acid (LPA) receptor-2 (LPA2) and LPA3 on the regulation of chemoresistance to anticancer drug in lung cancer 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.     

c-Src-mediated phosphorylation of TRIP6 regulates its function in lysophosphatidic acid-induced cell migration

TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA₂ receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130^cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.     

LPA-induced migration of ovarian cancer cells requires activation of ERM proteins via LPA1 and LPA2

Lysophosphatidic acid (LPA) has been implicated in the pathology of human ovarian cancer. This phospholipid elicits a wide range of cancer cell responses, such as proliferation, trans-differentiation, migration, and invasion, via various G-protein-coupled LPA receptors (LPARs). Here, we explored the cellular signaling pathway via which LPA induces migration of ovarian cancer cells. LPA induced robust phosphorylation of ezrin/radixin/moesin (ERM) proteins, which are membrane-cytoskeleton linkers, in the ovarian cancer cell line OVCAR-3. Among the LPAR subtypes expressed in these cells, LPA₁ and LPA₂, but not LPA₃, induced phosphorylation of ERM proteins at their C-termini. This phosphorylation was dependent on the Gα_12/13/RhoA pathway, but not on the Gα_q/Ca²⁺/PKC or Gα_s/adenylate cyclase/PKA pathway. The activated ERM proteins mediated cytoskeletal reorganization and formation of membrane protrusions in OVCAR-3 cells. Importantly, LPA-induced migration of OVCAR-3 cells was completely abolished not only by gene silencing of LPA₁ or LPA₂, but also by overexpression of a dominant negative ezrin mutant (ezrin-T567A). Taken together, this study demonstrates that the LPA₁/LPA₂/ERM pathway mediates LPA-induced migration of ovarian cancer cells. These findings may provide a potential therapeutic target to prevent metastatic progression of ovarian cancer.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Identification of Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Biomarker for Lysophosphatidic Acid Receptor Type 1 (LPA1) Activation in Human Breast and Prostate Cancers

Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA_1–6). LPA receptor type 1 (LPA₁) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA_1,2,3,6; MDA-MB-231: LPA_1,2; MCF-7: LPA_2,6). Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF) was inhibited by LPA_1–3 antagonists (Ki16425, Debio0719). Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁) and downregulated for LPA₁ (MDA-B02/shLPA₁), respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in quantifying LPA₁ activation state in patients receiving anti-LPA₁ therapies.     

Role of lysophosphatidic acid receptor LPA2 in the development of allergic airway inflammation in a murine model of asthma

Background

Lysophosphatidic acid (LPA) plays a critical role in airway inflammation through G protein-coupled LPA receptors (LPA_1-3). We have demonstrated that LPA induced cytokine and lipid mediator release in human bronchial epithelial cells. Here we provide evidence for the role of LPA and LPA receptors in Th2-dominant airway inflammation.

Methods

Wild type, LPA₁ heterozygous knockout mice (LPA₁^+/-), and LPA₂ heterozygous knockout mice (LPA₂^+/-) were sensitized with inactivated Schistosoma mansoni eggs and local antigenic challenge with Schistosoma mansoni soluble egg Ag (SEA) in the lungs. Bronchoalveolar larvage (BAL) fluids and lung tissues were collected for analysis of inflammatory responses. Further, tracheal epithelial cells were isolated and challenged with LPA.

Results

BAL fluids from Schistosoma mansoni egg-sensitized and challenged wild type mice (4 days of challenge) showed increase of LPA level (~2.8 fold), compared to control mice. LPA₂^+/- mice, but not LPA₁^+/- mice, exposed to Schistosoma mansoni egg revealed significantly reduced cell numbers and eosinophils in BAL fluids, compared to challenged wild type mice. Both LPA₂^+/- and LPA₁^+/- mice showed decreases in bronchial goblet cells. LPA₂^+/- mice, but not LPA₁^+/- mice showed the decreases in prostaglandin E2 (PGE2) and LPA levels in BAL fluids after SEA challenge. The PGE2 production by LPA was reduced in isolated tracheal epithelial cells from LPA₂^+/- mice. These results suggest that LPA and LPA receptors are involved in Schistosoma mansoni egg-mediated inflammation and further studies are proposed to understand the role of LPA and LPA receptors in the inflammatory process.