Lysophosphatidic Acid Degradation Research Articles

Alcohol Induced Oxidative Stress in neonatal Cardio myocytes

Alcohol Induced Oxidative Stress in neonatal Cardio myocytes

Abstract 541: LPP3 Deficiency Impairs Mitochondrial Function and Enhances Myocardial LPA Mediated Signaling

The enzyme autotaxin is responsible for the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA), and the enzyme lipid phosphate phosphatase 3 (LPP3) is responsible for the degradation of LPA. Recently, we have shown that LPP3 plays a prominent role in the heart compared to other isoforms of LPP(Redox Biol. 2018 Apr;14:261-271). LPP3 is localized on the Mitochondria-Associated Endoplasmic Reticulum Membranes (MEM), and changes in the activity of LPP3 in the MEM play a critical role in the transport of essential membranous lipids. Mitochondrial membranous lipids regulated by the MEM are necessary for the mitochondria to survive, as they are not produced in the mitochondria. Lack of LPP3 decreased both diacylglycerols (P < 0.001) and phosphatidylglycerol (P < 0.001) in the membrane fractions as assessed by mass spectrometry(MS/MS). Lpp3 Δ myocardium showed distorted intercalated disc with damaged mitochondria with disorganized cristae and amorphous matrix densities as compared to lpp3 fl in transmission electron microscopy. Lack of LPP3 resulted in deteriorating cardiac function. To further understand the mitochondrial regulation mediated by LPP3, we studied the effect of LPA on mitochondrial respiration under different conditions using the Seahorse XF24 analyzer to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cardiomyocytes. Our results show that the exposure to LPA significantly decreased OCR (P < 0.001) and increased ECAR (P < 0.001). The addition of autotaxin inhibitors PF8380 had a reverse effect on OCR and ECAR. The increase in OCR and reduction in ECAR was even more pronounced after blocking of LPA signaling using Brp-LPA receptor blocker (P < 0.001). Our real-time observation studies on mitochondrial oxidative phosphorylation status revealed mitochondrial dysfunction with increased production of superoxide (a normal byproduct of oxidative phosphorylation that is formed at increased rates when the electron transport chain is defective) in the cardiomyocytes that lack LPP3. The re-expression of LPP3 mitigated oxidative stress. This study is crucial for the understanding of the importance of LPP3 in the preservation of both cardiac and mitochondrial function.

Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

Lipid phosphate phosphatase inhibitors locally amplify lysophosphatidic acid LPA1 receptor signalling in rat brain cryosections without affecting global LPA degradation

BackgroundLysophosphatidic acid (LPA) is a signalling phospholipid with multiple biological functions, mainly mediated through specific G protein-coupled receptors. Aberrant LPA signalling is being increasingly implicated in the pathology of common human diseases, such as arteriosclerosis and cancer. The lifetime of the signalling pool of LPA is controlled by the equilibrium between synthesizing and degradative enzymatic activity. In the current study, we have characterized these enzymatic pathways in rat brain by pharmacologically manipulating the enzymatic machinery required for LPA degradation.ResultsIn rat brain cryosections, the lifetime of bioactive LPA was found to be controlled by Mg2+-independent, N-ethylmaleimide-insensitive phosphatase activity, attributed to lipid phosphate phosphatases (LPPs). Pharmacological inhibition of this LPP activity amplified LPA1 receptor signalling, as revealed using functional autoradiography. Although two LPP inhibitors, sodium orthovanadate and propranolol, locally amplified receptor responses, they did not affect global brain LPA phosphatase activity (also attributed to Mg2+-independent, N-ethylmaleimide-insensitive phosphatases), as confirmed by Pi determination and by LC/MS/MS. Interestingly, the phosphate analog, aluminium fluoride (AlFx-) not only irreversibly inhibited LPP activity thereby potentiating LPA1 receptor responses, but also totally prevented LPA degradation, however this latter effect was not essential in order to observe AlFx--dependent potentiation of receptor signalling.ConclusionsWe conclude that vanadate- and propranolol-sensitive LPP activity locally guards the signalling pool of LPA whereas the majority of brain LPA phosphatase activity is attributed to LPP-like enzymatic activity which, like LPP activity, is sensitive to AlFx- but resistant to the LPP inhibitors, vanadate and propranolol.

