br Author contributions br Competing interests br Transparen
Acknowledgments This research was funded by a TOP ZonMW grant (40-00812-98-10054) to R.O.E., a DFG grant (KR4391/1-1) and IZKF Erlangen grant (J36) to A.K. and supported in part by Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, and grants SAF 08/0412 and PI 12/01448, Ministerio de Economía y Competitvidad, Spain. We kindly thank Prof. J. Aoki for providing us with the rat monoclonal 4F1 anti-autotaxin antibody, Prof. W. Moolenaar for the stably ATX overexpressing cell line and Dr. D.R. de Waart for the HPLC bile salt analyses. We thank and honor Dr. C.M. van der Loos for his expert assistance in immunohistochemistry, before he unfortunately passed away.
Introduction Chronic viral hepatitis is a global health problem with at least 170 million hepatitis C virus (HCV) infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma (HCC). The recent availability of direct-acting antiviral agents can eliminate HCV in up to 90% of patients . However, the high cost of these drugs along with reports of viral genotype resistance, may limit their wide-spread use. Importantly, patients with liver cirrhosis cured of HCV may remain at risk of developing HCC, highlighting the need to understand host pathways playing a role in HCC development , . Autotaxin (ATX. Gene name: ENPP2) is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family of proteins that was identified as a motility-stimulating factor secreted from melanoma cells . ATX hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic PDK1 inhibitor synthesis (LPA), a growth factor that activates and signals via a family of six G-protein coupled LPA receptors (LPA1-6). The ATX-LPA signalling axis has been reported to play a tumorigenic role in a wide number of cancers and is a candidate for therapeutic intervention . Several studies have reported increased ATX and LPA levels in the plasma of HCV infected subjects that associates with liver fibrosis staging , , , . A recent prospective study showed that serum ATX predicts the severity of liver cirrhosis and prognosis of patients with cirrhosis . Mazzocca and colleagues reported that HCC secreted LPA increases the trans-differentiation of peritumoral fibroblasts to carcinoma associated fibroblasts that are considered to play a role in tumour proliferation and metastasis . ATX is expressed in many tissues and the mechanisms accounting for increased serum phospholipase activity in chronic hepatitis C and the impact on viral replication are not understood. We show that HCV infection of hepatocyte-derived cells or mice with humanized chimeric livers increases ATX mRNA and protein expression. Infection stabilizes hypoxia inducible factor-1α (HIF-1α) ,  and we show that low oxygen increases ATX transcripts in human liver slices, suggesting a pathway for HCV to regulate ATX. We demonstrate a positive association between ATX and hypoxia related gene expression in viral and non-viral HCC, providing an explanation for elevated ATX expression in tumours that are frequently hypoxic. Finally, we demonstrate that ATX-LPA signalling regulates HCV RNA replication via a phosphoinositide 3 kinase (PI3K) dependent pathway, demonstrating a role for lysophospholipids in viral infection. Our data support a model where HCV infection increases hepatocellular ATX expression that promotes viral replication and establishes a paracrine LPA signalling environment leading to fibrosis and HCC pathogenesis.
Materials and methods
Discussion Our study uncovers a role for the ATX-LPA signalling axis to positively regulate HCV RNA replication by activating PI3K and stabilizing HIF-1α (F). Inhibiting ATX activity or LPA signalling reduced HCV replication, providing evidence for an autocrine LPA-feedback loop to promote viral replication. PI3K signalling has been reported to positively regulate HCV replication  and suppressing this pathway inhibits HCV replication , . We previously reported that low oxygen stabilized HIF promotes HCV infection  and our current study showing that silencing HIF-1α limits HCV replication, suggests a role for this pathway in LPA-induced infection. Vassilaki et al. reported that low oxygen conditions stimulated HCV replication , however, the authors concluded that this phenotype was independent of HIF-1α or HIF-2α that may reflect the use of different HuH7 cell clones or partial HIF silencing. Our observation that LPA stabilized hepatocellular HIF-1α is consistent with reports showing a role for LPA to ‘rescue’ mesenchymal stromal cells  or human CD34+ cells  in ischemic disease and are most likely explained by its ability to activate HIF signalling.