ATX Expression Research Articles

Role of MNK/eIF4E pathway in CD4+ Th2 cytokine-induced lung fibrosis in chronic allergic asthma

Abstract Despite therapeutic advances in allergic asthma, the persistent challenge of airway remodeling still exists, necessitating further exploration. We aimed to investigate the influence of CD4+ Th2 cytokines on fibrosis associated with airway remodeling, with a focus on mesenchymal cell (MC) transformation. Using both human and mouse lung MCs, we investigated the pivotal role of MNK/eIF4E pathway in regulating fibrous protein expression driven by CD4+ Th2 cytokines. To assess this, we employed western blot and flow cytometry studies, while Luminex assays measured cytokine and chemokine secretion from activated lung MCs responding to Th2 cytokines. Treatment with Th2 cytokines significantly increased ATX expression in lung MCs, a response mitigated by the specific MNK1/2 inhibitor (eFT-508). Furthermore, this inhibitor not only abolished the phosphorylation of eIF4E but also reduced collagen levels within activated MCs induced by Th2 cytokines. Luminex assay results demonstrated a significant decrease in IL-6, CCL-2, and GM-CSF in the activated MCs upon inhibiting the MNK1/2 pathway. Additionally, eFT-508 exhibited a marked reduction in lymphocyte migration, utilizing the Boyden chamber transwell assay. Our study reveals that targeting the MNK/eIF4E pathway holds promise in mitigating airway remodeling associated with MCs fibrosis and provides valuable molecular insights for future therapeutic interventions related to fibrosis in the airway remodeling of allergic asthma.

A combined opposite targeting of p110δ PI3K and RhoA abrogates skin cancer

Malignant melanoma is the most aggressive and deadly skin cancer with an increasing incidence worldwide whereas SCC is the second most common non-melanoma human skin cancer with limited treatment options. Here we show that the development and metastasis of melanoma and SCC cancers can be blocked by a combined opposite targeting of RhoA and p110δ PI3K. We found that a targeted induction of RhoA activity into tumours by deletion of p190RhoGAP-a potent inhibitor of RhoA GTPase-in tumour cells together with adoptive macrophages transfer from δD910A/D910A mice in mice bearing tumours with active RhoA abrogated growth progression of melanoma and SCC tumours. Τhe efficacy of this combined treatment is the same in tumours lacking activating mutations in BRAF and in tumours harbouring the most frequent BRAF(V600E) mutation. Furthermore, the efficiency of this combined treatment is associated with decreased ATX expression in tumour cells and tumour stroma bypassing a positive feedback expression of ATX induced by direct ATX pharmacological inactivation. Together, our findings highlight the importance of targeting cancer cells and macrophages for skin cancer therapy, emerge a reverse link between ATX and RhoA and illustrate the benefit of p110δ PI3K inhibition as a combinatorial regimen for the treatment of skin cancers.

Insights into autotaxin- and lysophosphatidate-mediated signaling in the pancreatic ductal adenocarcinoma tumor microenvironment: a survey of pathway gene expression.