Lipid phosphate phosphohydrolase type 1 (LPP1) degrades extracellular lysophosphatidic acid in vivo

LPA (lysophosphatidic acid) is a lipid mediator that stimulates cell proliferation and growth, and is involved in physiological and pathological processes such as wound healing, platelet activation, angiogenesis and the growth of tumours. Therefore defining the mechanisms of LPA production and degradation are of interest in understanding the regulation of these processes. Extracellular LPA synthesis is relatively well understood, whereas the mechanisms of its degradation are not. One route of LPA degradation is dephosphorylation. A candidate enzyme is the integral membrane exophosphatase LPP1 (lipid phosphate phosphohydrolase type 1). In the present paper, we report the development of a mouse wherein the LPP1 gene (Ppap2a) was disrupted. The homozygous mice, which are phenotypically unremarkable, generally lack Ppap2a mRNA, and multiple tissues exhibit a substantial (35-95%) reduction in LPA phosphatase activity. Compared with wild-type littermates, Ppap2a(tr/tr) animals have increased levels of plasma LPA, and LPA injected intravenously is metabolized at a 4-fold lower rate. Our results demonstrate that LPA is rapidly metabolized in the bloodstream and that LPP1 is an important determinant of this turnover. These results indicate that LPP1 is a catabolic enzyme for LPA in vivo.

Lysophosphatidic acid receptors determine tumorigenicity and aggressiveness of ovarian cancer cells.

Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown. Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided. SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase. Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.

Lysophosphatidic acid protection against apoptosis in the human pre‐B‐cell line Nalm‐6

Lysophosphatidic acid (LPA) promotes survival, growth, differentiation, and motility in a variety of cell types, and has been reported to act as a cell survival and growth factor in B lymphocytes. Autotaxin (ATX), through its lysophospholipase D activity, generates LPA from lysophosphatidylcholine (LPC). In this study, we investigated the effects of LPA and also the expression of ATX and LPA receptor, in the human pre-B-cell line Nalm-6. It was found that LPA protects Nalm-6 cells against both spontaneous and staurosporine-induced apoptosis. Furthermore, ATX expression on the cell surface and ATX activity in the cell lysate were detected. No accumulation of LPA in the culture medium was, however, detected when the Nalm-6 cells were cultured with LPC. The pre-B cells were found to express the mRNA transcript for lipid phosphate phosphatase-1 and LPA degradation was inhibited in the presence of the phosphatase inhibitor vanadate, it was surmised that LPA production in the culture medium may have been masked by LPA degradation by this ecto-phosphatase. Abundant expression of LPA receptors, especially, LPA(4), was detected by a real-time polymerase chain reaction technique. Our results suggest an important and autocrine role of LPA in the survival of this well-established model cell line, although the direct involvement of ATX in the production of LPA in these cells was not confirmed.

The emerging role of lysophosphatidic acid in cancer.

The bioactive phospholipid lysophosphatidic acid (LPA) stimulates cell proliferation, migration and survival by acting on its cognate G-protein-coupled receptors. Aberrant LPA production, receptor expression and signalling probably contribute to cancer initiation, progression and metastasis. The recent identification of ecto-enzymes that mediate the production and degradation of LPA, as well as the development of receptor-selective analogues, indicate mechanisms by which LPA production or action could be modulated for cancer therapy.

Lysophosphatidic acid synthesis and release

Lysophosphatidic acid (LPA) is a bioactive phospholipid controlling numerous cellular responses through the activation of specific G-protein coupled transmembrane receptors. LPA is present in several biological fluids (serum, plasma, aqueous humor) and can be secreted by several cell types (platelets, fibroblasts, adipocytes, cancer cells). Whereas, multiple pathways of synthesis and degradation of LPA have been described, their relative contribution in extracellular secretion and biodisponibility is still a matter of debate. The first part of the present review is devoted to the description of the different enzymes involved in LPA synthesis (acyltransferases, phospholipases, kinases) and degradation (lysophospholipases, lipid-phosphatases), as well as to the molecules involved in LPA transport (albumin, fatty acid binding proteins, gelsolin, lipoproteins). In a second part, the different physio-pathological situations (aggregation, cancer, injuries) associated with LPA production, as well as the potential role played by LPA in genesis of certain diseases (cancer, obesity, arteriosclerosis) are listed and analyzed.

