Elisa Matas-Rico

LPA Is a Chemorepellent for B16 Melanoma Cells: Action through the cAMP-Elevating LPA5 Receptor

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA1–6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18∶1) being 10-fold more potent than acyl-LPA(18∶1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA2, LPA5 and LPA6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA5 receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

Steroid binding to Autotaxin links bile salts and lysophosphatidic acid signalling.

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

Loss of lysophosphatidic acid receptor LPA1 alters oligodendrocyte differentiation and myelination in the mouse cerebral cortex

Lysophosphatidic acid (LPA) is an intercellular signaling lipid that regulates multiple cellular functions, acting through specific G-protein coupled receptors (LPA1–6). Our previous studies using viable Malaga variant maLPA1-null mice demonstrated the requirement of the LPA1 receptor for normal proliferation, differentiation, and survival of the neuronal precursors. In the cerebral cortex LPA1 is expressed extensively in differentiating oligodendrocytes, in parallel with myelination. Although exogenous LPA-induced effects have been investigated in myelinating cells, the in vivo contribution of LPA1 to normal myelination remains to be demonstrated. This study identified a relevant in vivo role for LPA1 as a regulator of cortical myelination. Immunochemical analysis in adult maLPA1-null mice demonstrated a reduction in the steady-state levels of the myelin proteins MBP, PLP/DM20, and CNPase in the cerebral cortex. The myelin defects were confirmed using magnetic resonance spectroscopy and electron microscopy. Stereological analysis limited the defects to adult differentiating oligodendrocytes, without variation in the NG2+ precursor cells. Finally, a possible mechanism involving oligodendrocyte survival was demonstrated by the impaired intracellular transport of the PLP/DM20 myelin protein which was accompanied by cellular loss, suggesting stress-induced apoptosis. These findings describe a previously uncharacterized in vivo functional role for LPA1 in the regulation of oligodendrocyte differentiation and myelination in the CNS, underlining the importance of the maLPA1-null mouse as a model for the study of demyelinating diseases.

Glycerophosphodiesterase GDE2 Promotes Neuroblastoma Differentiation through Glypican Release and Is a Marker of Clinical Outcome

Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. A better understanding of neuroblastoma differentiation is essential for developing new therapeutic approaches. GDE2 (encoded by GDPD5) is a six-transmembrane-domain glycerophosphodiesterase that promotes embryonic neurogenesis. We find that high GDPD5 expression is strongly associated with favorable outcome in neuroblastoma. GDE2 induces differentiation of neuroblastoma cells, suppresses cell motility, and opposes RhoA-driven neurite retraction. GDE2 alters the Rac-RhoA activity balance and the expression of multiple differentiation-associated genes. Mechanistically, GDE2 acts by cleaving (in cis) and releasing glycosylphosphatidylinositol-anchored glypican-6, a putative co-receptor. A single point mutation in the ectodomain abolishes GDE2 function. Our results reveal GDE2 as a cell-autonomous inducer of neuroblastoma differentiation with prognostic significance and potential therapeutic value.

