LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4

The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1–LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC‐TERT. We find that hMSC‐TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum‐containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down‐regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC‐TERT, LPA induces a rise in both cAMP and Ca²⁺. The rise in Ca²⁺ is completely abolished by Ki16425, whereas LPA‐mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4‐deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4‐deficient mice have increased trabecular bone volume, number, and thickness. J. Cell. Biochem. 109: 794–800, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

TNF-alpha promotes LPA1- and LPA3-mediated recruitment of leukocytes in vivo through CXCR2 ligand chemokines

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid present in low concentrations in serum and biological fluids but in high concentrations at sites of inflammation. LPA evokes a variety of cellular responses via binding to and activation of its specific G protein-coupled receptors (GPCR), namely LPA(1-6). Even though LPA is a chemoattractant for inflammatory cells in vitro, such a role for LPA in vivo remains largely unexplored. In the present study, we used the murine air pouch model to study LPA-mediated leukocyte recruitment in vivo using selective LPA receptor agonist/antagonist and LPA(3)-deficient mice. We report that 1) LPA injection into the air pouch induced leukocyte recruitment and that both LPA(1) and LPA(3) were involved in this process; 2) LPA stimulated the release of the pro-inflammatory chemokines keratinocyte-derived chemokine (KC) and interferon-inducible protein-10 (IP-10) in the air pouch; 3) tumor necrosis factor-α (TNF-α) injected into the air pouch prior to LPA strongly potentiated LPA-mediated secretion of cytokines/chemokines, including KC, IL-6, and IP-10, which preceded the enhanced leukocyte influx; and 4) blocking CXCR2 significantly reduced leukocyte infiltration. We suggest that LPA, via LPA(1) and LPA(3) receptors, may play a significant role in inducing and/or sustaining the massive infiltration of leukocytes during inflammation.     

Lysophosphatidic acid (LPA) and its receptors: Role in airway inflammation and remodeling

Lysophosphatidic acid (LPA), a simple bioactive phospholipid, is present in biological fluids such as plasma and bronchoalveolar lavage (BAL). It appears to have both pro- and anti-inflammatory roles in inflammatory lung diseases. Exogenous LPA promotes inflammatory responses by regulating the expression of chemokines, cytokines, and cytokine receptors in lung epithelial cells. In addition to the modulation of inflammatory responses, LPA regulates cytoskeleton rearrangement and confers protection against lung injury by enhancing lung epithelial cell barrier integrity and remodeling. The biological effects of LPA are mediated through its cell surface G-protein coupled LPA_1–7 receptors. The roles of LPA receptors in lung fibrosis, asthma, and acute lung injury have been investigated using genetically engineered LPA receptor deficient mice and there appears to be a definitive role for endogenous LPA and its receptors in the pathogenesis of pulmonary inflammatory diseases. This review summarizes recent reports on the role of LPA and its receptors in the regulation of lung epithelial inflammatory responses and remodeling. This article is part of a Special Issue entitled: Advances in Lysophospholipid Research.     

Current progress in non-Edg family LPA receptor research

Lysophosphatidic acid (LPA) is the simplest phospholipid yet possesses myriad biological functions. Until 2003, the functions of LPA were thought to be elicited exclusively by three subtypes of the endothelial differentiation gene (Edg) family of G protein-coupled receptors — LPA₁, LPA₂, and LPA₃. However, several biological functions of LPA could not be assigned to any of these receptors indicating the existence of one or more additional LPA receptor(s). More recently, the discovery of a second cluster of LPA receptors which includes LPA₄, LPA₅, and LPA₆ has paved the way for new avenues of LPA research. Analyses of these non-Edg family LPA receptors have begun to fill in gaps to understand biological functions of LPA such as platelet aggregation and vascular development that could not be ascribed to classical Edg family LPA receptors and are also unveiling new biological functions. Here we review recent progress in the non-Edg family LPA receptor research, with special emphasis on the pharmacology, signaling, and physiological roles of this family of receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

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.     

Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s)were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 >LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitorY-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.     