Lysophosphatidate (LPA)-mediated signaling is a vital component of physiological wound healing, but the pathway is subverted to mediate chronic inflammatory signaling in many pathologies, including cancers. LPA, as an extracellular signaling molecule, is produced by the enzyme autotaxin (ATX, gene name ENPP2) and signals through six LPA receptors (LPARs). Its signaling is terminated by turnover via the ecto-activity of three lipid phosphate phosphatases (LPPs, gene names PLPP1-3). Many pharmacological developments against the LPA-signaling axis are underway, primarily against ATX. An ATX inhibitor against pancreatic ductal adenocarcinoma (PDAC), a very aggressive disease with limited systemic therapeutic options, is currently in clinical trials, and represents the first in-class drug against LPA signaling in cancers. In the present study, we surveyed the expression of ATX, LPARs, and LPPs in human PDACs and their clinical outcomes in two large independent cohorts, the Cancer Genome Atlas (TCGA) and GSE21501. Correlation among gene expressions, biological function and the cell composition of the tumor microenvironment were analysed using gene set enrichment analysis and cell cyber-sorting with xCell. ENPP2, LPAR1, LPAR4, LPAR5, LPAR6, PLPP1, and PLPP2 were significantly elevated in PDACs compared to normal pancreatic tissue, whereas LPAR2, LPAR3, and PLPP3 where downregulated (all P≤0.003). Only ENPP2 demonstrated survival differences, with overall survival favoring ENPP2-high patients (hazard ration 0.5-0.9). ENPP2 was also the only gene with enriched gene patterns for inflammatory and tissue repair gene sets. Epithelial (cancer) cells had increased LPAR2, LPAR5 and PLPP2 expression, and decreased ENPP2, LPAR1, PLPP1, and PLPP3 gene expression (all P<0.02). Tumor fibroblasts had increased ENPP2, LPAR2, LPAR4, PLPP1, and PLPP3 expression and decreased LPAR2, LPAR5, and PLPP2 expression in both cohorts (all P≤0.01). Immune cell populations were not well correlated to gene expression in PDACs, but across both cohorts, cytolytic scores were increased in high-expressing ENPP2, LPAR1, LPAR6, PLPP1, and PLPP3 tumors (P<0.01). Overall, in PDACs, ENPP2 may switch from an anti-to-pro tumor promoting gene with disease progression. LPAR2 and PLPP2 inhibition are also predicted to have potential therapeutic utility. Future multi-omics investigations are necessarily to validate which LPA signaling components are high-value candidates for pharmacological manipulation in PDAC treatment.

Infliximab, a Monoclonal Antibody against TNF-α, Inhibits NF-κB Activation, Autotaxin Expression and Breast Cancer Metastasis to Lungs.

An inflammatory milieu in the tumor microenvironment leads to immune evasion, resistance to cell death, metastasis and poor prognosis in breast cancer patients. TNF-α is a proinflammatory cytokine that regulates multiple aspects of tumor biology from initiation to progression. TNF-α-induced NF-κB activation initiates inflammatory pathways, which determine cell survival, death and tumor progression. One candidate pathway involves the increased secretion of autotaxin, which produces lysophosphatidate that signals through six G-protein-coupled receptors. Significantly, autotaxin is one of the 40-50 most upregulated genes in metastatic tumors. In this study, we investigated the effects of TNF-α by blocking its action with a monoclonal antibody, Infliximab, and studied the effects on autotaxin secretion and tumor progression. Infliximab had little effect on tumor growth, but it decreased lung metastasis by 60% in a syngeneic BALB/c mouse model using 4T1 breast cancer cells. Infliximab-treated mice also showed a decrease in proliferation and metastatic markers like Ki-67 and vimentin in tumors. This was accompanied by decreases in NF-κB activation, autotaxin expression and the concentrations of plasma and tumor cytokines/chemokines which are involved in metastasis. We also demonstrated a positive correlation of TNF-α -NF-κB and ATX expression in breast cancer patients using cancer databases. Studies in vitro showed that TNF-α-induced NF-κB activation increases autotaxin expression and the clone forming ability of 4T1 breast cancer cells. This report highlights the potential role of Infliximab as an additional approach to attenuate signaling through the autotaxin-lysophosphatidate-inflammatory cycle and decrease mortality from metastatic cancer.

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

Autotaxin promotes the degradation of the mucus layer by inhibiting autophagy in mouse colitis

Autotaxin (ATX or ENPP2) is an autocrine enzyme associated with the metabolism of various phospholipids. ATX has recently been identified as a regulatory factor in immune-related and inflammation-associated diseases, such as inflammatory bowel disease, but the exact mechanism is unclear. Here, we treated mice with recombinant ATX protein or an ATX inhibitor to investigate the effect of ATX on colitis in mice and the underlying mechanism. In a mouse model of colitis, ATX expression was increased, autophagy was impaired, and the mucus barrier was disrupted. Recombinant ATX protein promoted intestinal inflammation, inhibited autophagy, and disrupted the mucus barrier, while an ATX inhibitor had the opposite effect. Next, we treated mice that received ATX with an autophagy activator and an adenosine 5‘-monophosphate-activated protein kinase (AMPK) agonist. We observed that autophagy activator and AMPK agonist could repair the mucus barrier and alleviate intestinal inflammation in ATX-treated mice. In vitro, we obtained consistent results. Thus, we concluded that ATX could inhibit autophagy through the AMPK pathway, which consequently disordered the mucus barrier and aggravated intestinal inflammation.

