Role of phosphodiesterase 1 in the pathophysiology of diseases and potential therapeutic opportunities.

Cyclic nucleotide phosphodiesterases (PDEs) are superfamily of enzymes that regulate the spatial and temporal relationship of second messenger signaling in the cellular system. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin (CaM), resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. Thus, PDE1 enzymes play a critical role in the pathophysiology of diseases through the fundamental regulation of cAMP and cGMP signaling. This comprehensive review provides the current research on PDE1 and its potential utility as a therapeutic target in diseases including the cardiovascular, pulmonary, metabolic, neurocognitive, renal, cancers and possibly others.

Phosphodiesterase type 1, calcitonin gene-related peptide and vasoactive intestinal polypeptide are involved in the control of human vaginal arterial vessels.

OBJECTIVE: The vagina makes a major contribution to the normal female sexual response cycle. An increase in vaginal blood flow is considered a key event in the mechanism of sexual arousal. Recent research has focused mainly on the cyclic GMP pathway and phosphodiesterase type 5 (PDE5, cyclic GMP specific PDE) in the control of vaginal vascular smooth muscle, whereas only little is known on the role of other key proteins and mediators of cyclic nucleotide mediated signaling in this process. The aim of the present study was to evaluate in the human vagina, by means of immunohistochemistry, the expression and distribution of phosphodiesterase type 1 (PDE1, known to hydrolize both cyclic AMP and cyclic GMP) in relation to calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and protein gene product 9.5 (PGP 9.5). STUDY DESIGN: Sections of human vagina (full wall specimens) were incubated with antibodies directed against PDE1, CGRP, VIP, PGP 9.5 and alpha-actin, followed by exposure to fluorochrome-labelled secondary antibodies. Visualization was commenced by means of laser fluorescence microscopy. RESULTS: Microscopic examination revealed a dense meshwork of PGP 9.5-positive nerve fibers innervating the sections of vaginal wall. Small vessels interspersing the tissue presented dense staining for PDE1 in their smooth musculature. Blood vessels were seen surrounded by PDE1-immunoreactive longitudinal smooth muscle fibers. The vessels were also found innervated by PGP-positive varicose nerve fibers characterized by the expression of CGRP. Some fibers presented immunosignals specific for VIP. CONCLUSION: Key mediators of the cyclic AMP and cyclic GMP pathways are co-localized in nerves seen in close proximity to vascular smooth muscle expressing PDE1. These findings suggest that both signaling cascades are involved in the control of vaginal blood flow.

Growth Factors and their receptors in cancer metastases.

Metastatic, rather than primary tumours are responsible for ninety percent cancer deaths. Despite significant advances in the understanding of molecular and cellular mechanisms in tumour metastases, there are limitations in preventive treatment of metastatic tumours. Much evidence arising from laboratory and clinical studies suggests that growth factors and their receptors are implicated in cancer metastases development. We review the origin and production of growth factors and their receptors in all stages of cancer metastases including epithelial-mesenchymal transition, cancer cell invasion and migration, survival within the circulation, seeding at distant organs and metastatic tumour angiogenesis. The functions of growth factors and their receptors are also discussed. This review presents the efforts made in understanding this challenge to aid in the development of new treatment strategies for cancer metastases.

[Clinical introduction of lysophosphatidic acid and autotaxin assays].

The lysophospholipid mediator lysophosphatidic acid (LPA) has been shown to elicit a variety of (patho) physiological responses through specific cell-surface G protein-coupled receptors, which are now considered as promising targets for therapeutic purposes. On the other hand, determination of their concentrations in human samples, especially plasma, is clinically relevant and important for diagnostic purposes since these lysophospholipids mainly act extracellularly. LPA is predominantly and continuously produced in blood from lysophosphatidylcholine (LPC) through the plasma lysophospholipase D (lysoPLD) activity of autotaxin (ATX). Since the enzyme lysoPLD/ATX and its substrate LPC co-exist in the plasma, the level of plasma LPA changes easily in vitro after venepuncture. Laboratory testing of LPA for clinical purposes can be conducted reliably only when the samples are prepared under stringent conditions. Although it is postulated that LPA undergoes extensive dephosphorylation in vivo due to the action of lipid phosphate phosphatase, multiple regression analysis showed a strong positive correlation between the plasma LPA concentration and serum lysoPLD/ATX level. Since the serum ATX antigen level is stable, i.e., the preparation of clinical samples for this ATX measurement is easy and since its level is closely correlated to the plasma LPA concentration, the ATX assay seems to be promising for laboratory testing. In fact, the ATX level is significantly increased in several disorders, including chronic liver diseases and malignant lymphoma. The clinical significance of the LPA and lysoPLD/ATX assays will be discussed.

