Multidrug transporters and antibiotic resistance in Lactococcus lactis

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.     

溶血磷脂酸受体及下游信号通路在心脏细胞生长中的调节作用

溶血磷脂酸（1ysophosphatidic acid,LPA）是一种十分活跃的磷脂信号分子,具有广泛的生物学效应,包括诱导神经轴突回缩、应力纤维形成、促进血小板凝集、诱导平滑肌收缩、刺激血管平滑肌细胞增殖等。LPA通过其受体及耦联的G蛋白调节细胞内信号途径,介导各种生物学效应。心脏组织中存在多种LPA受体亚型,尤其受体LPAl亚型在心脏组织中的含量仅次于脑,位居第二,暗示LPA在心脏中有重要的生物学功能。本文着重对LPA的5种受体亚型的组织分布、与G蛋白的耦联和对第二信使的活性调节,以及LPA及其受体亚型对心脏细胞的生长调节作一综述。     

Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.     

Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.     

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.     

Proteome‐wide analysis of temporal phosphorylation dynamics in lysophosphatidic acid‐induced signaling

Most growth factor receptors trigger phosphorylation‐based signal transduction to translate environmental stimuli into defined biological responses. In addition to comprehensive and reliable assessment of growth factor‐induced phosphoregulation, temporal resolution is needed to gain insights into the organizing principles of the cellular signaling machinery. Here, we introduce a refined experimental design for MS‐based phosphoproteomics to reconcile the need for high comprehensiveness and temporal resolution with the key requirement of monitoring biological reproducibility. We treated SILAC‐labeled SCC‐9 cells with the seven transmembrane receptor ligand lysophosphatidic acid (LPA) and identified more than 17 000 phosphorylation sites. Filtering for biological replicate quantification yielded five‐time point profiles for 6292 site‐specific phosphorylations, which we analyzed for statistically significant regulation. Notably, about 30% of these sites changed significantly upon LPA stimulation, indicating extensive phosphoproteome regulation in response to this growth factor. Analysis of time series data identified distinct temporal profiles for different kinase substrate motifs, likely reflecting temporal orchestration of cellular kinase activities. Our data further indicated coordinated regulation of biological processes and phosphoprotein networks upon LPA stimulation. Finally, we detected regulation of functionally characterized phosphorylation sites not yet implicated in LPA signaling, which may foster a better understanding how LPA regulates cellular physiology on the molecular level.     

Regulation and biological activities of the autotaxin-LPA axis

Autotaxin (ATX), or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), is an exo-enzyme originally identified as a tumor cell autocrine motility factor. ATX is unique among the NPPs in that it primarily functions as a lysophospholipase D, converting lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA). LPA acts on specific G protein-coupled receptors to elicit a wide range of cellular responses, ranging from cell proliferation and migration to neurite remodeling and cytokine production. While LPA signaling has been studied extensively over the last decade, we are only now beginning to explore the properties and biological importance of ATX as the major LPA-producing phospholipase. In this review, we highlight recent advances in our understanding of the ATX-LPA axis, giving first an update on LPA action and then focusing on ATX, in particular its regulation, its link to cancer and its vital role in vascular development.     

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.     

Alkyl lysophosphatidic acid and fluoromethylene phosphonate analogs as metabolically-stabilized agonists for LPA receptors

We describe an efficient method for the synthesis of alkyl lysophosphatidic acid (LPA) analogs as well as alkyl LPA mono- and difluoromethylene phosphonate analogs. Each alkyl LPA analog was evaluated for subtype-specific LPA receptor agonist activity using a cell migration assay for LPA(1) activation in cancer cells and an intracellular calcium mobilization assay for LPA(2) and LPA(3) activation. Alkyl LPAs induced pronounced cell migration activity with equivalent or higher potency than sn-1-oleoyl LPA, while the alkyl LPA fluoromethylene phosphonates proved to be less potent agonists in this assay. However, each alkyl LPA analog activated Ca(2+) release by activation of LPA(2) and LPA(3) receptors. Interestingly, the absolute configuration of the sn-2 hydroxyl group of the alkyl LPA analogs was not recognized by any of the three LPA receptors. The use of alkyl LPA analogs further expands the scope of structure-activity studies, which will better define LPA-LPA receptor interactions.     