Values of ATX in predicting disease progression in patients with PBC and PBC related HCC

Objective: To clarify the values of autotaxin (ATX) in patients with primary biliary cholangitis (PBC) and PBC-related hepatocellular carcinoma (HCC). Methods: 179 patients with PBC were selected from prospective cohorts of autoimmune liver diseases at the time of first diagnosis of PBC in Department of Hepatology, the Fifth Medical Center of PLA General Hospital, from January 2016 to January 2018, all patients with PBC received UDCA therapy, primary endpoint was event of HCC, the follow-up period was censored at the date of HCC. The relationship between level of ATX and clinical features in patients with PBC and its potential value in predicting disease progression and PBC-related HCC were analyzed. Results: The ATX level in the peripheral blood of patients with PBC was significantly higher than that of alcoholic liver cirrhosis(ALC) (t = 3.278, P = 0.001) and healthy controls(HC) (t = 6.594, P < 0.001), however, when comparing PBC to non-PBC related HCC, no significant difference was found between the groups(t=-0.240, P = 0.811). Consistent with peripheral blood levels, histochemical staining indicated that ATX in the liver of patients with PBC was significantly higher than that of HC (Z=-3.633, P < 0.001) and ALC (Z=-3.283, P < 0.001), and the expression of ATX in PBC with advanced histological stage was significantly higher than PBC with early stage (Z=-2.018, P = 0.034). The baseline ATX level in PBC patients without developing to HCC during follow-up had significant difference to patients with developing to HCC (228.451 ± 124.093 ng/ml vs 301.583 ± 100.512 ng/ml, t = 2.339, P = 0.021). The result in multivariate logistic regression analysis showed that ATX were independent predictors of PBC related HCC(OR 1.245, 95%CI 1.097-1.413). The optimal critical value of peripheral blood ATX level at baseline for predicting HCC was 235.254 ng/ml, with the cut-off value of 0.714 in AUC of the ROC (95% CI was 0.597~ 0.857), sensitivity and specificity were 84.6% and 59.0%, respectively. Conclusion: ATX level was significantly higher in PBC patients over controls, and it's concentration was correlated with UDCA efficacy and fibrosis stage. ATX has potential values in predicting disease progression and PBC-related HCC.

Autotaxin signaling facilitates β cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency

Objectiveβ cell dedifferentiation may underlie the reversible reduction in pancreatic β cell mass and function in type 2 diabetes (T2D). We previously reported that β cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic β cell dedifferentiation in Sirt3-deficient β cells. MethodsWe applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on β cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the β cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. ResultsIn Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by β cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced β cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. ConclusionsSirt3 prevents β cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse β cell dysfunction associated with glucolipotoxicity.

Mesenteric Adipose Tissue Contributes to Intestinal Fibrosis in Crohn's Disease Through the ATX-LPA Axis.

Intestinal fibrostenosis is an important cause of surgical intervention in patients with Crohn's disease [CD]. Hypertrophic mesenteric adipose tissue [MAT] is associated with the disease process of CD. The purpose of this study was to investigate the contribution of MAT to intestinal fibrosis. MAT from surgical specimens of fibrostenotic CD patients and controls was collected for measurement of the levels of autotaxin [ATX] and lysophosphatidic acid [LPA]. ATX was inhibited in vivo in DNBS [dinitrobenzene sulfonic acid]-induced colitis mice, which were evaluated for colonic inflammation and fibrosis. 3T3-L1 cells and primary colonic fibroblasts were used in vitro to investigate the interaction between MAT and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. MAT adjacent to the fibrostenotic intestine in CD patients showed an activated ATX-LPA axis. An in vivo study indicated that inhibition of ATX was associated with the improvement of morphology and function of diseased MAT, which was combined with ameliorated intestinal inflammation and fibrosis in DNBS-instilled mice. In vitro studies showed that hypoxia stimulated adipocyte ATX expression and that LPA stabilized adipocyte HIF-1α protein, forming an ATX-LPA-HIF-1α amplification loop and aggravating adipocyte dysfunction. LPA secreted by adipocytes bound to LPA1 on the surface of fibroblasts, promoted their proliferation and differentiation, and increased the expression of fibrosis-related factors. The ATX-LPA axis regulated intestinal fibrosis by influencing the proliferation and differentiation of intestinal fibroblasts. Inhibiting this axis may be a therapeutic target for intestinal fibrosis in CD.

