Lysophosphatidic Acid Treatment Research Articles

The Japanese lamprey (Lethenteron camtschaticum) expresses functional lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) signaling plays diverse roles in the development of various vertebrates such as mammals and fish. The lamprey is a fish that retains ancestral features of vertebrates, but information regarding lamprey LPA receptor genes is limited. Here, using information from the lamprey genome database, we cloned two LPA receptor genes, Lpar1 and Lpar5, from the Japanese lamprey (Lethenteron camtschaticum). Lamprey Lpar1 had a high amino acid identity to mouse and medaka fish Lpar1, whereas Lpar5 amino acid sequences were more diverse between species. Our functional analyses using a heterologous expression system demonstrated that Lpar1 and Lpar5 responded to LPA treatment with G12/13-associated cellular responses, which are indicative of cytoskeletal actions. The existence of functional LPA receptors in the Japanese lamprey suggests that LPA receptor-dependent signals contribute to lamprey growth and development.

Elevated Permeability with Reduced Mitochondrial Function in Ischemic Stroke by ATX‐LPA axis

Introduction Stroke is a leading cause of death and a severe cause of long-term disability in the US. r-TPA is the only treatment available for stroke, and only 5% of patients receive it, as others have the risk of intracerebral hemorrhage. Ischemic reperfusion (I/R) following stroke exacerbates the condition and initiates various molecular signals, ultimately interrupting the blood-brain barrier so, better therapy is in need. Lysophosphatidic acid (LPA) is a bioactive lipid regulating various essential cellular functions. Autotaxin (ATX) produces LPA, which then regulates through its six G-protein coupled receptors. We hypothesize that the altered ATX-LPA axis could cause unfavorable signaling following ischemic stroke. Methods Transient middle cerebral artery occlusion (MCAO) with 90 minutes of ischemia and 24 hours of reperfusion was used as a stroke model in mice. LPA was analyzed in plasma with mass spectrometry and in tissue with immunohistochemistry. AR-2 probe fluorescence measured ATX activity. Seahorse analyzer measured the mitochondrial bioenergetics in brain and mouse brain microvascular endothelial cells (MBMEC). Permeability was studied with Electric Cell Substrate Impedance System. Results ATX was elevated 1.7 fold in mRNA level with increased activity following I/R in mice brain. LPA showed a significant (P<0.01) increase in the plasma of MCAO mice. LPA in the brain tissue of MCAO mice revealed an increased LPA following I/R injury. ATX produced LPA could be the cause for detrimental effects. Brain mitochondria isolated following I/R injury had significantly (P<0.01) reduced the mitochondrial oxygen consumption rate (OCR). Permeability measured in MBMECs showed that LPA treatment increases the permeability. The protein levels of junctional proteins such as βcatenin, Claudin5, VEcadherin, and Zo1 were significantly (P<0.05) decreased with LPA exposure. LPA, as a negative regulator of the BBB, caused permeability through LPA-LPAR1-ROCK dependent pathway. MBMECs show impaired mitochondrial function with decreased OCR and elevated glycolysis towards LPA treatment (P<0.01). Conclusion We have shownthat LPA and its regulator enzyme ATX play a significant role in ischemic stroke, increasing BBB permeability and reducing mitochondrial function. ATX inhibitors as therapy could improve the condition of stroke patients.

Lysophosphatidic acid improves oocyte quality during IVM by activating the ERK1/2 pathway in cumulus cells and oocytes.

