Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.     

Peritoneal fluids from patients with certain gynecologic tumor contain elevated levels of bioactive lysophospholipase D activity

Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.     

Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white

Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.     

Lysophospholipase D and its role in LPA production

Lysophosphatidic acid (LPA) is an important lipid mediator that binds to G-protein-coupled receptors of the Edg family, inducing proliferation and migration in many cell lines. Much has been learned about pathways involved in LPA signaling, but the pathways responsible for LPA production remain to be fully resolved. Several potential routes have been proposed for LPA production. One involves the sequential actions of phopholipase D (PLD) and phospholipase A(2) (PLA(2)). Another route involves the sequential actions of PLA(2) and lysophospholipase D (lysoPLD). LysoPLD is defined as an enzyme which hydrolyzes lysophospholipids to produce LPA. Two major forms of lysoPLD, microsomal and extracellular forms, have been reported. A microsomal lysoPLD plays an important role in the metabolism of platelet-activating factor (PAF) because of its preference for alkyl-phospholipids. The extracellular form of lysoPLD coexists with its substrate, lysophosphatidylcholine (LPC), in the extracellular compartment. LysoPLDs purified from the extracellular space have recently been shown to be molecularly identical to autotaxin (ATX). ATX, an enzyme previously known to possess 5'-nucleotide pyrophosphatase and phosphodiesterase (PDE) activities, was subsequently shown to have lysoPLD activity. The unexpected linkage of the extracellular lysoPLD with ATX has raised many interesting questions. The characterization and purification of lysoPLDs are reviewed here.     

Mechanisms of lysophosphatidic acid production

Lysophosphatidic acid is one of the most attractive phospholipid mediator with multiple biological functions and is implicated in various human diseases. In the past ten years much has been learned about the physiological roles of LPA through series of studies on LPA actions and its receptors. However, the molecular mechanisms of LPA have been poorly understood. LPA is produced in various conditions both in cells and in biological fluids, where multiple synthetic reactions occur. At least two pathways are postulated. In serum and plasma, LPA is mainly converted from lysophospholipids. By contrast, in platelets and some cancer cells, LPA is converted from phosphatidic acid. In each pathway, at least two phospholipase activities are required: phospholipase A1 (PLA1)/PLA2 plus lysophospholipase D (lysoPLD) activities are involved in the first pathway and phospholipase D (PLD) plus PLA1/PLA2 activities are involved in the second pathway. Now multiple phospholipases are identified that account for PLA1, PLA2, PLD, and lysoPLD activities. In the absence of specific inhibitors and genetically modified animals and individuals, the contribution of each phospholipase to LPA production can not be easily determined. However, apparently certain extracellular phospholipases such as secretory PLA2 (sPLA2-IIA), membrane-associated PA-selective PLA1 (mPA-PLA1), lecithin-cholesterol acyltransferase (LCAT), and lysoPLD are involved in LPA production.     

Phosphatase-resistant analogues of lysophosphatidic acid: agonists promote healing, antagonists and autotaxin inhibitors treat cancer

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and also biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues also are feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, a.k.a., autotaxin, ATX), a central regulator of invasion and metastasis. For cancer therapy, the optimal therapeutic profile would be a metabolically-stabilized, pan-LPA receptor antagonist that also inhibited lysoPLD. For protection of gastrointestinal mucosa and lymphocytes, LPA agonists would be desirable to minimize or reverse radiation or chemical-induced injury. Analogues of lysophosphatidic acid (LPA) that are chemically modified to be less susceptible to phospholipases and phosphatases show activity as long-lived receptor-specific agonists and antagonists for LPA receptors, as well as inhibitors for the lysoPLD activity of ATX.     

Role of lysophosphatidic acid in the regulation of uterine leiomyoma cell proliferation by phospholipase D and autotaxin

Phospholipase D (PLD) hydrolyzes phosphatidylcholine into phosphatidic acid (PA), a lipidic mediator that may act directly on cellular proteins or may be metabolized into lysophosphatidic acid (LPA). We previously showed that PLD contributed to the mitogenic effect of endothelin-1 (ET-1) in a leiomyoma cell line (ELT3 cells). In this work, we tested the ability of exogenous PA and PLD from Streptomyces chromofuscus (scPLD) to reproduce the effect of endogenous PLD in ELT3 cells and the possibility that these agents acted through LPA formation. We found that PA, scPLD, and LPA stimulated thymidine incorporation. LPA and scPLD induced extracellular signal-regulated kinase (ERK(1/2)) mitogen-activated protein kinase activation. Using Ki16425, an LPA(1)/LPA(3) receptor antagonist and small interfering RNA targeting LPA(1) receptor, we demonstrated that scPLD acted through LPA production and LPA(1) receptor activation. We found that scPLD induced LPA production by hydrolyzing lysophosphatidylcholine through its lysophospholipase D (lysoPLD) activity. Autotaxin (ATX), a naturally occurring lysoPLD, reproduced the effects of scPLD. By contrast, endogenous PLD stimulated by ET-1 failed to produce LPA. These results demonstrate that scPLD stimulated ELT3 cell proliferation by an LPA-dependent mechanism, different from that triggered by endogenous PLD. These data suggest that in vivo, an extracellular lysoPLD such as ATX may participate in leiomyoma growth through local LPA formation.     