Inhibition of autotaxin by bile salts and bile salt-like molecules increases its expression by feedback regulation

BackgroundAutotaxin is an enzyme that converts lysophospholipid into lysophosphatidic acid (LPA), a highly potent signaling molecule through a range of LPA receptors. It is therefore important to investigate which factors play a role in regulating ATX expression. Since we have reported that ATX levels increase dramatically in patients with various forms of cholestasis, we embarked on a study to reveal factors that influence the enzyme activity ATX as well as its expression level in vitro and in vivo. MethodsBile from cholestatic patients was fractionated by HPLC and analyzed for modulation of ATX activity. ATX expression was measured in fibroblasts upon stimulation or inhibition of LPA signaling. ResultsSurprisingly, ATX activity was stimulated by most forms of its product LPA, but it was inhibited by bile salts and bile salt-like molecules, particularly by 3-OH sulfated bile salts and sulfated progesterone metabolites that are known to accumulate during chronic cholestasis and cholestasis of pregnancy, respectively. Activation of fibroblasts by LPA decreased ATX expression by 72%. Conversely, inhibition of LPA signaling increased ATX expression 3-fold, indicating strong feedback regulation by LPA signaling. In fibroblasts, we could verify that inhibition of ATX activity by bile salts induces its expression. Furthermore, induction of cholestasis in mice causes increased plasma ATX activity. ConclusionsMultiple biliary compounds that accumulate in the systemic circulation during cholestasis inhibit ATX activity and thereby increase ATX expression through feedback regulation. This mechanism may contribute to increased serum ATX activity in patients with cholestasis.

Atx regulates skeletal muscle regeneration via LPAR1 and promotes hypertrophy

Muscle differentiation is a multifaceted and tightly controlled process required for the formation of skeletal muscle fibers. Satellite cells are the direct cellular contributors to muscle repair in injuries or disorders. Here, we show that autotaxin (Atx) expression and activity is required for satellite cell differentiation. Conditional ablation of Atx or its pharmacological inhibition impairs muscle repair. Mechanistically, we identify LPAR1 as the key receptor in Atx-LPA signaling. Myogenic gene array and pathway analysis identified that Atx-LPA signaling activates ribosomal protein S6 kinase (S6K), an mTOR-dependent master regulator of muscle cell growth via LPAR1. Furthermore, Atx transgenic mice show muscle hypertrophic effects and accelerated regeneration. Intramuscular injections of Atx/LPA show muscle hypertrophy. In addition, the regulatory effects of Atx on differentiation are conserved in human myoblasts. This study identifies Atx as a critical master regulator in murine and human muscles, identifying a promising extracellular ligand in muscle formation, regeneration, and hypertrophy.

Microglial lysophosphatidic acid promotes glioblastoma proliferation and migration via LPA1 receptor.

Glioblastomas (GBMs) are highly aggressive primary brain tumors characterized by cellular heterogeneity, insensitivity to chemotherapy and poor patient survival. Lysophosphatidic acid (LPA) is a lysophospholipid that acts as a bioactive signaling molecule and plays important roles in diverse biological events during development and disease, including several cancer types. Microglial cells, the resident macrophages of the central nervous system, express high levels of Autotaxin (ATX,Enpp2), an enzyme that synthetizes LPA. Our study aimed to investigate the role of LPA on tumor growth and invasion in the context of microglia-GBM interaction. First, through bioinformatics studies, patient data analysis demonstrated that more aggressive GBM expressed higher levels of ENPP2, which was also associated with worse patient prognosis with proneural GBM. Using GBM-microglia co-culture system we then demonstrated that GBM secreted factors were able to increase LPA1 and ATX in microglia, which could be further enhanced by hypoxia. On the other hand, interaction with microglial cells also increased ATX expression in GBM. Furthermore, microglial-induced GBM proliferation and migration could be inhibited by pharmacological inhibition of LPA1 , suggesting that microglial-derived LPA could support tumor growth and invasion. Finally, increased LPA1 expression was observed in GBM comparing with other gliomas and could be also associated with worse patient survival. These results show for the first time a microglia-GBM interaction through the LPA pathway with relevant implications for tumor progression. A better understanding of this interaction can lead to the development of new therapeutic strategies setting LPA as a potential target for GBM treatment.