Lysophosphatidic Acid-induced Mitogenesis Is Regulated by Lipid Phosphate Phosphatases and Is Edg-receptor Independent

Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors we have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found that LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, we have determined that the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7.

Surface LLP-1 Controls LPA Signaling

Lysophosphatidic acid (LPA) is a phospholipid growth factor found in serum. Many cell types respond to LPA through the activation of G protein-coupled receptors, EDG-2, EDG-4, or EDG-7. Xu et al . altered the cell surface levels of lipid phosphate phosphatase-1 (LLP-1) by overexpression or inhibition with antisense oligonucleotides and found that LPA degradation, LPA binding, and signal transduction through LPA-stimulated EDG-2 receptors were regulated. Overexpression of mouse LLP-1 in Rat2 fibroblasts increased LPA degradation, and decreased LPA binding to cells. Furthermore, increased LLP-1 blocked the LPA-induced decrease in cyclic adenosine monophosphate levels. Overexpression of LLP-1 also blocked LPA-induced increases in phospholipase D activity and mitogen-activated protein kinase activity and cell proliferation, as well as increases in intracellular Ca 2+ concentration. Inhibition of the expression of endogenous LLP-1 with antisense oligonucleotides had the opposite effects as overexpression in Rat2 cells, confirming a role for LLP-1 in regulating cellular responsiveness to exogenous LPA. Xu, J., Love, L.M., Singh, I., Zhang, Q.-X., Dewald, J., Wang, D.-A., Fischer, D.J., Tigyi, G., Berthiaume, L.G., Waggoner, D.W., and Brindley, D.N. (2000) Lipid phosphate phosphatase-1 and Ca 2+ control lysophosphatidate signaling through EDG-2 receptors. J. Biol. Chem. 275 : 27520-27530. [Abstract] [Full Text]

Depletion of lysophosphatidic acid triggers a loss of oriented detyrosinated microtubules in motile fibroblasts.

We reported earlier that isolated plasma membranes trigger a number of responses comprising contact inhibition of motility, including loss of oriented detyrosinated microtubules (Glu MTs) from the lamella of motile fibroblasts. In this study, we show that the membranes trigger this loss of Glu MTs, not by binding to cells, but by removing an essential component from the medium necessary to maintain oriented Glu MTs. Preincubation of membranes with medium containing serum followed by removal of the membranes by sedimentation rendered the membrane-treated medium capable of triggering the loss of oriented Glu MTs. Membrane activity was inhibited by high concentrations of serum and removal of serum from medium triggered the loss of oriented Glu MTs similar to that triggered by membranes. These results suggest that the membranes trigger the loss of Glu MTs by inactivating factors in serum that are required for the maintenance of oriented Glu MTs. By fractionating serum, we have identified lysophosphatidic acid (LPA) as the principal serum factor that is responsible for supporting oriented Glu MTs. The activity of LPA to maintain oriented Glu MTs upon serum withdrawal was half maximal at 100 nM and no activity was observed with structurally related phospholipids. Serum LPA levels were sufficient to account for the ability of serum to support oriented Glu MTs. Enzymatic degradation of serum LPA strongly reduced the ability of serum to support oriented Glu MTs. That membranes degrade LPA was shown by the ability of membranes to block LPA's ability to maintain oriented Glu MTs, and by direct measurement of the loss of radiolabeled LPA after incubation with membranes in vitro. These results show that isolated plasma membranes trigger the loss of Glu MTs from the lamella of motile cells by degrading serum LPA. Coupled with earlier results showing that membranes trigger a number of contact inhibition responses, our data suggest a new model for contact inhibition of motility in which local degradation of LPA and/or interference with LPA-stimulated signalling pathways initiates a contact inhibition response in colliding cells.