Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) regulate numerous biological processes through interaction with signaling effectors at the cell surface. As a unique feature, GPI-APs can be released from their anchors by multi-pass GPI-specific phospholipases (types A2, C, and D) to impact signaling networks, phenotype, and cell fate; however, many questions remain outstanding. Here, we discuss and expand our current understanding of the distinct GPI-specific phospholipases, their substrates, effector pathways, and emerging physiological roles, with a focus on the six-transmembrane ecto-phospholipases GDE2 (GDPD5) and GDE3 (GDPD2). We provide structural insight into their AlphaFold-predicted inner workings, revealing how transmembrane (TM) domain plasticity may enable GPI-anchor binding and hydrolysis. Understanding lipolytic cleavage of GPI-APs adds a new dimension to their signaling capabilities and biological functions.

Secreted autotaxin (ATX) promotes tumor progression by producing the pleiotropic lipid mediator lysophosphatidic acid (LPA). In a recent Nature Cancer paper, Bhattacharyya et al. show that ATX/LPA signaling suppresses CCL11-driven infiltration of eosinophils into the pancreatic tumor microenvironment to facilitate tumor progression, thus revealing a new ATX-mediated immune escape mechanism and highlighting the antitumor potential of eosinophils.

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr−/− mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr−/−/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr−/− mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

Ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family members (ENPP1-7) have been implicated in key biological and pathophysiological processes, including nucleotide and phospholipid signaling, bone mineralization, fibrotic diseases, and tumor-associated immune cell infiltration. ENPPs are single-pass transmembrane ecto-enzymes, with notable exceptions of ENPP2 (Autotaxin) and ENNP6, which are secreted and glycosylphosphatidylinositol (GPI)-anchored, respectively. ENNP1 and ENNP2 are the best characterized and functionally the most interesting members. Here, we review the structural features of ENPP1-7 to understand how they evolved to accommodate specific substrates and mediate different biological activities. ENPPs are defined by a conserved phosphodiesterase (PDE) domain. In ENPP1-3, the PDE domain is flanked by two N-terminal somatomedin B-like domains and a C-terminal inactive nuclease domain that confers structural stability, whereas ENPP4-7 only possess the PDE domain. Structural differences in the substrate-binding site endow each protein with unique characteristics. Thus, ENPP1, ENPP3, ENPP4, and ENPP5 hydrolyze nucleotides, whereas ENPP2, ENPP6, and ENNP7 evolved as phospholipases through adaptions in the catalytic domain. These adaptations explain the different biological and pathophysiological functions of individual members. Understanding the ENPP members as a whole advances our insights into common mechanisms, highlights their functional diversity, and helps to explore new biological roles.

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

SummaryAutotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+T cellsex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

BackgroundAutotaxin (ATX) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). The expression of both ATX and LPA is elevated in most solid tumors and plasma. LPA signaling directly modulates tumor cell function and contributes to the development of the fibrotic tumor microenvironment, a mechanism by which tumors evade host immunity and impairs response to therapy. IOA-289 is a potent, orally available autotaxin inhibitor which is being developed as a novel treatment of solid tumours burdened with a high degree of fibrosis.MethodsInhibition of ATX activity in human plasma was determined by measuring reduction in LPA species as quantified by LC-MS/MS. In vitro activity on biomarkers of fibrosis was assessed using the BioMAP screen and fibroblast cell cultures. T cell migration was measured using 48-well chemotaxis chambers. PK/PD studies were performed following a single oral dose of IOA-289 in mice, and plasma LPA was used as a PD biomarker. In vivo efficacy was studied in two models of breast cancer, 4T1 and E0771. Bioinformatics used TCGA and GTEX publicly available datasets.ResultsIOA-289 inhibits plasma LPA18:2 with an IC50 of 36nM, with similar results for other LPA species. IOA-289 inhibited fibrosis relevant factors in the BioMAP phenotypic screen, including sIL-6, MCP-1, αSMA, collagen-III, and sVEGF. In further studies, IOA-289 inhibited the secretion of PAI-1 and IL-6 by stimulated fibroblasts. LPA and cancer cell conditioned media inhibited T cell chemotaxis in vitro and the effect was overcome in the presence of IOA-289. The efficacious human dose of IOA-289 was determined following PK/PD studies using plasma LPA as a biomarker of response to ATX inhibition. In vivo studies showed that IOA-289 inhibited metastasis of 4T1 cells, enhanced the infiltration of T cells into 4T1 s.c. implanted tumors and prevented the growth of primary, orthotopically implanted E0771 tumors. Bioinformatics analysis demonstrated elevated ATX expression in pancreatic cancer (PDAC), and PDAC patient plasma showed a correlation of ATX levels with CA-19-9.ConclusionsThe ATX/LPA pathway represents a novel target for anti-cancer therapy with actions on the tumor, immune cell and stromal environment. IOA-289 is a highly potent and selective inhibitor of ATX with demonstrated monotherapy activity in cancer models. Based on the mechanism of action we are investigating combinations of IOA-289 with chemotherapy, immunotherapy and novel agents in ongoing preclinical studies. An acceptable safety and PK profile support the clinical development of IOA-289 which is currently in a phase I clinical trial.Ethics ApprovalThe 4T1 study was approved by The University Claude Bernard Lyon 1 Ethics Board; approval number DR2014-38 (vM). The E0771 study was reviewed and approved by the Institutional Animal Care and Use Committee of the contract research organization (Covance, Ann Arbor, MI, USA), an AAALAC International accredited program.