Oocyte IVM technology is an option for fertility preservation in some groups of patients, such as those with polycystic ovary syndrome, patients with ovarian hyperstimulation syndrome, and for patients with cancer. However, the developmental potential of oocytes from IVM still needs to improve. Several previous studies have reported that lysophosphatidic acid (LPA) promotes glucose metabolism, cumulus cell (CC) expansion, and oocyte nuclear maturation. However, the effect of LPA on oocyte cytoplasmic maturation, particularly mitochondrial function, has rarely been studied and the underlying mechanism is largely unknown, which impedes (pre)clinical applications of LPA. In this study, cumulus-oocyte complexes (COCs) and cumulus-denuded germinal vesicle oocytes (DOs) were treated with various concentrations of LPA during IVM, in the presence or absence of the oxidative stressor cyclophosphamide (CTX). In both normal and CTX-damaged COCs, the 25 μM LPA group exhibited improved CC expansion capacity, a higher nuclear maturation rate, and superior mitochondrial function, compared to no LPA treatment. When the concentration of LPA was over 40 μM, detrimental effects of LPA on oocyte maturation occurred. Compared with COCs, the addition of LPA slightly improved oocyte nuclear and cytoplasmic maturation of DOs, but this was not statistically significant. We observed that LPA promotes the activation of extracellular signal-regulated kinase (ERK)1/2, although this was not statistically significant in DOs. Furthermore, LPA could not reverse the negative effect of CC expansion and mitochondrial function after inactivation of ERK1/2 by U0126. RNA-sequencing and RT-PCR results showed that LPA upregulated several ERK1/2 downstream genes related to CC expansion, such as Areg, Cited4, and Ptgs2. This study demonstrates that LPA improves oocyte quality during IVM through the activation of ERK1/2 pathway CCs and oocytes, which provides evidence for the potential addition of LPA to IVM medium.

LPA1 Receptor Promotes Progesterone Receptor Phosphorylation through PKCα in Human Glioblastoma Cells.

Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.

High Expression of Lysophosphatidic Acid Induces Nerve Injury in LSS Patients via AKT Mediated NF-κB p65 Pathway.

Lumbar spinal stenosis (LSS) is a spinal degenerative disease, complicated with nerve injury. Lysophosphatidic acid (LPA), a kind of glycerophospholipid molecule is elevated in the initial stages of neural injury. This research aimed to investigate the patho-mechanism of nerve injury caused by LPA in LSS patients. Twenty-five LSS patients and fifteen idiopathic scoliosis patients (without neurological symptoms) were recruited from Xianyang Central Hospital of Shanxi Province. We measured the concentration of LPA in cerebrospinal fluid samples of all subjects. Different concentrations (0.1, 1, and 10 mol/L) of LPA were used to stimulate Rat Neurons-spinal cord (RN-SC) cells. The effects of LPA on cell injury was detected by MTT and LDH (lactate dehydrogenase) assay. Cell apoptosis was determined by FCM (flow cytometry) and TUNEL staining. The changes in the expression of key proteins involved in Akt mediated NF-κB p65 pathway intervened by LPA were determined by western blot. RN-SC cells were pretreated with JSH-23 (NF-κB inhibitor) before LPA exposure, followed by cell apoptosis measurement. The concentration of LPA in LSS patients was notably higher than that in control patients (p < 0.01). The level of LPA was positively correlated with the severity of LSS. LPA treatment induced RN-SC cells displaying oval or rounded cell body with degenerated protrusion dose dependently. In addition, LPA decreased RN-SC cell viability and promoted cell apoptosis in a dose-dependent manner. LPA initiated Akt phosphorylation, IKB phosphorylation, and NF-κB nuclear translocation in a dose-dependent manner. However, JSH-23 (NF-κB inhibitor) pre-treatment prevented effects of LPA. The high levels of LPA induced nerve injury by reducing the viability of RN-SC cells and promoted cell apoptosis through Akt mediated NF-κB p65 signaling pathway. LPA might be a new therapeutic target for relieving nerve injury in LSS patients.

Abstract P773: Autotaxin and Lysophosphatidic Acid Impair Blood Brain Barrier and Mitochondria Following Ischemic Stroke