Neuronal differentiation through GPI-anchor cleavage

The cell surface of virtually all cell types harbors a great variety of glycosylphosphatidylinositol (GPI)-anchored proteins with diverse biological functions. GPI-anchoring is a complex posttranslational modification that anchors proteins in the outer leaflet of the plasma membrane. GPI-anchoring may allow for lateral movement and some structural flexibility of membrane proteins, but otherwise the biological function of GPI anchors has long remained a mystery. Certain GPI-anchored proteins are involved in signal transduction but, by their very nature, they lack intrinsic signaling capacity. Instead, they must rely on direct communication with nearby partners. For example, by binding growth factors or/and through lateral interactions with transmembrane receptors, GPI-anchored proteins can function as co-receptors to modulate signaling pathways. Some GPIanchored proteins are released from their anchor and detected in body fluids, implying involvement of putative GPI-specific phospholipases. But the mechanism and physiological significance, if any, of GPI-anchor cleavage has long remained unexplored. A unique GPI-specific phospholipase D (GPI-PLD) has often been assumed to release GPI-anchored proteins, but this enzyme does not function on native membranes. Recent studies have advanced the field by showing that selective cleavage of GPI-anchored proteins by an ecto-phosphodiesterase, termed GDE2, leads to neuronal differentiation. GDE2 (encoded by GDPD5) is a member of the glycerophosphodiesterase family and characterized by 6 transmembrane domains and a large catalytic ectodomain. GDE2 was originally identified as a retinoic acid-inducible gene that promotes spinal motor neuron differentiation by inhibiting Notch signaling in adjacent neuronal progenitors. Mechanistically, the Notch transmembrane receptor is activated by binding membrane-bound ligands on contacting cells. GDE2 was reported to cleave and release a GPI-anchored Notch regulator, termed RECK, that normally inhibits protease-dependent shedding of a Notch ligand. By releasing RECK, GDE2 inactivates the ligand-Notch signaling axis and thereby promotes differentiation in contacting neuronal cells (Fig. 1). Thus, in the developing spinal cord, GDE2 promotes neurogenesis in a noncell-autonomous manner, requiring cell-cell contact. In our laboratory, GDE2 was identified and tested as a candidate ecto-phospholipase D that might produce the lipid mediator lysophosphatidic acid (LPA) (unpublished work). LPA has long been known to induce growth cone collapse and neurite retraction in immature neuronal cells through activation of RhoA, one of the master small-GTPases that regulate the actin cytoskeleton and cellular phenotype. Unexpectedly, however, GDE2 was found to oppose rather than promote LPA-induced neurite retraction and to induce phenotypic differentiation of neuroblastoma cells. In this case, GDE2 acted in a cell-autonomous manner, not requiring cell-cell contact. Through overexpression and knockdown studies, GDE2 was found to initiate a neuronal differentiation program that involves a change in the Rac-RhoA activity balance leading to cell spreading, neurite outgrowth, inhibition of RhoA-driven neurite retraction and reduced cell motility in both mouse and human neuroblastoma cells. In addition, GDE2 was found to regulate multiple differentiation-associated genes, including the transcription factor NEUROD1, a key player in nervous system development. We discovered that GDE2 promotes neuronal differentiation through GPI-anchor cleavage of the heparan sulfate proteoglycan glypican-6 (GPC6) (Fig. 1). While little is known about the normal biological function of GPC6, heparan sulfate proteoglycans such as the glypicans play key roles in neurodevelopment, being capable of recruiting and distributing growth factors at the cell surface, dictated by the structure of their glycosaminoglycan chains. Glypican release may thus result in growth factor redistribution with either stimulatory or inhibitory effects on receptor signaling pathways. As we discussed (ref. 3), glypicans may also serve as membrane-tethered ligands in their own right by directly interacting with transmembrane receptors, such as the type-II receptor tyrosine phosphatases. In the latter scenario, GDE2-induced GPC6 release could trigger phosphotyrosine-based signaling events to impact neuronal differentiation, an attractive hypothesis that warrants further study. The new findings are of clinical relevance, since elevated GDPD5 expression was strongly associated with improved

Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.

Glycerophosphodiesterase GDE2/GDPD5 affects pancreas differentiation in zebrafish

Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One regulator of the Notch pathway is GDE2 (or GDPD5), a transmembrane ecto-phosphodiesterase that cleaves GPI-anchored proteins at the plasma membrane, including a Notch ligand regulator. Here we report that Gdpd5-knockdown in zebrafish embryos leads to developmental defects, particularly, impaired motility and reduced pancreas differentiation, as shown by decreased expression of insulin and other pancreatic markers. Exogenous expression of human GDE2, but not catalytically dead GDE2, similarly leads to developmental defects. Human GDE2 restores insulin expression in Gdpd5a-depleted zebrafish embryos. Importantly, zebrafish Gdpd5 orthologues localize to the plasma membrane where they show catalytic activity against GPI-anchored GPC6. Thus, our data reveal functional conservation between zebrafish Gdpd5 and human GDE2, and suggest that strict regulation of GDE2 expression and catalytic activity is critical for correct embryonic patterning. In particular, our data uncover a role for GDE2 in regulating pancreas differentiation.

