Lysophosphatidic acid (LPA) stimulates mouse mammary epithelial cell growth

LPA (lysophosphatidic acid) is a bioactive phospholipid having diverse effects on various types of tissues. When NMuMG (normal murine mammary gland) cells were cultured in the presence of 0-10 μM LPA, cell numbers were increased by dose dependency for the 6-day culture periods (P<0.05). In DNA synthesis assay, 10 μM LPA induced 4.5-fold more DNA synthesis compared with control (P<0.05). In addition, the cultured cell density in the given area was increased by LPA treatment. MMP (matrix metalloproteinase) inhibitor GM6001 and EGFR [EGF (epidermal growth factor) receptor] tyrosine kinase inhibitor AG1478 [tyrphostin AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline] significantly decreased LPA-induced DNA synthesis and cell growth without cell death (P<0.05). To test the hypothesis that LPA-induced cell growth is mediated through LPA subtype receptors, LPA subtype receptor gene expressions were amplified by PCR. NMuMG cells expressed LPA1 and LPA2 receptor genes in the presence of 10% FBS (fetal bovine serum). LPA treatments increased ERK1/2 (extracellular-signal-regulated kinase) phosphorylation at 30 min and then dephosphorylated at 2 h after treatment. LPA treatment phosphorylated at tyrosine residues on a variety of Gi and PI3-dependent signal transducers in NMuMG cells. These results suggest that LPA subtype receptors play a role as the active transactivator of EGFR-associated kinases as well as direct growth regulator in mammary tissues.     

Lysophosphatidic acid (LPA) receptors: Signaling properties and disease relevance

Lysophosphatidic acid (LPA), a water-soluble phospholipid, has gained significant attention in recent years since the discovery that it acts as a potent signaling molecule with wide-ranging effects on many different target tissues. There are currently five identified G protein-coupled receptors for LPA and more are undergoing validation. The complexity of the expression pattern and signaling properties of LPA receptors results in multiple influences on developmental, physiological, and pathological processes. This review provides a summary of LPA receptor signaling and current views on the potential involvement of this pathway in human diseases that include cardiovascular, cancer, neuropathic pain, neuropsychiatric disorders, reproductive disorders, and fibrosis. The involvement of LPA signaling in these processes implicates multiple, potential drug targets including LPA receptor subtypes and LPA metabolizing enzymes. Modulation of LPA signaling may thus provide therapeutic inroads for the treatment of human disease.     

Lysophosphatidic acid stimulates neuronal differentiation of cortical neuroblasts through the LPA1-G(i/o) pathway

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates cortical development. Here we examined how LPA influences the cell fate of cortical neuroblasts using a neurosphere culture system. We generated neurospheres in the presence of basic fibroblast growth factor (bFGF). Treatment with LPA throughout the culture period significantly reduced the number of cells in the neurospheres. When dissociated single cells derived from neurospheres were induced to differentiate by adherence on coverslips, the proportion of MAP2-positive neurons was higher in LPA-treated neurospheres than in those treated with bFGF alone, and the proportion of myelin basic protein-positive oligodendrocytes was lower. Consistent with this finding, LPA raised the ratio of beta-tubulin type III-positive young neurons and reduced the ratio of CD140a-positive oligodendrocyte precursors in neurospheres. These effects of LPA were inhibited by pretreatment of neurospheres with pertussis toxin or an LPA(1)-preferring antagonist, Ki16425. Moreover, LPA-induced enhancement of neuronal differentiation was not observed in neurospheres derived from lpa(1)-null mice. These results suggest that LPA promotes the commitment of neuroblasts to the neural lineage through the LPA(1)-G(i/o) pathway.     

Lysophosphatidic acid (LPA) in malignant ascites stimulates motility of human pancreatic cancer cells through LPA1

Cytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA1 and LPA3), but not by an LPA3-selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells.     

Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure-activity relationship of cloned LPA receptors

We examined the structure-activity relationship of cloned lysophosphatidic acid (LPA) receptors (endothelial cell differentiation gene (EDG) 2, EDG4, and EDG7) by measuring [Ca(2+)](i) in Sf9 insect cells expressing each receptor using LPA with various acyl chains bound at either the sn-1 or the sn-2 position of the glycerol backbone. For EDG7 the highest reactivity was observed with LPA with Delta9-unsaturated fatty acid (oleic (18:1), linoleic (18:2), and linolenic (18:3)) at sn-2 followed by 2-palmitoleoyl (16:1) and 2-arachidonoyl (20:4) LPA. In contrast, EDG2 and EDG4 showed broad ligand specificities, although EDG2 and EDG4 discriminated between 14:0 (myristoyl) and 16:0 (palmitoyl), and 12:0 (lauroyl) and 14:0 LPAs, respectively. EDG7 recognizes the cis double bond at the Delta9 position of octadecanoyl residues, since 2-elaidoyl (18:1, trans) and 2-petroselinoyl (18:1, cis-Delta12) LPA were poor ligands for EDG7. In conclusion, the present study demonstrates that each LPA receptor can be activated differentially by the LPA species.     

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.     

Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors

Lysophosphatidic acid (LPA) exerts multiple biological functions through G protein-coupled receptors (EDG2/LPA(1), EDG4/LPA(2), and EDG7/LPA(3)) and is present in serum where it is associated with albumin. In this study we examined LPA activity in various biological fluids by measuring the LPA-induced increase in the intracellular concentration of calcium ion in three types of Sf9 insect cells, each expressing one of the LPA receptors. Using this system, we found that EDG2 and EDG4, but not EDG7, were activated strongly by addition of incubated plasma. By contrast, LPA detected in seminal plasma, which contains a low concentration of albumin, selectively activated EDG7. After LPA in these samples was extracted and reconstituted, it activated all three receptors. We also found that serum albumin readily inhibits the activation of EDG7 but not the activation of EDG2 or EDG4. In addition, plasma from Nagase analbuminemic rats but not plasma from control Sprague-Dawley rats was found to strongly activate EDG7, although the plasma of these two types of rats contained equal amounts of LPA and activated both EDG2 and EDG4. The present study shows that serum albumin can negatively regulate EDG7 but not EDG2 or EDG4, and suggests that protein factors are present in seminal plasma and deliver LPA efficiently to EDG7 but not to EDG2 or EDG4.     

Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.     

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules

Journal of Physiology and Biochemistry - Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma...     

Lysophosphatidic acid receptor-2 (LPA2)-mediated signaling enhances chemoresistance in melanoma cells treated with anticancer drugs

Molecular and Cellular Biochemistry - Parkinson’s disease (PD) is the second common age-related neurodegenerative disease. It is characterized by control loss of voluntary movements control,...     

Lysophosphatidic acid and calcitriol co-operate to promote human osteoblastogenesis: requirement of albumin-bound LPA

Lysophosphatidic acid (LPA), a pleiotropic signalling lipid is assuming growing significance in osteoblast biology. Although committed osteoblasts from several mammalian species are receptive to LPA far less is known about the potential for LPA to influence osteoblast formation from their mesenchymal progenitors. An essential factor for both bone development and post-natal bone growth and homeostasis is the active metabolite of vitamin D3, calcitriol (D3). Previously we reported how a combination of LPA and D3 synergistically co-operated to enhance the differentiation of immature human osteoblasts. Herein we provide evidence for the formation of human osteoblasts from multiple, primary human bone marrow derived stromal (stem) cells (hBMSCs). Importantly osteoblast development from hBMSCs only occurred when LPA was administered as a complex with albumin, its natural carrier. Collectively our findings support a co-operative role of LPA and D3 in osteoblastogenesis, findings which may aid the development of novel treatment strategies for bone repair.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.     

The MLK-related Kinase (MRK) Is a Novel RhoC Effector That Mediates Lysophosphatidic Acid (LPA)-stimulated Tumor Cell Invasion

The small GTPase RhoC is overexpressed in many invasive tumors and is essential for metastasis. Despite its high structural homology to RhoA, RhoC appears to perform functions that are different from those controlled by RhoA. The identity of the signaling components that are differentially regulated by these two GTPases is only beginning to emerge. Here, we show that the MAP3K protein MRK directly binds to the GTP-bound forms of both RhoA and RhoC in vitro. However, siRNA-mediated depletion of MRK in cells phenocopies depletion of RhoC, rather than that of RhoA. MRK depletion, like that of RhoC, inhibits LPA-stimulated cell invasion, while depletion of RhoA increases invasion. We also show that active MRK enhances LPA-stimulated invasion, further supporting a role for MRK in the regulation of invasion. Depletion of either RhoC or MRK causes sustained myosin light chain phosphorylation after LPA stimulation. In addition, activation of MRK causes a reduction in myosin light chain phosphorylation. In contrast, as expected, depletion of RhoA inhibits myosin light chain phosphorylation. We also present evidence that both RhoC and MRK are required for LPA-induced stimulation of the p38 and ERK MAP kinases. In conclusion, we have identified MRK as a novel RhoC effector that controls LPA-stimulated cell invasion at least in part by regulating myosin dynamics, ERK and p38.     

Lysophosphatidic acid as a novel cell survival/apoptotic factor

Lysophosphatidic acid (LPA) activates its cognate G protein-coupled receptors (GPCRs) LPA(1-3) to exert diverse cellular effects, including cell survival and apoptosis. The potent survival effect of LPA on Schwann cells (SCs) is mediated through the pertussis toxin (PTX)-sensitive G(i/o)/phosphoinositide 3-kinase (PI3K)/Akt signaling pathways and possibly enhanced by the activation of PTX-insensitive Rho-dependent pathways. LPA promotes survival of many other cell types mainly through PTX-sensitive G(i/o) proteins. Paradoxically, LPA also induces apoptosis in certain cells, such as myeloid progenitor cells, hippocampal neurons, and PC12 cells, in which the activation of the Rho-dependent pathways and caspase cascades has been implicated. The effects of LPA on both cell survival and apoptosis underscore important roles for this lipid in normal development and pathological processes.     

Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1

Autotaxin (ATX) is a tumor cell motility-stimulating factor originally isolated from melanoma cell supernatant that has been implicated in regulation of invasive and metastatic properties of cancer cells. Recently, we showed that ATX is identical to lysophospholipase D, which converts lysophosphatidylcholine to a potent bioactive phospholipid mediator, lysophosphatidic acid (LPA), raising the possibility that autocrine or paracrine production of LPA by ATX contributes to tumor cell motility. Here we demonstrate that LPA and ATX mediate cell motility-stimulating activity through the LPA receptor, LPA(1). In fibroblasts isolated from lpa(1)(-/-) mice, but not from wild-type or lpa(2)(-/-), cell motility stimulated with LPA and ATX was completely absent. In the lpa(1)(-/-) cells, LPA-stimulated lamellipodia formation was markedly diminished with a concomitant decrease in Rac1 activation. LPA stimulated the motility of multiple human cancer cell lines expressing LPA(1), and the motility was attenuated by an LPA(1)-selective antagonist, Ki16425. The present study suggests that ATX and LPA(1) represent potential targets for cancer therapy.     

Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA3 receptor

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA(1) receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA(3) receptor-expressing Sf9 cells but poorly in LPA(2) receptor-expressing cells. Guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA(3) receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA(3) but not the LPA(1) or LPA(2) receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA(1)/LPA(3) receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA(3)-selective agonist. OMPT binding to the LPA(3) receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.