Gintonin, a ginseng-derived novel ingredient, evokes long-term potentiation through N-methyl-D-aspartic acid receptor activation: Involvement of LPA receptors

Ginseng has been shown to have memory-improving effects in human. However, little is known about the active components and the molecular mechanisms underlying its effects. Recently, we isolated novel lysophosphatidic acids (LPAs)-ginseng protein complex derived from ginseng, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity. Gintonin activated Ca²⁺-activated Clchannels in Xenopus oocytes through the activation of endogenous LPA receptor. In the present study, we investigated whether the activation of LPA receptor by gintonin is coupled to the regulation of N-methyl-d-aspartic acid (NMDA) receptor channel activity in Xenopus oocytes expressing rat NMDA receptors. The NMDA receptor-mediated ion current (I _NMDA ) was measured using the two-electrode voltage-clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, gintonin enhanced I _NMDA in a concentration-dependent manner. Gintonin-mediated I _NMDA enhancement was blocked by Ki16425, an LPA1/3 receptor antagonist. Gintonin action was blocked by a PLC inhibitor, IP₃ receptor antagonist, Ca²⁺ chelator, and a tyrosine kinase inhibitor. The site-directed mutation of Ser1308 of the NMDA receptor, which is phosphorylated by protein kinase C (PKC), to an Ala residue, or co-expression of receptor protein tyrosine phosphatase with the NMDA receptor attenuated gintonin action. Moreover, gintonin treatment elicited a transient elevation of [Ca²⁺]_i in cultured hippocampal neurons and elevated longterm potentiation (LTP) in both concentration-dependent manners in rat hippocampal slices. Gintonin-mediated LTP induction was abolished by Ki16425. These results indicate that gintonin-mediated I _NMDA potentiation and LTP induction in the hippocampus via the activation of LPA receptor might be responsible for ginseng-mediated improvement of memory-related brain functions.     

Modulation of NMDA Receptor Subunits Expression by Concanavalin A

The processes of N-methyl-d-aspartate (NMDA) receptor subunits expression were examined in cortical neurons and rat brain in order to investigate how the concanavalin A (Con A) modulates neuronal cells. Con A modulated the expression of NMDA receptor subunits in cultured cortical cells. Con A augmented the level of intracellular Ca²⁺ by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). We determined whether activation of AMPA receptors was involved in the regulation of NMDA receptor expression with Con A by blocking the desensitization of AMPA receptors. The results showed that AMPA receptor antagonists suppressed NMDA receptor subunits expression in Con A-treated cortical neuronal cells. PMA elevated the expression of NMDA receptor subunits, while PKC inhibitor and tyrosine kinases inhibitor suppressed the expression of NMDA receptor subunits. Furthermore, it was shown that NMDA receptor subunits expression was modulated in a region-specific manner after the sustained microinfusion of Con A into the cerebroventricle of the rat brain. Collectively, it could be presumed that the AMPA receptor activation was involved in Con A-induced modulation of NMDA receptor subunits expression.     

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.     

Modulation of LPA Receptor Expression in the Human Brain Following Neurotrauma

Lysophosphatidic acid (LPA) is involved in physiological and pathological states, including in neural development and inflammation. We assessed the expression pattern of the LPA receptors 1-3 and of LPA-producing enzyme autotaxin in post-mortem human brain tissue, both in normal individuals and in individuals who died following traumatic brain injury. We found that LPA receptors and autotaxin are weakly expressed in the normal control adult brain. Quantitative PCR for the LPA receptors and autotaxin mRNA showed an increase of LPAR₂ and a decrease of autotaxin mRNA expression in the cortex following brain injury. Immunohistochemical analysis showed that LPAR₁ colocalized with astrocytes and that LPAR₂ is present on the ependymal cells lining the lateral ventricle in the brain samples from individuals who died following severe head injury. This work shows for the first time that key components of the LPA pathway are modulated following TBI in humans.     

Effect of NDP-α-MSH on PPAR-γ and –β Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia

Brain inflammation plays a central role in numerous brain pathologies. Microglia and astrocytes are the main effector cells that become activated when an inflammatory process takes place within the central nervous system. α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with proven anti-inflammatory properties. It binds with highest affinity to the melanocortin receptor 4 (MC4R), which is present in astrocytes and upon activation triggers anti-inflammatory pathways. The aim of this research was to identify anti-inflammatory mediators that may participate in the immunomodulatory effects of melanocortins in glial cells. Since peroxisome proliferator-activated receptors (PPARs) have recently been implicated in the modulation of inflammation, we investigated the effect of an α-MSH analog, [Nle⁴, D-Phe⁷]-α-MSH (NDP-α-MSH), on PPAR-β and PPAR-γ gene and protein expression in rat primary astrocytes and microglia. We initially demonstrated that rat primary microglia express MC4R and showed that treatment with NDP-α-MSH increases PPAR-γ protein levels and strongly decreases PPAR-β levels in both astrocytes and microglia. We also showed that extracellular signal-regulated kinase 1/2 (ERK1/2)–mediated signaling is partially involved in these effects in a cell-specific fashion. Finally, we showed that NDP-α-MSH stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β from microglia and astrocytes, respectively. The presented data suggest a role for IL-10 and TGF-β in the protective action of melanocortins and a connection between MC4R pathway and that of the nuclear receptor PPAR-γ. This is the first report providing evidence that MC4R is expressed in rat primary microglia and that melanocortins modulate PPAR levels in glial cells. Our findings provide new insights into the mechanisms underlying the activation of glial MC4R and open perspectives for new therapeutic strategies for the treatment of inflammation-mediated brain diseases.     

Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

Action mechanism of lipopolysaccharide (LPS), interleukin-1β (IL-1β), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1β resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1β precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1β treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1β treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1β was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1β-treated cells. These results suggest that IL-1β inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.     

Lysophosphatidic Acid Alters the Expression Profiles of Angiogenic Factors,Cytokines, and Chemokines in Mouse Liver Sinusoidal Endothelial Cells

Background and Aims

Lysophosphatidic acid (LPA) is a multi-function glycerophospholipid. LPA affects the proliferation of hepatocytes and stellate cells in vitro, and in a partial hepatectomy induced liver regeneration model, the circulating LPA levels and LPA receptor (LPAR) expression levels in liver tissue are significantly changed. Liver sinusoidal endothelial cells (Lsecs) play an important role during liver regeneration. However, the effects of LPA on Lsecs are not well known. Thus, we investigated the effects of LPA on the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs.

Methods

Mouse Lsecs were isolated using CD31-coated magnetic beads. The mRNA expression levels of LPAR’s and other target genes were determined by quantitative RT-PCR. The protein levels of angiogenesis factors, cytokines, and chemokines were determined using protein arrays and enzyme immunoassay (EIA). Critical LPAR related signal transduction was verified by using an appropriate chemical inhibitor.

Results

LPAR1 and LPAR3 mRNA’s were expressed in mouse LPA-treated Lsecs. Treating Lsecs with a physiological level of LPA significantly enhanced the protein levels of angiogenesis related proteins (cyr61 and TIMP-1), cytokines (C5/C5a, M-CSF, and SDF-1), and chemokines (MCP-5, gp130, CCL28, and CXCL16). The LPAR1 and LPAR3 antagonist ki16425 significantly inhibited the LPA-enhanced expression of cyr61, TIMP-1, SDF-1, MCP-5, gp130, CCL28, and CXCL16, but not that of C5/C5a or M-CSF. LPA-induced C5/C5a and M-CSF expression may have been through an indirect regulation mechanism.

Conclusion

LPA regulated the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs that was mediated via LPAR1 and LPAR3 signaling. Most of the factors that were enhanced by LPA have been found to play critical roles during liver regeneration. Thus, these results may prove useful for manipulating LPA effects on liver regeneration.     

Deficiency of α1,6-fucosyltransferase promotes neuroinflammation by increasing the sensitivity of glial cells to inflammatory mediators

Background

α1,6-Fucosyltransferase-deficient (Fut8^?/?) mice displayed increased locomotion and schizophrenia-like behaviors. Since neuroinflammation is a common pathological change in most brain diseases, this study was focused on investigating the effects of Fut8 in microglia and astrocytes.

Functional lysophosphatidic acid receptors expressed in Oryzias latipes

Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1–Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA_5b respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling.     

Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway

Lysophosphatidic acid (LPA) plays important roles in many biological processes, such as brain development, oncogenesis and immune functions, via its specific receptors. We previously demonstrated that LPA-primed astrocytes induce neuronal commitment of cerebral cortical progenitors (Spohr et al. 2008). In the present study, we analyzed neurite outgrowth induced by LPA-treated astrocytes and the molecular mechanism underlying this event. LPA-primed astrocytes increase neuronal differentiation, arborization and neurite outgrowth of developing cortical neurons. Treatment of astrocytes with epidermal growth factor (EGF) ligands yielded similar results, suggesting that members of the EGF family might mediate LPA-induced neuritogenesis. Furthermore, treatment of astrocytes with LPA or EGF ligands led to an increase in the levels of the extracellular matrix molecule, laminin (LN), thus enhancing astrocyte permissiveness to neurite outgrowth. This event was reversed by pharmacological inhibitors of the MAPK signaling pathway and of the EGF receptor. Our data reveal an important role of astrocytes and EGF receptor ligands pathway as mediators of bioactive lipids action in brain development, and implicate the LN and MAPK pathway in this process.