Introduction: Ischemic stroke is a leading cause of death and a serious cause of long-term disability in the US. r-TPA is the only treatment available for stroke. Only 5% of patients receive it, as others have the risk of intracerebral hemorrhage. Ischemic reperfusion (I/R) following stroke exacerbates the condition and initiates various molecular signals, ultimately interrupting blood-brain barrier BBB so, better therapy is in need. Lysophosphatidic acid (LPA) is a bioactive lipid regulating various essential cellular functions such as migration, proliferation, adhesion. Autotaxin (ATX) produces LPA, which then regulates through its six G-protein coupled receptors. Methods: Transient middle cerebral artery occlusion (MCAO) with 90 minutes of ischemia and 24 hours of reperfusion was used as a stroke model in mice. LPA was analyzed in plasma with mass spectrometry and in tissue with immunohistochemistry. AR-2 probe fluorescence measured ATX activity. Seahorse analyzer measured the mitochondrial bioenergetics in brain and mouse brain microvascular endothelial cells (MBMEC). Permeability was studied with Electric Cell Substrate Impedance System. Results: ATX was elevated in mRNA level with 1.7 fold increased activity following I/R. LPA showed a significant (P&lt;0.01) increase in the plasma of MCAO mice. LPA in the brain tissue of MCAO mice revealed an increased synthesis of LPA by ATX following I/R injury. Brain mitochondria isolated following I/R injury had significantly (P&lt;0.01) reduced the mitochondrial oxygen consumption rate (OCR). Permeability measured in MBMECs showed LPA treatment increases the permeability. Also, the protein levels of junctional proteins such as βcatenin, Claudin5, VEcadherin, and Zo1 were significantly (P&lt;0.05) decreased with LPA exposure. LPA, as a negative regulator of the BBB, caused permeability through LPA-LPAR1-ROCK dependent pathway. MBMECs show impaired mitochondrial function with decreased OCR and elevated glycolysis towards LPA treatment (P&lt;0.01). Conclusion: We have shown that LPA and its regulator enzyme ATX play a significant role in ischemic stroke, increasing BBB permeability, and elevating mitochondrial dysfunction. ATX inhibitors as therapy could improve the condition of stroke patients.

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2–related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.

CMTM8 as an LPA1-associated partner mediates lysophosphatidic acid-induced pancreatic cancer metastasis.

BackgroundLysophosphatidic acid (LPA) is known to promote cancer cell invasiveness through LPA1, but the downstream signaling cascades are still not fully clarified. The CKLF-like MARVEL transmembrane domain-containing (CMTM) family regulates aggressive phenotype in many cancers.MethodsWe performed LPA1 co-immunoprecipitation combined with mass spectrometry to search for LPA1-associated proteins. The role of CMTM8 in mediating the pro-invasive activity of LPA was investigated in pancreatic cancer.ResultsWe identified CMTM8 as an LPA1-interacting protein. LPA1 and CMTM8 were co-localized in pancreatic cancer cells. LPA treatment led to stabilization of CMTM8 protein, which was impaired by knockdown of LPA1. Depletion of CMTM8 significantly suppressed the migration and invasion of pancreatic cancer cells. Conversely, ectopic expression of CMTM8 enhanced the migratory and invasive capacity of pancreatic cancer cells. CMTM8 depletion blocked the formation of metastatic lesions in the lung. Knockdown of CMTM8 attenuated LPA-induced migration and invasion in pancreatic cancer cells. CMTM8 overexpression stimulated β-catenin activation through reduction of GSK3β. In addition, knockdown of β-catenin dramatically antagonized CMTM8-mediated migration and invasion in pancreatic cancer cells.ConclusionsCMTM8 serves as a key mediator of LPA-induced invasiveness in pancreatic cancer. The interaction between CMTM8 and LPA1 leads to activation of oncogenic β-catenin signaling. CMTM8 represents a potential therapeutic target for pancreatic cancer.

Roles of endothelial cells in the regulation of cell motility via lysophosphatidic acid receptor-2 (LPA2) and LPA3 in osteosarcoma cells

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of biological responses. In tumor microenvironment, endothelial cells promote cancer cell functions. In this study, we investigated the roles of endothelial cells in the regulation of cell motile activity via LPA2 and LPA3 in human osteosarcoma MG-63 cells. In cell motility assay, the cell motile activity of MG-63 cells was markedly increased by the supernatants of endothelial F2 cells. MG-63 cell motility elevated by the supernatants was enhanced by GRI-977143 (LPA2 agonist) and reduced by (2S)-OMPT (LPA3 agonist). LPAR2 and LPAR3 expressions were increased in highly migratory MG63-CR7(F2) cells, which were generated from MG-63 cells by co-culture with F2 cell supernatants. MG63-CR7(F2) cell motility was stimulated by LPA treatment. In the presence of F2 cell supernatants, MG63-CR7(F2) cell motility was markedly enhanced by GRI-977143 and suppressed by (2S)-OMPT. Autotaxin (ATX) enzymatically converts lysophosphatidylcholine (LPC) to LPA. ATX expression was higher in MG63-CR(F2) cells than in MG-63 cells. MG63-CR7(F2) cell motility was markedly increased by LPC in comparison with MG-63 cells. In addition, MG63-CR(F2) cell motility was significantly stimulated by the supernatants of LPC treated F2 cells. The present results suggest that the activation of LPA signaling via LPA2 and LPA3 by endothelial cells is involved in the modulation of cell motile activity of MG-63 cells.

