Dysregulation of methionine metabolism of hepatocytes induced epithelial-mesenchymal transition of hepatocytes and abnormal growth of cholangiocytes in biliary atresia

Abstract

Biliary atresia (BA) is the most prevalent serious neonatal biliary obstructive disorder characterized by rapidly progressive biliary and liver fibrosis. However, the patho-mechanisms underlying the disease initiation and progression of BA are not known. Few studies have investigated whether liver deterioration, despite surgical treatment, represents a primary event affecting hepatocytes or reactive changes secondary to bile duct injuries. A heterozygous de novo G to A mutation in exon 8 of the MAT1A gene was identified in a BA patient, which generated a frameshift and a premature stop codon. The mutant MAT was prone to degradation, and the level of MAT in patient’s liver was only 70% of that in normal control livers. The MAT1A gene encodes Methionine adenosyltransferase enzyme 1A in hepatocytes, which catalyzes the biosynthesis of S-adenosylmethionine (SAMe) from methionine and ATP. Induced pluripotent stem cells (iPSCs) generated from this patient’s peripheral blood (BA638C) exhibited an epithelial-mesenchymal transition (EMT) behaviorduring hepatocyte differentiation, accompanied by mitochondrial membrane potential disruption and elevation of oxidative stress. scRNA seq data also showed the emergence of fibroblast clusters and disappearance of hepatocyte cluster in BA638C derived hepatocyte differentiation process. These effects could be reversed by supplementing SAMe and glutathione (GSH), or by MG132, which inhibited the mutant MAT degradation. Methionine also trigger an EMT like behavior in healthy control generated iPSC cell line (HKUPS001) derived hepatocytes and cholangiocyte organoids. Its toxicity was further verified in mice, which showed spleen enlargement, disrupted bile flow, lipid and glucose metabolic dysfunction, and bile duct damage. A co-culture system was established to support the physiology of both hepatocytes and cholangiocyte organoids. When HKUPS001 derived cholangiocyte organoids were co-cultured with BA638C derived hepatocytes, their growth was inhibited, resulting in fewer and smaller organoids. Additionally, these organoids exhibited altered morphology and elevated expression of hepatic marker genes. Bulk RNA sequencing of the organoids revealed cell cycle arrest, reduced proliferation, and stress response, with transcriptomics resembling those of BA liver tissue-derived organoids. In conclusion, this study indicates that (i) the MAT1A mutation induced EMT- like behavior of hepatocytes, potentially promoting liver fibrosis in disease progression; (ii) patients’ hepatocytes induced abnormal cholangiocyte development, which suggests that hepatocyte metabolic dysfunction can lead to cholangiocyte/bile duct injury in the initiation of BA.