ABSTRACT
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.
Subject(s)
Cell Movement/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , Ethionine/pharmacology , Liver/cytology , Receptors, Lysophosphatidic Acid/metabolism , Animals , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , Receptors, Lysophosphatidic Acid/geneticsABSTRACT
Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 µM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.
Subject(s)
Cell Movement/drug effects , Epithelial Cells/drug effects , Hydrogen Peroxide/pharmacology , Lysophospholipids/pharmacology , Receptors, Lysophosphatidic Acid/genetics , Animals , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Liver/cytology , Liver/drug effects , Liver/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , Rats , Real-Time Polymerase Chain Reaction , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/metabolism , Signal Transduction/drug effectsABSTRACT
Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA(2) using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA(2) increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA(2) may be enhanced by oncogenic K-ras.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Cell Movement/genetics , Genes, ras/physiology , Pancreatic Neoplasms/genetics , Receptors, Lysophosphatidic Acid/physiology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cricetinae , Diphosphates/pharmacology , Gene Knockdown Techniques , Genes, ras/genetics , Glycerol/analogs & derivatives , Glycerol/pharmacology , Lysophospholipids/pharmacology , Neoplasm Invasiveness , Pancreatic Neoplasms/pathology , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Up-RegulationABSTRACT
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.
Subject(s)
Cell Movement/genetics , Neoplasms , Receptors, Lysophosphatidic Acid , Sarcoma , Cell Line, Tumor , Humans , Lysophospholipids/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Sarcoma/genetics , Sarcoma/metabolism , Sarcoma/pathology , Signal TransductionABSTRACT
Lysophosphatidic acid (LPA) interacts with G protein-coupled transmembrane LPA receptors (LPA receptors; LPA(1)-LPA(6)). Recently, we demonstrated that each LPA receptor acts as a positive or negative regulator of cell migration ability. It is known that estrogens indicate a variety of biological functions, including cell motility. In the present study, to assess whether LPA signaling is involved in cell motile activity stimulated by estrogens, we measured cell motile activity and LPA receptor expressions of rat liver epithelial WB-F344 cells treated with 17ß-estradiol (E(2)), ethinyl estradiol (EE) and diethylstilbestrol (DES) at concentrations of 0.1 and 1.0 µM for 48 h. The cell motility of E(2) and EE treated cells was significantly higher than that of untreated cells. By contrast, DES markedly inhibited cell motile activity. Using quantitative real time RT-PCR analysis, Lpar1 and Lpar3 expressions in E(2) treated cells were significantly higher than those in untreated cells. In EE treated cells, Lpar3 expression was markedly elevated, whereas Lpar1 expression was decreased. On the other hand, Lpar1 expression was significantly increased in DES treated cells. Interestingly, the effects of E(2), EE and DES on cell motility were suppressed by Lpar1 or Lpar3 knockdown. These results suggest that the different induction of LPA receptors by estrogens may regulate cell motile activity of WB-F344 cells.