GDE2 (also known as GDPD5) is a multispanning membrane phosphodiesterase with phospholipase D-like activity that cleaves select glycosylphosphatidylinositol (GPI)-anchored proteins and thereby promotes neuronal differentiation both in vitro and in vivo. GDE2 is a prognostic marker in neuroblastoma, while loss of GDE2 leads to progressive neurodegeneration in mice; however, its regulation remains unclear. Here, we report that, in immature neuronal cells, GDE2 undergoes constitutive endocytosis and travels back along both fast and slow recycling routes. GDE2 trafficking is directed by C-terminal tail sequences that determine the ability of GDE2 to cleave GPI-anchored glypican-6 (GPC6) and induce a neuronal differentiation program. Specifically, we define a GDE2 truncation mutant that shows aberrant recycling and is dysfunctional, whereas a consecutive deletion results in cellsurface retention and gain of GDE2 function, thus uncovering distinctive regulatory sequences. Moreover, we identify a C-terminal leucine residue in a unique motif that is essential for GDE2 internalization. These findings establish a mechanistic link between GDE2 neuronal function and sequence-dependent trafficking, a crucial process gone awry in neurodegenerative diseases. This article has an associated First Person interview with the first author of the paper.

Lysophosphatidic acid (LPA) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid Receptors [54, 18, 80, 125]) are activated by the endogenous phospholipid LPA. The first receptor, LPA1, was identified as ventricular zone gene-1 (vzg-1) [39], leading to deorphanisation of members of the endothelial differentiation gene (edg) family as other LPA receptors along with sphingosine 1-phosphate (S1P) receptors. Additional LPA receptor GPCRs were later identified. Gene names have been codified as LPAR1, etc. to reflect the receptor function of proteins. The crystal structure of LPA1 was solved and demonstrates extracellular LPA access to the binding pocket, consistent with proposed delivery via autotaxin [12]. These studies have also implicated cross-talk with endocannabinoids via phosphorylated intermediates that can also activate these receptors. The identified receptors can account for most, although not all, LPA-induced phenomena in the literature, indicating that a majority of LPA-dependent phenomena are receptor-mediated. Binding affinities of unlabeled, natural LPA and AEAp to LPA1 were measured using backscattering interferometry (pKd = 9) [81, 102]. Binding affinities were 77-fold lower than than values obtained using radioactivity [124]. Targeted deletion of LPA receptors has clarified signalling pathways and identified physiological and pathophysiological roles. Independent validation by multiple groups has been reported in the peer-reviewed literature for all six LPA receptors described in the tables, including further validation using a distinct read-out via a novel TGFα "shedding" assay [47]. LPA LPA has been proposed to be a ligand for GPCR35 [92], supported by a recent study revealing that LPA modulates macrophage function through GPR35 [53]. However CXCL17 is reported to be a ligand for GPR35/CXCR8 [74]. Moreover, LPA has also been described as an agonist for the transient receptor potential (Trp) ion channel TRPV1 [85] and TRPA1 [57]. All of these proposed non-GPCR receptor identities require confirmation and are not currently recognized as bona fide LPA receptors.

Autotaxin (ATX) is a secretory lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA) and thereby regulates diverse cellular functions such as migration, survival, proliferation and metabolism. Adipocytes are known as significant source of systemic LPA under physiological conditions. Evidence suggests that LPA regulates glucose levels, but the role of ATX in glucose homeostasis is unknown. Here we investigated the role of hepatocellular ATX in glucose homeostasis using mice with heptocyte-specific Atx knockout (Atxh-/h-) generated by crossing mice with floxed Atx gene and AlbCre mice. The data indicate that the blood levels of LPA in Atxh-/h- mice were reduced by approximately 50% as compared to AlbCre mice. Basal glucose levels were similar in normally fed Atxh-/h- and AlbCre mice, whereas Atxh-/h- mice showed an increased disposal rate of injected glucose from the blood. Atxh-/h- mice showed a stronger increase of the blood glucose concentration in response to glucagon, a higher gluconeogenic capacity, and higher fasting glucose levels. This study provides the first evidence that hepatocellular ATX is the major source of systemic LPA under physiological conditions. It affects glucometabolism by two divergent mechanisms: by inhibiting glucose removal from the circulation and lowering the glycemic effect of glucagon, implying a novel role in hepatic glucose regulation.

