Prohibitin1 (PHB1) is a mitochondrial chaperone with diverse functions that include cell proliferation, apoptosis, and mitochondrial homoeostasis. Liver-specific Phb1 knockout (KO) mice develop spontaneous injury and hepatocellular carcinoma (HCC). Our previous work demonstrated that PHB1 negatively regulates the H19-insulin-like growth factor 2 (IGF2)-H19-IGF2 axis signaling pathway and E-box activity in hepatocytes and HCC cells. Phb1 KO livers exhibited increased expression of multiple wingless/integrated (WNT) target genes compared to control littermates. Therefore, we hypothesized that PHB1 is a negative regulator of WNT-beta-catenin signaling in the liver. Analysis of livers from Phb1 KO mice demonstrated an activation of the WNT-beta-catenin pathway as determined by phosphorylation of glycogen synthase kinase 3 (GSK3)betaserine [Ser]9 and protein kinase B (AKT)Ser473. Phb1 KO livers showed increased messenger RNA (mRNA) levels of multiple WNT ligands, with Wnt7a (79-fold), Wnt10a (12-fold), and Wnt16 (48-fold) being most highly overexpressed compared to control littermates. Subcellular fractionation of liver cells from Phb1 KO mice indicated that hepatocytes are the main source of WNT ligands. Immunostaining and cellular colocalization analysis of Phb1 KO livers demonstrated expression of WNT7a, WNT10a, and WNT16 in hepatocytes. Chromatin immunoprecipitation revealed increased binding of transcription factor E2F1 (E2F1) to the Wnt10a promoter in Phb1 KO livers and WNT9A in HepG2 cells. PHB1 silencing in HepG2 cells activated WNT signaling, whereas its overexpression caused inactivation of this pathway. PHB1 silencing in HepG2 cells induced the expression of multiple WNT ligands of which WNT9A induction was partly regulated through E2F1. Conclusion: PHB1 acts as a negative regulator of WNT signaling, and its down-regulation causes the induction of multiple WNT ligands and downstream activation of canonical WNT-beta-catenin signaling in murine liver and human HCC cells, in part through E2F1.

Background and aims

Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge.

Approach and results

We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC.

Conclusions

Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.