Lysophosphatidic Acid Prevents Ischemia Reperfusion Injury but does not Prevent Tubular Dysfunction

Lysophosphatidic acid (LPA) protects the kidneys from tissue ischemic reperfusion injury (IRI), but its impact on renal function is primarily limited to glomerular function. We estimated the status of renal function by a complete glomerular and tubular functional analysis to test the hypothesis that LPA treatment during ischemia-reperfusion (I/R) protects renal function by attenuating IRI. Male Wistar rats were subjected to bilateral kidney I/R. Along with ischemia, LPA was administered. LPA increased levels of plasma LPA, downregulated LPA2R, prevented interstitial fibronectin and TGF-β1 accumulation, and prevented a decrease in glomerular filtration rate (GFR). I/R increased urine volume and proteinuria and decreased fractional Na+ excretion (FENa) and urine osmolality. These effects were not prevented by LPA. The reduction in FENa was attributed to disruption in tubular Na+ transport and downregulation of protein kinase C (PKC) activity. LPA treatment maintained (Na++K+)ATPase activity to the control level, due to a sustained sensitivity to PLC/PKC pathway. Na+-ATPase activity was insensitivity to LPA treatment. This ineffectiveness was associated with downregulation of LPA2R, resulting in low FENa. Altogether, LPA treatment maintained normal kidney structure and prevented the reduction of glomerular function. However, even in the setting of preserved GFR, impaired tubular function may present a high risk for silent progression of kidney disease.

Lysophosphatidic acid induces thrombospondin-1 production in primary cultured rat cortical astrocytes.

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA1 receptor antagonist, and Ki16425, LPA1/3 receptors antagonist, but not by H2L5146303, LPA2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA1 and LPA3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders.

Different effects of lysophosphatidic acid receptor-2 (LPA2) and LPA5 on the regulation of chemoresistance in colon cancer cells

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits several biological functions by binding to G-protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). The present study aimed to evaluate whether LPA signaling via LPA2 and LPA5 is involved in the chemoresistance to anticancer drugs in colon cancer DLD1 cells. In cell survival assay, cells were treated with fluorouracil (5-FU) every 24 h for 2 days. The cell survival rate to 5-FU of DLD1 cells was significantly decreased by LPA treatment. In the presence of LPA, the cell survival rate to 5-FU was significantly elevated by LPA5 knockdown. Before initiation of the cell survival assay, cells were pretreated with an LPA2 agonist, GRI-977143. The cell survival rate to 5-FU was markedly increased in DLD1 cells treated with GRI-977143. In the presence of GRI-977143, the elevated cell survival rate of DLD1 cells was reduced by LPA2 knockdown. To assess the effects of LPA2 and LPA5 on the enhancement of chemoresistance, long-term 5-FU treated (DLD-5FU) cells were generated from DLD1 cells. The cell survival rate to 5-FU of DLD-5FU cells were significantly elevated by LPA5 knockdown. GRI-977143 treatment increased the cell survival rate to 5-FU of DLD-5FU cells. These results suggest that LPA2 promotes and LPA5 suppresses the acquisition of chemoresistance in colon cancer cells treated with anticancer drugs.

Abstract B63: Lysophosphatidic acid as a mediator of ovarian cancer cell stemness