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with 1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

Introduction Glycerophosphodiester phosphodiesterase 2 (GDE2) is a multi-pass membrane protein that promotes neuronal differentiation through the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins at the cell surface. High GDE2 expression is associated with favourable outcome in neuroblastoma, while loss of GDE2 in mice leads to neuronal pathologies similar to human neurodegenerative diseases. Thus, enhancing GDE2 activity could be an attractive therapeutic strategy for neuroblastoma and related pathologies. However, the regulation of GDE2 is poorly understood. Material and methods We employed TIRF microscopy to study GDE2 subcellular localization in neuronal cell lines. Membrane internalisation of GDE2 was detected by confocal microscopy, and confirmed biochemically by biotin labelling assays. To determine the nature of the GDE2-containg intracellular compartments, we examined co-localization of GDE2 with endosome markers by both confocal microscopy and immunoprecipitation assays. Results and discussions When expressed at relatively low levels in N1E-115, Neuro2A and SH-SY5Y neuronal cell lines, GDE2 localises to discrete membrane microdomains, as well as in high-turnover intracellular vesicles. We corroborated this intracellular trafficking by biotin labelling assays, which showed that GDE2 undergoes constitutive endocytosis and recycling back to the plasma membrane in a serum-independent manner. In addition, GDE2 was found to co-localise with well-established early-endosome markers, namely EEA1 and Rab5, indicating that GDE2 internalises from the plasma membrane. GDE2 also localised partially to Rab7-positive late endosomes, but was hardly detected in lysosomes (LAMP1, LysoTracker). Furthermore, GDE2 localised to Rab11-positive recycling endosomes, pinpointing the long recycling pathway. When co-expressed with labelled ubiquitin, GDE2 was heavily poly-ubiquitinated, which takes place non-specifically in the four cytosolic lysine residues of GDE2. Lastly, sequential truncations in the N- and C-terminal cytosolic tails of GDE2 highlighted a 10-amino acid stretch that potentially regulates intracellular trafficking. Conclusion Here we report that, in neuronal cells, GDE2 is constitutively internalised and undergoes endocytic recycling along both the short and, in particular, the long recycling pathways. This process appears to be regulated by a stretch of 10 residues in the cytosolic C-terminal tail, which could be related to non-specific ubiquitin ligation.

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1(-/-) and MT mice, suggesting B cells as a major ATX-producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El-Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Introduction The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that promotes tissue remodelling and tumour progression. uPAR is highly expressed in many cancers and correlates with poor prognosis. uPAR mediates matrix degradation through protease recruitment and enhances tumour cell migration and signalling through vitronectin binding and interaction with integrins. Full-length uPAR is released from the cell surface, resulting in a soluble form (suPAR), but the mechanism and functional significance of uPAR shedding have been elusive. Material and methods Cell biological and biochemical assays; super-resolution microscopy; homology modelling; knockdown/knockout studies; xenograft model; patient survival analysis. Results and discussions We find that uPAR is released from the cell surface through GPI-anchor cleavage by a multi-pass membrane glycerophosphodiesterase, termed GDE3, acting as a GPI-specific phospholipase C (PLC), leading to loss of uPAR function. By contrast, GDE3’s closest relative GDE2 fails to cleave uPAR. By shedding uPAR from the cell surface, GDE3 abrogates uPAR-driven cell adhesion, spreading and lamellipodia formation on vitronectin. In breast cancer cells, high GDE3 expression depletes uPAR form distinct basolateral membrane microdomains resulting in a less transformed phenotype, as revealed by reduced matrix degradation, cell motility and colony formation. Furthermore, elevated GDE3 expression reduces tumour progression in a xenograft model and correlates with higher survival probability in breast cancer patients. Our results establish GDE3 as a cell-intrinsic GPI-specific PLC that sheds uPAR to attenuate malignant cell behaviour. Conclusion GDE3 is the first mammalian GPI-specific PLC that negatively regulates the uPAR signalling network, thereby suppressing the malignant phenotype of uPAR-positive cancer cells. Future studies should address how GDE3 activity and its substrate specificity are regulated.

CLIC4 is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by G12/13-coupled receptor agonists and then partly co-localizes with β1 integrins. Receptor-mediated CLIC4 translocation depends on actin polymerization, but the mechanism and functional significance of CLIC4 trafficking are unknown. Here we show that RhoA activation by either LPA or EGF is necessary and sufficient for CLIC4 translocation, with a regulatory role for the RhoA effector mDia2, an inducer of actin polymerization. We find that CLIC4 directly interacts with the G-actin-binding protein Profilin-1 via conserved residues that are required for CLIC4 trafficking and lie in a concave surface. Consistently, silencing of Profilin-1 impaired CLIC4 trafficking induced by either LPA or EGF. CLIC4 knockdown promoted the formation of long integrin-dependent filopodia, a phenotype rescued by wild-type CLIC4 but not by trafficking-incompetent CLIC4(C35A). Our results establish CLIC4 as a Profilin-1-binding protein and suggest that CLIC4 translocation provides a feedback mechanism to modulate mDia2/Profilin-1-driven cortical actin assembly and membrane protrusion.