Autotaxin-Lysophosphatidic Acid Axis Blockade Improves Inflammation by Regulating Th17 Cell Differentiation in DSS-Induced Chronic Colitis Mice.

Autotaxin-lysophosphatidic acid (ATX-LPA) axis is closely associated with several inflammation-related diseases. In the colonic mucosa of patients with chronic ulcerative colitis (UC), the expression of ATX and the percentage of Th17 cells are found to increase. However, it is unclear whether ATX-LPA axis affects the differentiation of Th17 cells in chronic UC. To investigate whether ATX-LPA axis contributes to Th17 cell differentiation, a mouse model of chronic UC was established by drinking water with DSS at intervals. ATX inhibitor was used as an intervention. The disease active index (DAI), colonic weight to length ratio, colon length, colon histopathology, and MAdCAM-1 were observed. Additionally, the expression of ATX, LPA receptor, CD34, IL-17A, IL-21, IL-6, ROR-γt, STAT3 in colonic tissue, and the percentage of Th17 cells in spleens and mesenteric lymph nodes (MLNs) were measured using different methods. ATX blockade was able to relieve symptoms and inflammatory response of DSS-induced chronic colitis. The DAI and colonic weight to length ratio were apparently decreased, while the colon length was increased. The pathological damage and colitis severity were lighter in the inhibitor group than that in the DSS group. Inhibiting ATX reduced the expression of ATX, LPA receptor, and CD34 and also decreased the percentages of Th17 cells in spleens and MLNs and the expressions of IL-17A and IL-21, as well as the factors in Th17 cell signaling pathway including IL-6, ROR-γt, and STAT3 in colonic tissue. ATX-LPA axis blockade could alleviate inflammation by suppressing Th17 cell differentiation in chronic UC.

Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

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 μMT 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.

Hepatic autotaxin overexpression in infants with biliary atresia.

Background Autotaxin (ATX) is a secreted glycoprotein that is involved in the development of hepatic fibrogenesis via the enzymatic production of lysophosphatidic acid. The aim of this study was to investigate hepatic expression of ATX in biliary atresia (BA) compared with non-BA liver controls and to examine the association between ATX expression and clinical outcome in BA.MethodsLiver specimens from BA infants (n = 20) were compared with samples from infants who underwent liver biopsy for reasons other than BA (n = 14) and served as controls. Relative mRNA and protein expression of ATX were quantified using real-time polymerase chain reaction (PCR) and immunohistochemistry. Masson’s Trichrome staining was performed to determine the degree of liver fibrosis.ResultsQuantitative real-time PCR demonstrated overexpression of ATX mRNA in BA livers. In immunohistochemical evaluation, ATX was positively stained on the hepatic parenchyma and the biliary epithelium in BA patients, as compared to non-BA controls. The immunostaining score of ATX in BA livers was also significantly higher than that observed in non-BA livers (P < 0.001). Subgroup analysis revealed that ATX expression in the patients with poor outcomes was significantly greater than in those with good outcomes (P = 0.03). Additionally, there was a positive correlation between hepatic ATX expression and Metavir fibrosis stage in BA livers (r = 0.79, P < 0.001).DiscussionThis study found that mRNA and protein expression of ATX were increased in BA livers. High hepatic ATX expression at the time of Kasai operation was associated with liver fibrosis and outcome in BA, suggesting that ATX may serve a role as a promising biomarker of the prognosis in biliary atresia.

Role of the placenta in serum autotaxin elevation during maternal cholestasis.