Autotaxin and lysophospholipids in rheumatoid arthritis.

Autotaxin (ATX) is an autocrine motility-stimulating factor and an extracellular enzyme that catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). Although ATX can also hydrolyze sphingosylphosphorylcholine (SPC) to sphingosine-1-phosphate (S1P), the major source of extracellular S1P originates from the intracellular phosphorylation of sphingosine by sphingosine kinases (SphKs). LPA and S1P are well-characterized bioactive lysophospholipid mediators, which have critical roles in multiple cellular processes through binding and activating GPCRs. These two lipids have been implicated in various physiological (eg, cell growth, differentiation, migration and survival) and pathological (eg, angiogenesis, metastasis and autoimmunity) processes. The roles of LPA and S1P in autoimmune diseases, including rheumatoid arthritis (RA), have recently emerged. This review discusses recent findings suggesting that the LPA- and S1P-induced cellular functions of synoviocytes from patients with RA may contribute to the pathophysiology of the disease by exacerbating the disease process. ATX and the lysophospholipid mediators are potential targets for the treatment of patients with RA.

Lysophosphatidic acid production and action: critical new players in breast cancer initiation and progression.

Lysophosphatidic acid (LPA) is a potent lipid mediator that acts on a series of specific G protein-coupled receptors, leading to diverse biological actions. Lysophosphatidic acid induces cell proliferation, survival and migration, which are critically required for tumour formation and metastasis. This bioactive lipid is produced by the ectoenzyme lysophospholipase D or autotaxin (ATX), earlier known as an autocrine motility factor. The ATX-LPA signalling axis has emerged as an important player in many types of cancer. Indeed, aberrant expression of ATX and LPA receptors occurs during the development and progression of breast cancer. Importantly, expression of either ATX or LPA receptors in the mammary gland of transgenic mice is sufficient to induce the development of a high frequency of invasive and metastatic mammary cancers. The focus of research now turns to understanding the mechanisms by which ATX and LPA promote mammary tumourigenesis and metastasis. Targeting the ATX-LPA signalling axis for drug development may further improve outcomes in patients with breast cancer.

Evidence for de novo synthesis of lysophosphatidic acid in the spinal cord through phospholipase A2 and autotaxin in nerve injury-induced neuropathic pain.

We previously reported that lysophosphatidic acid (LPA) initiates nerve injury-induced neuropathic pain and its underlying mechanisms. In addition, we recently demonstrated that intrathecal injection of LPA induces de novo LPA production through the action of autotaxin (ATX), which converts lysophosphatidylcholine to LPA. Here, we examined nerve injury-induced de novo LPA production by using a highly sensitive biological titration assay with B103 cells expressing LPA1 receptors. Nerve injury caused high levels of LPA production in the ipsilateral sides of the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve, or sciatic nerve. Nerve injury-induced LPA production reached its maximum at 3 h after injury, followed by a rapid decline by 6 h. The LPA production was significantly attenuated in ATX heterozygous mutant mice, whereas the concentration and activity of ATX in cerebrospinal fluid were not affected by nerve injury. On the other hand, the activities of cytosolic phospholipase A2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2) were enhanced, with peaks at 1 h after injury. Both de novo LPA production and neuropathic pain-like behaviors were substantially abolished by intrathecal injection of arachidonyl trifluoromethyl ketone, a mixed inhibitor of cPLA2 and iPLA2, or bromoenol lactone, an iPLA2 inhibitor, at 1 h after injury. However, administration of these inhibitors at 6 h after injury had no significant effect on neuropathic pain. These findings provide evidence that PLA2- and ATX-mediated de novo LPA production in the early phase is involved in nerve injury-induced neuropathic pain.

Autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells.

Drug resistance remains a barrier to the effective long term treatment of ovarian cancer. We have established an RNAi-based screen to identify genes which confer resistance to carboplatin or paclitaxel. To validate the screen we showed that siRNA interfering with the apoptosis regulators FLIP and Bcl-X(L) conferred sensitivity to paclitaxel and carboplatin respectively. The expression of 90 genes which have previously been shown to be over-expressed in drug-resistant ovarian cancer was inhibited using siRNA and the impact on sensitivity to carboplatin and paclitaxel was assessed. ENPP2 was identified as a candidate gene causing drug resistance. ENPP2 encodes autotaxin, a phospholipase involved in the synthesis of the survival factor lysophosphatidic acid. siRNA directed to ENPP2 resulted in earlier apoptosis following treatment with carboplatin. 2-carbacyclic phosphatidic acid (ccPA 16:1), a small molecule inhibitor of autotaxin, also accelerated apoptosis induced by carboplatin. Stable ectopic expression of autotaxin in OVCAR-3 cells led to a delay in apoptosis. When serum was withdrawn to remove exogenous LPA, ccPA caused a pronounced potentiation of apoptosis induced by carboplatin in cells expressing autotaxin. These results indicate that autotaxin delays apoptosis induced by carboplatin in ovarian cancer cells.

Lysophosphatidic acid-3 receptor-mediated feed-forward production of lysophosphatidic acid: an initiator of nerve injury-induced neuropathic pain.

BACKGROUND: We previously reported that intrathecal injection of lysophosphatidylcholine (LPC) induced neuropathic pain through activation of the lysophosphatidic acid (LPA)-1 receptor, possibly via conversion to LPA by autotaxin (ATX). RESULTS: We examined in vivo LPA-induced LPA production using a biological titration assay with B103 cells expressing LPA1 receptors. Intrathecal administration of LPC caused time-related production of LPA in the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve or sciatic nerve. LPC-induced LPA production was markedly diminished in ATX heterozygotes, and was abolished in mice that were deficient in LPA3, but not LPA1 or LPA2 receptors. Similar time-related and LPA3 receptor-mediated production of LPA was observed following intrathecal administration of LPA. In an in vitro study using spinal cord slices, LPA-induced LPA production was also mediated by ATX and the LPA3 receptor. Intrathecal administration of LPA, in contrast, induced neuropathic pain, which was abolished in mice deficient in LPA1 or LPA3 receptors. CONCLUSION: These findings suggest that feed-forward LPA production is involved in LPA-induced neuropathic pain.

Autotaxin.

Autotaxin is a protein of approximately 900 amino acids discovered in the early 1990s. Over the past 15 years, a strong association between cancer cells and autotaxin production has been observed. Recent publications indicate that autotaxin and the capacity of cancer to metastasise are intimately linked. The discovery of new molecular targets in pharmacology is a mixture of pure luck, hard work and industrial strategy. Despite a crucial and desperate need for new therapeutic tools, many targets are approached in oncology, but only a few are validated and end up at the patient bed. Outside the busy domain of kinases, few targets have been discovered that can be useful in treating cancer, particularly metastatic processes. The fortuitous relationship between autotaxin and lysophosphatidic acid renders the results of observations made in the diabetes/obesity context considerably important. The literature provides observations that may aid in redesigning experiments to validate autotaxin as a potential oncology target.

Autotaxin protects MCF-7 breast cancer and MDA-MB-435 melanoma cells against Taxol-induced apoptosis.

