Developmental Changes in the Expression of Lysophosphatidic Acid Receptor 4 in the Mouse Brain

Integrated Human Evaluation of the Lysophosphatidic Acid Pathway as a Novel Therapeutic Target in Atherosclerosis

751a Opt-80 Versus Vancomycin in Clostridium difficile Infection: Results of a Randomized Clinical Trial

751c Effectiveness of the Third Eye Retroscope for Detecting Polyps in the Right Colon

Breast cancer(BC) generally exhibits poor prognosis owing to its invasive and metastatic characteristics and is the leading cause of cancer-related deaths in women worldwide. Lysophosphatidic acid receptor2 (LPA2) and hypoxia inducible factor-1α (HIF-1α) were found to be correlated with BC invasion and metastasis, respectively. However, the effect of LPA2 on BC in Chinese women has not yet been reported, nor have the overall survival and prognostic significance of LPA2 or its association with HIF-1α in BC. In the present study, we assessed the effect of LPA2 on HIF-1α expression, on overall survival and prognostic significance in BC in Chinese women, and on cell proliferation, migration and invasion in MCF-7 BC cells invitro. The data showed that LPA2 and HIF-1α protein expression levels were higher in the BC tissue specimens and that LPA2 expression was significantly associated with menopausal status (postmenopausal), nodal metastasis and tumor-node-metastasis (TNM) stage, whereas HIF-1α expression was significantly associated with estrogen receptor status, nodal metastasis and TNM stage. Furthermore, LPA2 and HIF-1α expression were positively correlated (r=0.562; P<0.001). LPA2 and HIF-1α protein levels were associated with shorter overall patient survival according to univariate analysis (log-rank test; P<0.001), and nodal metastasis, TNM stage, LPA2 and HIF-1α expression were independent prognostic predictors in patients as determined by multivariate analysis (P<0.05). Invitro, the data suggested that LPA2 affected HIF-1α expression and LPA2 overexpression or knockdown resulted in increased or inhibited tumor cell proliferation, migration and invasion, respectively. In conclusion, our data demonstrated that LPA2 expression was associated with HIF-1α expression and that a high level of LPA2 was associated with shorter overall survival and was an independent prognostic predictor for BC in Chinese women. Furthermore, LPA2 showed the ability to regulate the cell proliferation, migration and invasion of BC cells.

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

BackgroundThe aim of this study was to evaluate lysophosphatidic acid receptor-2 (LPA2) and Krüppel-like factor 5 (KLF5) protein expression in gastric adenocarcinoma and their correlation with patient clinicopathological characteristics and prognosis.Material/MethodsFifty-one gastric adenocarcinoma tissue samples, 21 gastric intraepithelial neoplasia (GIN) samples, and 13 normal gastric tissue samples were collected to test for LPA2 and KLF5 expression by tissue microarray and immunohistochemistry assay. LPA2 and KLF5 positive expression rate between gastric adenocarcinoma, GIN, and normal gastric tissue were compared. The relationship between LPA2 expression, KLF5 expression, and patients’ clinicopathological characteristics and prognosis were evaluated.ResultsThe positive expression rate of LPA2 and KLF5 were statistical different in gastric adenocarcinoma, GIN, and normal gastric tissue (P<0.05). LPA2 positive expression was associated with tumor invasion depth, Lauren type, vascular invasion, local lymph node metastasis, and clinical stage (P<0.05). There was no correlation between LPA2 expression (hazard ratio [HR]=1.84, 95% confidence interval [CI]: 0.89–3.80, P>0.05), KLF5 expression (HR=1.13, 95% CI: 0.53–2.36, P>0.05), and gastric cancer patients’ overall survival.ConclusionsLPA2 and KLF5 protein expressions were differently expressed in gastric adenocarcinoma, GIN, and normal gastric tissue, and differences were correlated with patients’ clinical characteristic. However, LPA2 and KLF5 expressions were not correlated with the patients’ prognosis.