Abstract Epithelial ovarian cancer (OC) is the deadliest gynecologic malignancy. Recurrence of advanced, multidrug-resistant metastatic disease after initially good response to standard lines of chemotherapy occurs in the majority of OC patients. Chemoresistant, recurrent OC is essentially fatal. Disease relapse is due to survival and expansion of a small pool of chemorefractory ovarian cancer stem cells (OCSCs). OC evolution to a platinum-resistant state is associated with aberrant epigenetic modifications, including altered DNA methylation and histone modifications. In addition, OC cells residing in the peritoneal cavity are exposed to a variety of external cues arising from the ascitic microenvironment, including the bioactive molecule lysophosphatidic acid (LPA), which potentially contribute to cell stemness and chemoresistance. LPA in the intraperitoneal fluid of OC patients has been strongly implicated in ovarian carcinogenesis and metastasis through a variety of mechanisms. However, regulation of OC cells by LPA is incompletely understood, and the role of LPA in OCSC acquisition/chemoresistance remains unclear. In the current study, we tested the hypothesis that LPA is a mediator of OCSC promotion and survival. Expression of aldehyde dehydrogenase (ALDH), an accepted functional marker of cancer stem cells, was assessed in a panel of high-grade serous ovarian cancer (HGSOC) cell lines (OVSAHO, OVCAR3, OVCAR5, PEO1, and Kuramochi) using an Aldefluor assay. Treatment of HGSOC cells with increasing (0-80uM, 72h) doses of LPA enriched the ALDH+ population in a cell line-specific dose-dependent manner. In PEO1 and Kuramochi cells, intermediate LPA doses (1-10uM) resulted in a 1.5-2.5-fold increase (P&amp;lt;0.05) of ALDH+ cells (from ∼4% to ∼8% in PEO1 and from ∼4% up to ∼10% in Kuramochi, respectively). However, 80uM LPA treatment was necessary to increase (P&amp;lt;0.05) the percentage ALDH+ cells in OVCAR3 (from ∼17% to 34%) and OVSAHO cells (from ∼10% up to 29%). In accordance with the results of the flow cytometry analysis, cancer stem cell properties after LPA treatment (0-80uM, every 72hr, up to 21 days) were enhanced (P&amp;lt;0.05), based on clonogenic assay. In particular, moderate (10-20uM) LPA treatment increased (P&amp;lt;0.05) cell clonogenic survival of PEO1 and Kuramochi, while a greater (80uM) LPA dose was needed for OVCAR3 and OVSAHO. Furthermore, LPA (0-80uM, 14 days) augmented spheroid formation ability in PEO1 cells (3-4-fold increase; P&amp;lt;0.05). Global transcriptomic and epigenomic assessment of altered genes/pathways induced by LPA are currently being examined to assess stemness-promoting pathways and epigenetic changes, which we believe will identify new biologic insights and potential therapeutic targets to eradicate OCSC in patients. Citation Format: Yuliya Klymenko, Kenneth P. Nephew. Lysophosphatidic acid as a mediator of ovarian cancer cell stemness [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B63.

Engineering self-assembled neomenisci through combination of matrix augmentation and directional remodeling

Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. This study describes an approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties. Self-assembled fibrocartilages were treated with TGF-β1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed TCL) in addition to lysophosphatidic acid (LPA). TCL + LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink content per dry weight, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilizes a combination of bioactive stimuli for use in tissue engineering studies, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. Statement of SignificanceThis study utilizes a scaffold-free approach, which strays from the tissue engineering paradigm of using scaffolds with cells and bioactive factors to engineer neotissue. While self-assembled neomenisci have attained compressive properties akin to native tissue, tensile properties still require improvement before being able to deploy engineered neomenisci as functional tissue repair or replacement options. In order to augment tensile properties, this study utilized bioactive factors known to augment matrix content in combination with a soluble factor that enhances matrix organization and anisotropy via cell traction forces. Using a bioactive factor to enhance matrix organization mitigates the need for bioreactors used to apply mechanical stimuli or scaffolds to induce proper fiber alignment.

Lysophosphatidic acid enhances intestinal tuft cell differentiation in Myo5b KO mice independent of Pou2f3

