We demonstrate a role for

We demonstrate a role for low oxygen to regulate ATX mRNA in hepatocyte-derived HuH7 Lomustine and human liver slices, consistent with reports of increased ATX expression in a variety of tumours that are frequently hypoxic. Importantly, we show a positive association between elevated ATX mRNA levels in HCC and the hypoxia gene score. Transient overexpression of HIF-1α in HuH7 cells increases ATX mRNA, suggesting an activating role for this transcription factor. However, low oxygen had a minimal effect on ATX promoter activity, in agreement with the lack of HRE sites in this region and suggesting that enhancer regions beyond the published 1.2kb promoter may bind HIFs or that low oxygen regulated factors increase ATX mRNA half-life and/or protein stability. Wu and colleagues reported that TNFα induced a modest 3-fold increase in ATX mRNA levels in HepG2 cells via NF-κβ activation [7]. However, we failed to see any evidence for TNFα modulation of ATX promoter activity in HuH7 cells or human liver slices, suggesting that this may be cell type dependent. ATX is expressed in many tissues, however the source of elevated ATX in the sera of chronic hepatitis C patients is unknown. Our studies with chimeric liver uPA-SCID mice show that hepatocytes express ATX and HCV infection induces its expression in the absence of any inflammatory response. We confirmed increased ATX transcript levels in HCC tumour tissues from subjects with either HCV, HBV or ALD aetiologies, demonstrating that increased ATX expression is not unique to HCV infection. Reports that HBV can stabilize HIF [31] and that ALD is associated with hepatic HIF expression [32] lend support to our model that HIFs regulate hepatic ATX expression. In the healthy liver, ATX is most likely removed from the circulation by sinusoidal endothelial cells [33], however, during fibrosis phenotypic changes in the sinusoidal endothelium [34] are likely to impair ATX clearance that may account for the increased expression reported in the fibrotic liver. However, these morphological changes are unlikely to account for the increase in ATX mRNA observed in this study. It is interesting to note that Epstein Barr virus (EBV) infection of Hodgkin lymphoma cells induces ATX expression that augments their proliferation and survival [35]. EBV is an oncogenic virus associated with B-lymphoid and non-lymphoid malignancies that is known to stabilize HIF-1α [36], suggesting a common pathway for viruses to activate the ATX-LPA signalling axis. LPA is not a single entity and exists in several forms with differing acyl chain lengths and degrees of saturation that interact with specific LPA receptors and regulate physiological responses. For example 18:1 LPA activates all receptors, whereas 20:4 LPA shows a higher potency to activate LPA3 [37]. HuH7 cells expressed a range of LPA molecular species and HA130 showed differential effects on the genesis of some LPA species. These results highlight potential differences in the role of LPA molecular species in the viral life cycle, however, this variability may reflect differences in LPC substrate availability and/or lipid phosphate phosphatases that may selectively degrade LPA species. LPA signals through binding to a family of G-protein coupled receptors can activate signalling pathways including PI3 kinase and adenylyl cyclase to induce physiological changes including cellular proliferation, anti-apoptosis and migration. Whilst LPA receptor overexpression studies suggest that individual receptors can regulate physiological responses, our understanding of tissue-specific LPA signalling is limited. PHHs and HuH7 express all of the LPARs at the mRNA level with the exception of LPA4 in HuH7 cells. The ability of LPA1/3 antagonist Ki16425 [20] to limit HCV infection suggests a direct role for LPA1 or LPA3 in viral replication. LPA signalling has been reported to drive chronic wound healing leading to fibrosis and LPA modulators are in development for treating fibrosis [38]. A recent study reported a role for LPA6 in maintaining the proliferative capacity and tumorigenic phenotype of HCC via the transcriptional activation of proto-oncogene Pim-3[39], highlighting the value of LPA receptor-targeted therapies for treating HCC.