Autotaxin (ATX) promotes cancer cell survival, growth, migration, invasion and metastasis. ATX converts extracellular lysophosphatidylcholine (LPC) into lysophosphatidate (LPA). As these lipids have been reported to affect cell signaling through their own G-protein-coupled receptors, ATX could modify the balance of this signaling. Also, ATX affects cell adhesion independently of its catalytic activity. We investigated the interactions of ATX, LPC and LPA on the apoptotic effects of Taxol, which is commonly used in breast cancer treatment. LPC had no significant effect on Taxol-induced apoptosis in MCF-7 breast cancer cells, which do not secrete significant ATX. Addition of incubation medium from MDA-MB-435 melanoma cells, which secrete ATX, or recombinat ATX enabled LPC to inhibit Taxol-induced apoptosis of MCF-7 cells. Inhibiting ATX activity blocked this protection against apoptosis. We conclude that LPC has no significant effect in protecting MCF-7 cells against Taxol treatment unless it is converted to LPA by ATX. LPA strongly antagonized Taxol-induced apoptosis through stimulating phosphatidylinositol 3-kinase and inhibiting ceramide formation. LPA also partially reversed the Taxol-induced arrest in the G2/M phase of the cell cycle. Our results support the hypothesis that therapeutic inhibition of ATX activity could improve the efficacy of Taxol as a chemotherapeutic agent for cancer treatment.

Lysophosphatidic acids, cyclic phosphatidic acids and autotaxin as promising targets in therapies of cancer and other diseases.

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently lysophosphatidic acids (LPA) have been demonstrated to act on specific G protein-coupled receptors. The widespread expression of LPA receptors and coupling to several classes of G proteins allow LPA-dependent regulation of numerous processes, such as vascular development, neurogenesis, wound healing, immunity, and cancerogenesis. Lysophosphatidic acids have been found to induce many of the hallmarks of cancer including cellular processes such as proliferation, survival, migration, invasion, and neovascularization. Furthermore, autotaxin (ATX), the main enzyme converting lysophosphatidylcholine into LPA was identified as a tumor cell autocrine motility factor. On the other hand, cyclic phosphatidic acids (naturally occurring analogs of LPA generated by ATX) have anti-proliferative activity and inhibit tumor cell invasion and metastasis. Research achievements of the past decade suggest implementation of preclinical and clinical evaluation of LPA and its analogs, LPA receptors, as well as autotaxin as potential therapeutic targets.

Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.

The extracellular lysophospholipase D autotaxin (ATX) and its product, lysophosphatidic acid, have diverse functions in development and cancer, but little is known about their functions in the immune system. Here we found that ATX had high expression in the high endothelial venules of lymphoid organs and was secreted. Chemokine-activated lymphocytes expressed receptors with enhanced affinity for ATX, which provides a mechanism for targeting the secreted ATX to lymphocytes undergoing recruitment. Lysophosphatidic acid induced chemokinesis in T cells. Intravenous injection of enzymatically inactive ATX attenuated the homing of T cells to lymphoid tissues, probably through competition with endogenous ATX and exertion of a dominant negative effect. Our results support the idea of a new and general step in the homing cascade in which the ectoenzyme ATX facilitates the entry of lymphocytes into lymphoid organs.

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia.

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-gamma 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-gamma 1 and villin, both of which participate in the initiation of protrusion.

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.

Submucosal connective tissue-type mast cells contribute to the production of lysophosphatidic acid (LPA) in the gastrointestinal tract through the secretion of autotaxin (ATX)/lysophospholipase D (lysoPLD).

Lysophosphatidic acid (LPA) is involved in a broad spectrum of biological activities, including wound healing and cancer metastasis. Autotaxin (ATX), originally isolated from a melanoma supernatant as a tumor cell motility-stimulating factor, has been shown to be molecularly identical to lysophospholipase D (lysoPLD), which is the main enzyme in the production of LPA. Although ATX/lysoPLD is known to be widely expressed in normal human tissues, the exact distribution of ATX-producing cells has not been fully investigated. In this study, we evaluated ATX/lysoPLD expression by immunohistochemical staining using a rat anti-ATX mAb in the human gastrointestinal tract and found that submucosal mast cells (MC) highly expressed this enzyme. This was confirmed by immunofluorescent double staining using mAbs to tryptase and chymase. Then, we isolated MC from human gastric tissue by an immunomagnetic method using CD117-microbeads and showed that a subpopulation of CD203c-positive MC showed positive staining for intracellular ATX/lysoPLD on flowcytometry. This was confirmed by Western blotting of the isolated cells. Moreover, a significant level of ATX/lysoPLD release could be detected in the culture supernatants of human MC by Western blot analysis. Our data suggest that submucosal MC play significant roles in various aspects of pathophysiology in the gastrointestinal tract by locally providing bioactive LPA through the production of ATX/lysoPLD.