We have reported that adult tamoxifen‐induced, intestine‐specific, Myosin Vb knockout (Myo5b KO) mice recapitulate malabsorption syndrome in a congenital diarrheal disease, microvillus inclusion disease, and that daily treatment with lysophosphatidic acid (LPA) stimulated brush border maturation and sodium/glucose absorption in the Myo5b KO mice. Influence of Myo5b loss and LPA in sensory cell differentiation had not been studied. The present study evaluated the global changes in gene expression following Myo5b loss and LPA treatment, focusing on the differentiation of a nutrient sensor cell lineage, tuft cell. VillinCreERT2;Myo5bflox/flox mice were treated with LPA or vehicle for 4 days after a single tamoxifen injection. Immunostaining on small intestinal sections and whole‐mounted enteroids was used to quantify tuft cell density. RNA sequencing was performed on isolated jejunal epithelial cells. The sections of entire small intestine were analyzed by whole‐slide scanning and digital analysis with Cell Profiler. The frequency of DCLK1+ tuft cell was decreased by 80% with Myo5b loss, whereas enteroendocrine cells showed no difference between Myo5b KO mice and littermate controls. Paneth cell numbers were significantly increased in Myo5b KO, correlating with hyperproliferation in crypts. LPA did not alter the increased Paneth cells or Ki67+ cells. Intraperitoneal LPA treatment re‐established the frequency of tuft cells in Myo5b KO mice, indicating that a tuft cell‐specific differentiation mechanism was activated by LPA. In contrast, enteroids that were generated from VillinCreERT2;Myo5Bflox/flox jejunum had no difference in tuft cell number between hydroxytamoxifen‐treated (iKO) and control enteroids. The disrupted tuft cell differentiation by Myo5b loss was not represented in in vitro system, although microvillus formation was immature in iKO enteroids. RNA‐seq revealed that 844 genes in jejunal epithelial cells were significantly down‐regulated, and 657 genes were up‐regulated by tamoxifen‐induced loss of Myo5b compared to control. Three stem cell transcription factors were significantly increased in KO, whereas two tuft cell‐specific transcription factors (Spib and Pou2f3) were decreased. Epithelial Myo5b loss demonstrated a broad impact on epithelial cell differentiation and maturation. Treatment of Myo5b KO mice with ip LPA up‐regulated only 9 genes and had no effect on the expression of transcription factors, suggesting that LPA may modulate transporter trafficking to stimulate nutrient absorption rather than change the transcription. Myo5b loss abolished the differentiation of a sensory tuft cell, and in vivo treatment with LPA re‐established the tuft cell population independent of Pou2f3 expression. These findings suggest unexplored functions for Myo5b and LPA signaling in cell lineage choice in the intestinal epithelial cells, interacting with non‐epithelial cells.Support or Funding InformationR01 DK48370 and R01 DK70856 and a gift from the Christine Volpe Fund to J.R.G. This work was supported by core resources of the Vanderbilt Digestive Disease Center (P30 DK058404), the Vanderbilt‐Ingram Cancer Center (P30 CA68485), VUMC Digital Histology Shared Resource (supported by a VA Shared Equipment Grant 1IS1BX003097).

Regulation of PLPP3 gene expression by NF-κB family transcription factors

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Homer1/mGluR1-mediated ER stress contributes to lysophosphatidic acid-induced neurotoxicity in cortical neurons

Lysophosphatidic acid (LPA) is a glycerophospholipid that can be detected in serum, saliva and cerebrospinal fluid. However, the effect of LPA on neuronal death and survival has not been fully determined. In the present study, we investigated the potential neurotoxic effect of LPA in primary cultured cortical neurons. Treatment with LPA (0.5, 1 and 5 μM) markedly decreased neuronal viability, increased lactate dehydrogenase (LDH) release and promoted apoptosis in cortical neurons. The results of western blot showed that LPA increased the expression of endoplasmic reticulum (ER) stress associated factors, and the protein misfolding inhibitor 4-phenylbutyric acid (4-PBA) attenuated LPA-induced toxicity. In addition, treatment with LPA did not alter the expression and distribution of Homer1 in cortical neurons. The protein levels of metabotropic glutamate receptor 1 (mGluR1), but not metabotropic glutamate receptor 5 (mGluR5), were significantly increased by LPA at 12 and 24 h after treatment. Knockdown of Homer1 using specific siRNA partially prevented the LPA-induced neurotoxicity and ER stress. Furthermore, the results of Ca2+ imaging showed that treatment with LPA induced intracellular Ca2+ release, which could be partially prevented by 4-PBA and downregulation of Homer1. The LPA-induced intracellular Ca2+ release was associated with ER Ca2+ release through the Homer1-mGluR1 pathway. In summary, our results showed that LPA treatment induced ER stress and apoptosis in cortical neurons, and its neurotoxicity was partially mediated by Ca2+ release from the ER via the Homer1/mGluR1 pathway.