Rho-dependent, Rho kinase-independent inhibitory regulation of Rac and cell migration by LPA1 receptor in Gi-inactivated CHO cells

Lysophosphatidic acid (LPA) is a major serum lysophospholipid that stimulates cell migration in diverse cell types including ovarian cancer cells. We report here that in the absence of Gi function, LPA induces inhibition, rather than stimulation, of cellular Rac activity, lamellipodium formation, and cell migration in response to insulin like growth factor I (IGF-I) in Chinese hamster ovary (CHO) cells, which solely express LPA1 as a LPA receptor. The inhibitory effects of LPA are abrogated by the expression of either Galpha13 C-terminal peptide or C3 toxin pretreatment, but not a Rho kinase inhibitor. Without PTX pretreatment, LPA stimulates Rac and cell migration yet similarly activates Rho, indicating that Rho activation by itself is not sufficient for inhibition of cell migration. Conversely, the expression of a dominant negative Rac mutant sufficiently mimics the LPA inhibition of cell migration. LPA inhibits IGF I-induced Akt activation by only 40% in a manner dependent on Rho kinase. These results demonstrate that inhibition of Gi function converts LPA regulation on Rac and cell migration to an inhibitory mode, which is mediated by G13 and Rho but not Rho kinase, and raise a possibility of Gi as a new therapeutic target for LPA-dependent tumor progression.     

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.     

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.     

Identification of residues responsible for ligand recognition and regioisomeric selectivity of lysophosphatidic acid receptors expressed in mammalian cells

The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA(3) receptor has been proposed to be selectively activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA(1) and LPA(2) showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA sn-1 regioisomer. To identify residues involved in ligand recognition of LPA(3), we developed and validated computational models of LPA(3) complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 of LPA(3) form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA(3) underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC(50) of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.     

Lysophosphatidic acid inhibits serum deprivation-induced autophagy in human prostate cancer PC-3 cells

Lysophosphatidic acid (LPA) is a platelet-enriched bioactive lysophospholipid. By binding to its cognitive G protein-coupled receptors, which are encoded by endothelial differentiation genes (edgs), LPA regulates various cellular activities including proliferation, survival, and migration. Currently, little is known about the influences of LPA on autophagy, a pivotal mechanism for cell survival during conditions of starvation. Herein we present data indicating that LPA attenuates starvation-induced autophagy, by monitoring the percentage of LC3-II, an autophagy indicator, in human prostate PC-3 cells. In addition, by using cells stably expressing EGFP-LC3, LPA is shown to inhibit the formation of autophagosomes in serum-starved conditions. Our results suggest that in these conditions, LPA inhibits autophagy, which might facilitate early cancer development.     

自分泌运动因子-溶血磷脂酸轴的生物学功能与调控

自分泌运动因子(autotaxin,ATX)也称作磷酸二酯酶Iα,是核苷酸焦磷酸酶/磷酸二酯酶家族(nucleotide pyrophosphatases,NPPs)中的一员,因而也称作NPP2.ATX是NPPs中唯一具有溶血磷脂酶D(lysophospholipase D,lysoPLD)活性的成员,它可以将溶血磷脂...     

An endogenous redox molecule, thioredoxin, regulates transactivation of epidermal growth factor receptor and activation of NF-kappaB by lysophosphatidic acid

Lysophosphatidic acid (LPA) is the smallest and simplest of all the glycerophospholipids that activates a specific GTP-binding protein coupled receptor to evoke multiple cellular responses. In this paper, we have demonstrated that LPA stimulates nuclear factor (NF)-kappaB-dependent gene induction in a neuronal cell line, NG108-15 and that this is under redox regulation by an endogenous molecule, thioredoxin. We also have shown that redox-sensitive transactivation of epidermal growth factor receptor by LPA confers NF-kappaB activation and small GTPase proteins are involved in this pathway.     

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.     

Dynamic actin remodeling in response to lysophosphatidic acid

Abstract

Lysophosphatidic acid (LPA) is a multifunctional regulator of actin cytoskeleton that exerts a dramatic impact on the actin cytoskeleton to build a platform for diverse cellular processes including growth cone guidance, neurite retraction and cell motility. It has been implicated in the formation and dissociation of complexes between actin and actin binding proteins, supporting its role in actin remodeling. Several studies point towards its ability to facilitate formation of special cellular structures including focal adhesions and actin stress fibres by phosphoregulation of several actin associated proteins and their multiple regulatory kinases and phosphatases. In addition, multiple levels of crosstalk among the signaling cascades activated by LPA, affect actin cytoskeleton-mediated cell migration and chemotaxis which in turn play a crucial role in cancer metastasis. In the current review, we have attempted to highlight the role of LPA as an actin modulator which functions by controlling activities of specific cellular proteins that underlie mechanisms employed in cytoskeletal and pathophysiological events within the cell. Further studies on the actin affecting/remodeling activity of LPA in different cell types will no doubt throw up many surprises essential to gain a full understanding of its contribution in physiological processes as well as in diseases.

Communicated by Ramaswamy H. Sarma