Intrahepatic cholestasis of pregnancy (ICP) is frequently accompanied by pruritus, whose etiology has been associated with an enhanced production of lysophosphatidic acid (LPA) by the combined action of phospholipase A1/A2 (PLA1/PLA2) and autotaxin (ATX). Here, we have investigated whether the placenta is involved in LPA release to maternal circulation during ICP. Serum levels of ATX and LPA (determined by ELISA) were elevated in women with ICP, and a correlation between both parameters was found. No relationship between serum levels of ATX or LPA and bile acids was found. Expression levels of ATX and PLA2 were determined by RT-qPCR and Western blot. Placenta ATX but not PLA2 was significantly upregulated in ICP, and a tendency to increase was found at the protein level. A correlation between serum ATX and placental ATX mRNA levels was found. In human placenta at term, ATX was clearly detected (by immunofluorescence) in Hofbauer cells, but only faintly in trophoblast cells. In pregnant rats, the expression of Atx and Pla2 in placenta was lower than in liver. When obstructive cholestasis was imposed by bile duct ligation from day 14 of gestation until term, placenta Atx and Pla2 expression was markedly enhanced, and overexpression was confirmed at the protein level for Pla2, whereas Atx protein was not detected. In conclusion, the placenta substantially participates in LPA production during gestation. This contribution is markedly higher during maternal cholestasis and hence, may be involved in ICP-associated pruritus. NEW & NOTEWORTHY Fetal placental macrophages and, to a lesser extent, trophoblast cells express high levels of autotaxin at term. An increased expression of mRNA and protein autotaxin, the key secretory enzyme responsible for the production of lysophosphatidic acid in serum, has been observed in placentas of women with cholestasis of pregnancy, which supports that the placenta can contribute to an increased production of this pruritogenic compound in women suffering from this liver disease.

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication

Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

The Bulk of Autotaxin Activity Is Dispensable for Adult Mouse Life.

Autotaxin (ATX, Enpp2) is a secreted lysophospholipase D catalysing the production of lysophosphatidic acid, a pleiotropic growth factor-like lysophospholipid. Increased ATX expression has been detected in a number of chronic inflammatory diseases and different types of cancer, while genetic interventions have proven a role for ATX in disease pathogenesis. Therefore, ATX has emerged as a potential drug target and a large number of ATX inhibitors have been developed exhibiting promising therapeutic potential. However, the embryonic lethality of ATX null mice and the ubiquitous expression of ATX and LPA receptors in adult life question the suitability of ATX as a drug target. Here we show that inducible, ubiquitous genetic deletion of ATX in adult mice, as well as long-term potent pharmacologic inhibition, are well tolerated, alleviating potential toxicity concerns of ATX therapeutic targeting.

OP0241 Autotaxin is Over-Expressed in Systemic Sclerosis (SSC) Skin, Mediates Bleomycin-Induced Dermal Fibrosis via IL-6, and is A Target for SSC Therapy

BackgroundAutotaxin (ATX) is an enzyme present in biological fluids that is responsible for the production of the lipid mediator, lysophosphatidic acid (LPA). We previously implicated LPA and its receptor, LPA1...

Pro-fibrotic activity of lysophosphatidic acid in adipose tissue: In vivo and in vitro evidence

Lysophosphatidic acid (LPA) is a pro-fibrotic mediator acting via specific receptors (LPARs) and is synthesized by autotaxin, that increases with obesity. We tested whether LPA could play a role in adipose tissue (AT)-fibrosis associated with obesity. Fibrosis [type I, III, and IV collagens (COL), fibronectin (FN), TGFβ, CTGF and αSMA] and inflammation (MCP1 and F4/80) markers were quantified: (i) in vivo in inguinal (IAT) and perigonadic (PGAT) AT from obese-diabetic db/db mice treated with the LPAR antagonist Ki16425 (5mg/kg/day ip for 7weeks); and (ii) in vitro in human AT explants in primary culture for 72h in the presence of oleoyl-LPA (10μM) and/or Ki16425 (10μM) and/or the HIF-1α inhibitor YC-1 (100μM). Treatment of db/db mice with Ki16425 reduced Col I and IV mRNAs in IAT and PGAT while Col III mRNAs were only reduced in IAT. This was associated with reduction of COL protein staining in both IAT and PGAT. AT explants showed a spontaneous and time-dependent increase in ATX expression and production of LPA in the culture medium, along with increased levels of Col I and III, TGFβ and αSMA mRNAs and of COL protein staining. In vitro fibrosis was blocked by Ki16425 and was further amplified by oleoyl-LPA. LPA-dependent in vitro fibrosis was blocked by co-treatment with YC1. Our results show that endogenous and exogenous LPA exert a pro-fibrotic activity in AT in vivo and in vitro. This activity could be mediated by an LPA1R-dependent pathway and could involve HIF-1α.