The heterotrimeric G protein subunits Gα(q) and Gβ(1) have lysophospholipase D activity

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gβ(1) respectively. When FLAG-tagged Gα(q) or Gβ(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gβ(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

Autotaxin induces lung epithelial cell migration through lysoPLD activity-dependent and -independent pathways

Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through G(αi)-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated association of ATX with the epithelial cell-surface LPA receptor and integrin β4.     

Serum lysophosphatidic acid is produced through diverse phospholipase pathways

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A(2) (sPLA(2)-IIA) and phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA(1), sPLA(2)-IIA, LCAT, and lysoPLD.     

Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of In vitro fertilization patients.

Lysophosphatidic acids (LPAs) are known to be normal constituents of mammalian serum, and they mimic some biological effects of the serum. We previously reported that lysophospholipase D (LPLD) was involved in the accumulation of LPAs in incubated rat plasma and serum. In this study we detected, by gas-liquid chromatography, various molecular species of LPA in follicular fluids collected from women programmed for in vitro fertilization. When the follicular fluid was incubated at 37 degrees C for 48 h, persistent increases in the amounts of LPAs were observed concomitant with decreases in the amounts of the corresponding lysophosphatidylcholines (LPCs), although the concentrations of saturated LPCs increased in the first 6 h of incubation. These results suggest that human follicular fluid has LPLD activity, and this was confirmed by experiments with follicular fluids mixed with an exogenous radioactive LPC. The LPLD showed preference for unsaturated over saturated LPCs, similar to plasma LPLD, indicating that it originated from the circulation.     

Increased formation of lysophosphatidic acids by lysophospholipase D in serum of hypercholesterolemic rabbits

Lysophosphatidic acid (LPA) is a biologically active phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Previously, we found that mammalian plasma and serum contain a lysophospholipase D, which hydrolyzes lysophosphatidylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37 degree C. In this study, we examined whether lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feeding rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rabbits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor-alpha. These results indicated that increases in the levels of LPAs generated by lysophospholipase D in the blood of hypercholesterolemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis.     

Autotaxin-LPA轴在肥胖及其相关疾病中的作用

溶血磷脂酸(lysophosphatidic acid,LPA)是一种结构简单的生物活性脂质分子,可通过与细胞膜上的LPA受体(lysophosphatidic acid receptors,LPARs)结合参与调控细胞生命活动,在多种生理和病理过程中发挥作用.分泌型糖蛋白Autotaxin(ATX)具溶血磷脂酶D(l...     

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production

Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.     

Role of the autotaxin-lysophosphatidic acid axis in glaucoma,aqueous humor drainage and fibrogenic activity

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1β and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.     

Constitutively active lysophosphatidic acid receptor-1 enhances the induction of matrix metalloproteinase-2

Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA(1) enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA(1) underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1Δ-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1Δ-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1Δ-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1Δ-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA(1) may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells.     

Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.     

Autotaxin的结构与功能

Autotaxin(ATX)是一个分泌型糖蛋白,具有磷酸二酯酶(PDE)活性,是胞外焦磷酸酶/磷酸二酯酶(ENPP)家族的一员.ATX还具有溶血磷脂酶D(lysoPLD)活性,能够以溶血磷脂酰胆碱(lysophosphatidylcholjne,LPC)为底物催化生成溶血磷脂酸(lysophosphatidic acid,LPA).ATX在很多肿瘤细胞中都有高表达,在肿瘤的发生、发展过程中有着重要作用,被认为是肿瘤治疗中一个可能的靶位.此外,ATX在神经系统、免疫系统中也发挥重要作用.目前已经建立了一系列快速检测ATX活性的方法,并在此基础上研发了相关疾病的诊断技术.基于ATX的多功能性,对其表达调控机理的研究和抑制剂的开发成为当前的研究热点.