GDE2/GDPD5 in neuroblastoma

Neuroblastoma is an embryonal cancer of early childhood that originates from the developing sympathetic nervous system, usually arising in the adrenal gland. It is the most common malignancy during infancy, accounting for about 10% of childhood cancer mortality, and there is an urgent need for new therapeutic approaches. Neuroblastoma is a highly heterogeneous disease, characterized by relatively few recurrent somatic mutations [1]. In some cases, at very young age, the tumor can undergo spontaneous regression through poorly understood mechanisms [2]. In many cases, however, neuroblastoma progresses into a high-risk metastatic disease. Neuroblastoma originates from the neural crest and is characterized by aberrant growth and impaired neuronal differentiation of immature neuroblasts. In general, patient survival depends on the degree of neuronal differentiation in the primary tumors and is inversely correlated with a motile phenotype. Some high-risk neuroblastomas are associated with mutations in Rho GTPase pathway genes that normally regulate the differentiated phenotype and are implicated in neuritogenesis and cell motility [3]. Unfortunately, treatment options are very limited for high-risk neuroblastoma. To identify new therapeutic targets, it will be essential to gain a better understanding of neuroblastoma differentiation at the molecular level. Indent in a recent study, we have uncovered a previously unknown mechanism of neuroblastoma differentiation with prognostic significance [4]. The new mechanism involves the action of GDE2 (encoded by GDPD5), a transmembrane glycosylphosphatidylinositol (GPI)specific glycerophosphodiesterase and member of a larger GDE family [5]. GDE2 was earlier reported to promote the differentiation of spinal motor neurons by cleaving a GPI-anchored Notch ligand regulator, resulting in down-regulation of Notch signaling in adjacent neural progenitors [6]. In our study, GDE2 induced neuroblastoma cell differentiation in a cell-autonomous manner through cleavage and release of a GPI-anchored heparan sulfate proteoglycan, termed glypican-6 (GPC6). By releasing GPC6, GDE2 was found to initiate a cellintrinsic differentiation program involving altered Rac/ Rho activity and transcription of multiple differentiationassociated genes. Phenotypically, GDE2-induced GPC6 release led to increased cell-matrix adhesion, cell spreading, neurite outgrowth, blocked neurite retraction, and reduced cell motility [4], as illustrated in Figure 1. Strikingly, high expression of GDPD5 was strongly associated with favorable clinical outcome, while low GDPD5 expression correlated with poor outcome in independent patient cohorts [4] (Figure 1). Conversely, high expression of GDE2 substrate GPC6 was associated with poor prognosis. GDPD5 maps to chromosome 11q13, a region often showing loss of heterozygosity in high-risk neuroblastoma. Based on these results, GDPD5 would qualify as a potential tumor suppressor gene. However, no mutations or deletions were detected in the tumor samples analyzed [4]. Furthermore, GDPD5 expression in patients with an 11q deletion was similar to those with a normal chromosome 11, neither was there an inverse correlation with amplification of the MYCN oncogene. While the

PO-228 Regulation of gde2 membrane localization and trafficking

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.

Negative regulation of uPAR activity by a GPI-specific phospholipase C

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins and other receptors. Full-length uPAR is released from the cell surface, but the mechanism and functional significance of uPAR release remain obscure. Here we show that transmembrane glycerophosphodiesterase GDE3 is a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of the proteolytic and non-proteolytic activities of uPAR. In breast cancer cells, high GDE3 expression depletes endogenous uPAR resulting in a less transformed phenotype, correlating with higher survival probability in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, more generally, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.