The central nervous system (CNS) is characterized by a remarkably elaborate cellular architecture comprising large numbers of glial and neuronal cells with enormous functional diversity, organized into highly complex and specific networks. During development, the various neural cell types must first be correctly specified, then assume their appropriate positions through carefully choreographed cellular migration, and finally establish and refine their functional connections, often over long distances. The end result of all these processes is an extraordinarily intricate anatomical structure, able to receive, integrate, and store information and orchestrate appropriate responses. The molecular mechanisms of the developing CNS are only poorly understood, and due to its outstanding complexity in adulthood, only little regeneration or repair mechanisms occur. The wiring of the normal adult CNS has classically been seen as stable and permanent, but this is not completely true. The neuronal network of the adult CNS does retain a limited capacity for growth and structural change. A large number of regeneration factors have been identified in the recent past, but a general solution for the induction of repair mechanisms after damage is still missing. So far, the main focus of neural regeneration research has been based on investigating proteins and their signaling cascades. However, the field of lipidomics has been successful in providing information on the crucial involvement of bioactive lipids, such as lysophosphatidic acid (LPA) or sphingosine-1-phosphate, as signaling molecules and regulators in physiological and pathophysiological neuronal processes. These novel findings raise the question, whether neuronal lipid metabolism could be the future target for therapeutic approaches addressing neural regeneration. One successful example of such a therapy approach is the drug fingolimod (Gilenya®). Marketed in 2010 as an oral treatment for relapsing-remitting multiple sclerosis, it has become the first drug to modulate the sphingolipid signaling pathway. Fingolimod is a substrate of sphingosine kinases, generating fingolimod phosphate which acts as an agonist at sphingosine-1-phosphate receptors. However, this interaction prompts the internalization of the receptors from the membrane, resulting in functional antagonistic action of fingolimod. It was initially discovered for its immunomodulative effects, preventing experimental autoimmune encephalitis in rats by reducing the number of lymphocytes in the blood and CNS. However, pathology improving effects were also observed in lymphocyte-independent multiple sclerosis models, indicating additional CNS specific actions of fingolimod (reviewed in Brinkmann et al. (2010)). Nevertheless, traumatic injuries of the CNS remain a major challenge and no effective drugs for stimulating regeneration processes are so far in use. The extracellular environment, however, allows neurite elongation only under specific molecular conditions. Molecules involved in neurite outgrowth, such as semaphorins, netrins and ephrins, are able to transduce outgrowth-regulating signals to elongate axons via specific receptors. A phospholipid–rich environment normally inhibits outgrowth of fibers. The bioactive lipid LPA is present in the extracellular space and acts via the LPA receptors involving intracellular activation of small G-proteins that mediate neurite retraction (Yung et al., 2014). Crack et al. showed elevated levels of the pro-inflammatory LPA in cerebrospinal fluid samples from patients with traumatic brain injuries and of mice subjected to control cortical impact injury (Crack et al., 2014). Interestingly, blocking LPA with a LPA-specific antibody improved the neurological outcome in control cortical impact injury mice, by reducing lesion size and behavioral deficits (Crack et al., 2014). These findings suggest a substantial role of LPA in restraining neural regeneration processes in the adult CNS after injury, making it a highly interesting target lipid. LPA can bind to at least six known G-protein coupled receptors (LPA1-6). Each receptor can couple with multiple types of G proteins (G12/13, Gi/o, Gq/11, Gs) to activate a range of downstream signaling pathways inducing pleiotropic effects inside the cell. For example, activation of phospholipase C, Rho, and Akt, and phosphatidylinositol 3-kinase pathways or inhibition of adenylyl cyclase (reviewed in Yung et al. (2014)). LPA receptor gene products are detectable in most mammalian tissues (reviewed in Yung et al. (2014)). In our recent study, we showed the dynamic temporal and spatial expression of LPA1, LPA2, LPA4 and LPA6 receptors in the developing mouse brain and in differentiation of neuronal cells (Suckau et al. (2019) and Table 1). This dynamic receptor expression proposes a significant role of LPA signaling during fundamental neurodifferentiation processes, like astrogenesis and oligogenesis, axon and dendrite growth or synapse formation and maturation. With this dynamic expression pattern, a highly complex regulation mechanism is generated that further complicates the investigation of neuronal LPA metabolism. The LPA-induced effects may result from differences in concentration and differential expression of various LPA receptor subtypes. Kingsbury et al. showed that LPA exposure to cortical hemisphere cultures induces folding and widening of the cerebral wall, which was absent in cortical hemispheres of LPA1/LPA2 double-null-mice, indicating a receptor mediated effect (Kingsbury et al., 2003). Zheng et al. on the other hand demonstrated that, depending on the concentration, LPA can act as both a survival and an apoptotic factor in cultured cortical neurons (Zheng et al., 2004).Table 1: Reported mRNA expression of lysophosphatidic acid (LPA) receptors and plasticity-related genes (PRGs) in adult mouse brain areas and murine brain cell typesThese inconsistent