Abstract: Background and aims: Acyl-CoA oxidase 2 (ACOX2) is a key enzyme in cholic acid biosynthesis. Its partial deficiency (APD) results in accumulation of trihydroxycholestanoic acid (THCA) and has been recently described as a cause of asymptomatic hypertransaminasemia. Here we have explored the mechanisms controlling ACOX2 expression and the pathogenic consequences of the absence of its enzymatic activity. Methods: Human hepatocytes, either immortalized or freshly isolated, and human hepatoma cells (HepG2 and Huh7) were cultured in the presence of bile acids (BA), FXR or PPAR agonists, recombinant FGF19, oncostatin M (OSM), IL-6, IL-1beta and TNF-alpha. ACOX2 expression was determined by quantitative real-time PCR and immunoblot. Specific inhibitors were used to investigate the signalling pathways involved in gene regulation. Predicted genetic variants in ACOX2 ORF were generated by site-directed mutagenesis and overexpressed in Huh7 cells using lentiviral vectors. Their ability to metabolize THCA was measured by HPLC-MS/MS. Oxidative stress was determined by flow cytometry using specific probes. Endoplasmic reticulum (ER) stress was studied by measuring CHOP and GRP78 up-regulation plus spliced XBP1 generation by immunoblot and PCR, respectively. Results: Several genetic variants of ACOX2 were able to retain its enzymatic activity, although others partially or completely blocked THCA biotransformation resulting in oxidative and ER stress. In human hepatocytes, ACOX2 expression was downregulated by BA, FGF19 and OSM. Similar but weaker effect was exerted by IL-6, IL-1beta and TNF-alpha, whereas PPAR agonists had no effect. After washing-out OSM from de culture, ACOX2 down-regulation persisted for more than 24 h, which was associated to strong OSM adhesion to extracellular collagen. Abrogation of the Janus kinase activity completely blocked the suppressive effect of OSM, while inhibition of the MAPKs p38 and JNK had no effect. Inhibition of ERK1/2 pathway partially overcame OSM-induced ACOX2 down-regulation, whereas it was able to completely block FGF19-mediated effect. Conclusion: Inflammatory processes and cholestasis may affect ACOX2 expression. Moreover, the presence of inactivating mutations in ACOX2 gene leads to altered BA metabolism and enhanced oxidative and ER stress.

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