Lysophosphatidic acid induces the migration and invasion of SGC-7901 gastric cancer cells through the LPA2 and Notch signaling pathways

Lysophosphatidic acid (LPA), a simple water-soluble glycerophospholipid with growth factor-like activity, regulates certain behaviors of multiple cancer types by binding to its receptor, LPA receptor 2 (LPA2). Notch1 is a key mediator in multiple human cancer cell types. The association between LPA2 and Notch1 in gastric cancer cells is not well known. The present study aimed to investigate the function of LPA2 and Notch1 in controlling the migration and invasion activities of SGC-7901 gastric cancer cells following stimulation with LPA. It was revealed that LPA may stimulate the expression of Notch1 and Hes family bHLH transcription factor 1, and the phosphorylation of protein kinase B which belongs to the Notch pathway. Furthermore, by performing transwell migration and invasion assays, immunofluorescent staining, analyzing the expression of markers for the epithelial-mesenchymal transition (EMT) and downregulating LPA2 and Notch1 expression, it was verified that LPA2 and Notch1 mediated the metastasis, invasion, EMT and rebuilding of the cytoskeleton of SGC-7901 cells upon LPA treatment. An immunoprecipitation assay revealed that LPA2 interacted with Notch1 in SGC-7901 cells. The present study may provide novel ideas and an experimental basis for identifying the factors that affect the functions of SGC-7901 cells.

527 – Lysophosphatidic Acid Treatment Ameliorates the Function of Apical Sglt1, But Does Not Prevent the Formation of Microvillus Inclusions Or Loss of Tuft Cell Differentiation in Myo5B Deficient Mice

527 – Lysophosphatidic Acid Treatment Ameliorates the Function of Apical Sglt1, But Does Not Prevent the Formation of Microvillus Inclusions Or Loss of Tuft Cell Differentiation in Myo5B Deficient Mice

Lysophosphatidic acid signaling restores mislocalization of brush border proteins in MYO5B deficient mice

Background &amp; AimMyosin Vb (MYO5B) is an essential trafficking protein for membrane protein recycling in gastrointestinal (GI) epithelial cells and its mutation causes the congenital diarrheal disease, microvillus inclusion disease (MVID). Our group has reported that MYO5B loss results in a decrease in the brush border localization of nutrient transporters and enzymes, but CFTR expression is maintained, causing malabsorption and secretory diarrhea in MVID patients and MYO5B KO mice. Activation of lysophosphatidic acid (LPA) receptors (LPA2 and LPA5) can inhibit CFTR and activate NHE3 in the intestinal epithelium. This study tested whether LPA treatment reverses the functional expression of apical proteins in MYO5B knockout (KO) mice.MethodsAdult tamoxifen‐induced, intestine‐specific, MYOB KO mice (VilCreERT2;Myo5bflox/flox) and littermate control mice were treated with LPA (18:1 at 1 or 3 mg/kg/day) or vehicle, orally or intraperitoneally for 4 days after a single injection of tamoxifen (2 mg/mouse, i.p.). Apical transporter activities in jejunum and ileum were measured by an Üssing chamber system and the localization of apical proteins were evaluated immunostaining with whole‐slide imaging and digital image analysis.ResultsFollowing tamoxifen injection, F‐actin‐positive inclusions appeared in the upper small intestine of VilCreERT2;Myo5bflox/flox mice and the brush border localization of phospho‐ezrin, SGLT1, NHE3, DRA, PEPT1, alkaline phosphatase, and DPPIV were lost throughout the intestine. Treatment with LPA partially restored apical localization of phospho‐ezrin, SGLT1, NHE3, alkaline phosphatase, and DPPIV particularly in villus epithelial cells. Consistent with these observations, SGLT1‐dependent short‐circuit current (Isc) was 3 times higher in LPA (3 mg/kg)‐treated jejunum and ileum than that of vehicle‐treated VilCreERT2;Myo5bflox/flox mice. LPA dose‐dependently decreased CFTR‐dependent Isc in the steady state, but had no effect on CFTR dependence in carbachol‐ and forskolin‐stimulated Isc, which was increased by MYO5B loss.ConclusionsThese results suggest that LPA signaling may modulate a MYO5B‐independent trafficking mechanism for some brush border proteins in the small intestine, demonstrating a therapeutic potential for LPA in the treatment of MVID.Support or Funding InformationSupported by The Christine Volpe FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.