results demonstrate the complexity and ubiquity of the LPA metabolism during neuronal de- and regeneration processes. It also stresses that more research on the underlying fundamental mechanisms is needed and that an overall understanding is not yet in sight. Moreover, for therapeutic approaches targeting the LPA metabolism, this deeper understanding is vitally important, as due to the ubiquitous actions of LPA, severe side effects can occur, and these must be more assessable. The complexity of the regulation mechanisms might represent the largest problem in intervention of LPA metabolism. This begins right from LPA synthesis: it can be generated through different metabolic pathways with two major routes of synthesis. One of them is the conversion of lysophospholipids, like lysophosphatidylcholine, lysophosphatidylethanolamine or lysophosphatidylserine via enzymatic action of Autotaxin. In the other one, LPA is derived from membrane phospholipids trough the actions of phospholipases. Consequently, LPA synthesis involves the conversion of precursor phospholipids, like phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine and generates lysophospholipids and phosphatidic acid as intermediate lipid products (reviewed in Yung et al. (2014)). These phospholipids are also involved in other cellular processes and must be considered when interfering with LPA metabolism. As already pointed out, an important regulation mechanism is the localization and composition of the LPA receptor molecules. As we showed in our recent study, the expression of this high number of specific receptors is a dynamic and complex regulation tool, used for controlling LPA actions over temporal processes. The expression of LPA receptors changes depending on the developmental stage of the mouse brain. We detected only LPA1, LPA2, LPA4, and LPA6 receptor mRNA transcripts in the developing mouse brain, with different dynamic expression patterns. LPA3 and LPA5 meanwhile remained below the detection level (Suckau et al. (2019) and Table 1). To give an example, the LPA2 receptor showed high expression levels in all examined brain regions until birth, followed by an expression decrease except for in the hippocampus region. The hippocampal formation is involved in learning and memory, and here the LPA2 receptor remained at a high expression level until adulthood (Suckau et al., 2019). These findings are consistent with others, which show that the LPA2 receptor is presynaptically localized and plays an important role in the modulatory control of hippocampal excitability (Trimbuch et al., 2009). We also examined LPA receptor expression in the maturation of different brain cells by analyzing mRNA expression in primary cultured cells. All four detected receptors were expressed in primary cultured neurons and increased expression during the maturation process, with LPA6 showing the highest expression levels. LPA1 and LPA6 mRNA was strongly detectable in cultured astrocytes and only LPA6 showed high expression in cultured microglia. LPA1 receptor expression increased during maturation of cultured oligodendrocytes, whereas the other three receptors were expressed weakly or not at all (Suckau et al. (2019) and Table 1). The balancing and interfering of LPA signaling could be mediated by receptor inactivation, or by metabolizing and caging of its ligand. The latter is controlled by lipid phosphate phosphatases (LPPs), an enzyme family which is not neuron-specifically expressed. These ecto-phosphatases can control the extracellular availability and thus the signaling of LPA and other phospholipids and can in turn also be regulated by their expression pattern. A structural homologue to LPPs and a highly brain-specific class of proteins, the plasticity-related genes (PRGs), were shown to be involved in both regeneration processes and attenuation of LPA-induced effects (reviewed in Bräuer and Nitsch (2008)). Five PRGs have been identified so far, but their distinct roles are understood partially or not at all. Nevertheless, individual expression patterns during brain development in mice have given rise to the assumption that PRGs have different regulatory mechanisms and neuronal functions in the CNS. They interfere with lipid phosphate signaling through various mechanisms (Bräuer and Nitsch (2008), Velmans et al. (2013) and Table 1). PRG1 can enhance axon outgrowth during development and after appearance of lesions, and reduces LPA-induced axon collapse (Bräuer and Nitsch, 2008). It also modulates the LPA-mediated control of neuronal transmission specifically at glutamatergic synapsis via the presynaptic LPA2 receptor (Trimbuch et al., 2009). However, while phosphatase activity has been shown in LPPs, PRGs lack critical amino acids within the conserved domains. This indicates that PRGs are not able to dephosphorylate LPA by the same mechanism that has been proposed for the LPPs. Another member of the family, PRG5, promotes spine formation in primary cultured hippocampal neurons, proposing a specific role in neurodifferentiation processes that are also essential for effective neural regeneration (Coiro et al., 2014). Brain trauma, cancer and chronic inflammatory diseases leave irreparable damage to the CNS with only limited therapeutical options. Modulating LPA receptor activity can be a tool for addressing the problem of neural regeneration and previous results point to a high number of opportunities. On the other hand, LPA metabolism is characterized by high complexity and a multitude of regulation mechanisms that are still far from being understood. Thus, a key understanding of LPA induced processes and regulation mechanisms is of vital importance before lipid-mediated therapies can be expanded and used as a reliable and effective tool in neural regeneration. The authors thank Eric Ahlberg for proofreading the paper as a native speaker.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

The Absence of LPA2 Attenuates Tumor Formation in an Experimental Model of Colitis-Associated Cancer

Mammary Tumorigenesis through LPA Receptor Signaling

Non-Edg family lysophosphatidic acid (LPA) receptors

Differential anatomical and cellular expression of lysophosphatidic acid receptor 1 in adult mouse brain

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Lysophosphatidic acid receptor 1 (LPAR1) has been identified as a biomarker in various cancer types. However, its biological function in papillary thyroid carcinoma (PTC) remains unknown. LPAR1 was identified as a key regulator of epithelial-mesenchymal transition (EMT) in PTC cells through bioinformatics analysis of TCGA and GEO datasets. PPI analysis and correlation with immune infiltrates were also conducted. LPAR1 expression was evaluated using Gepia2 and GTEx, and miRNA target gene prediction was done with multiMiR. To assess the expression of LPAR1, we extracted total RNA from both the BCPAP cell line and the normal human thyroid epithelial cell line Nthy-ori 3-1. The levels of LPAR1 expression were then measured using quantitative real-time polymerase chain reaction (qRT-PCR) in the BCPAP cell line, with a comparison to the Nthy-ori 3-1 cell line. 1081 genes were upregulated, and 544 were downregulated compared to normal tissue. LPAR1 was identified as a key candidate by analyzing the TCGA and GEO datasets. PPI data analysis showed interactions with metastasis-related proteins. Functional enrichment analysis indicated involvement in signaling pathways like phospholipase D and actin cytoskeleton regulation. LPAR1 expression correlated positively with immune infiltrates such as CD4+ T cells, macrophages, neutrophils, and myeloid dendritic cells but negatively with B cells. Additionally, miR-221-5p was predicted to target LPAR1 in PTC. Furthermore, our experimental data demonstrated that LPAR1 was under-expressed in the PTC cell line compared to the nonmalignant one (P < .01). LPAR1 suppresses metastasis and is linked to EMT, as evidenced by the decreased LPAR1 expression and increased miR-221-5p in PTC. This suggests its potential as a biomarker for diagnosis and prognosis and as a therapeutic target for EMT.

Objective To evaluate the differential expression of lysophosphatidic acid receptor (LPAR) in breast cancer(BC), and its relationship with clinicopathological features of BC patients. Methods The qRT-PCR and immunohistochemical staining were used to detect the LPAR expression in 37 normal tissues, 55 benign disease tissues and 82 BC tissues, besides, the correlation of LPAR expression with clinicopathological data was also analyzed. Results The expression levels of LPAR2 and LPAR3 mRNA and protein in BC tissues were higher than those in normal benign tissues (all P 0.05). LPAR1 expression was not associated with clinicopathological features in BC tissues (P>0.05). LPAR2 expression in postmenopausal patients was higher than that in premenopausal patients (χ2=4.821, P<0.05). LPAR3 expression was significantly associated with nodal metastasis, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in BC tissues (all P<0.05). Conclusion LPAR in BC tissues has differential expression, which is associated with nodal metastasis, ER, PR and HER2. Key words: Breast neoplasms; Lysophospholipase receptor; Clinicopathological features