Loss of the glycine N-methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice

Abstract

Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice.

Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.

Fig. 1. Deletion of GNMT leads to steatosis, fibrosis, and HCC

(A) At 3 months of age, macro and microvesicular steatosis (white droplets) could be seen through the hepatic lobule in GNMT-knockout (GNMT-KO) mice compared with wild-type (WT) animals. Collagen deposits (stained red) indicate moderate liver fibrosis. By 8 months of age, liver steatosis and fibrosis in mutant mice were more prominent. At least 10 animals per group were examined. (B) At 8 months of age, livers from GNMT-KO mice also had multifocal HCC. Liver cords up to 5 cells thick, lined by endothelial cells, could be seen, with occasional pseudogland formation. Staining with the nuclear proliferation marker Ki67 (red spots) indicates a higher proportion of cells in the cell cycle compared with WT cells. At least 10 animals per group were examined (E&H, eosin and hematoxylin staining; SR, Sirius red staining). Original magnification ×40.

Fig. 2. Activation of Ras and JAK/STAT pathways in GNMT-mutant mice liver

(A) Representative Western blot analysis of Ras downstream effectors (pRAF, pMEK1/2, and pERK1/2). Phosphorylation of RAF, MEK1/2, and ERK1/2 was markedly increased in liver tumors from GNMT-knockout (GNMT-KO) mice as compared with wild-type (WT) animals. One representative data set is shown out of 4 experiments performed. (B) Ras activity, assessed by immunoprecipitation with anti-pan Ras antibody and probed with anti-RAF-1 antibody, was markedly increased in livers from 3-month-old GNMT mice and in liver tumors from 8-month-old GNMT-mutant mice. Four animals from each subgroup were analyzed (WT mice, open bars; GNMT-KO mice, filled bars). (C) Representative Western blot analysis of JAK/STAT downstream effectors (pJAK1/2, pSTAT1, pSTAT3, cyclin D1 and D2, and Bcl-xL). Phosphorylation of JAK1/2, STAT1, and STAT 3 and protein levels of the JAK/STAT target genes, including the proliferation proteins cyclin D1 and cyclin D2 and the antiapoptotic protein Bcl-xL, were markedly increased in liver tumors from GNMT-KO mice. One representative data set is shown of 4 experiments performed. Unless otherwise indicated, animals were all 8 months old.

Fig. 3. Deletion of GNMT induces DNA and histone hypermethylation

(A) Quantification of global DNA methylation. Total cytosine and 5-methylcytosine (5mC) content was determined in genomic DNA samples isolated from livers of wild-type (WT) mice at 3 and 8 months of age, in livers from 3-month-old GNMT-knockout (GNMT-KO) mice, and in liver tumors from 8-month-old knockout animals, and relative 5mC content, expressed as the percentage of total cytosine content (methylated and nonmethylated), was determined. Data represent means ± SDs from 5 animals per subgroup (*P < 0.05 versus WT). (B) Analysis of sequence-specific DNA methylation. CpG DNA methylation of a subtelomeric DNA region at chromosome 1 was determined by PCR analysis after bisulfite modification in samples isolated from livers of 3- and 8-month-old WT mice, in liver samples from 3-month-old GNMT-knockout mice, and in liver tumors from 8-month-old knockout animals. Data represent means ± SDs. from 5 animals per subgroup (*P < 0.05 versus WT mice). (c–e) Analysis of RASSF1 and SOCS2 promoter methylation. (C) To determine the CpG DNA methylation status of RASSF1 and SOCS2 promoters in liver samples from 3-month-old mice, we employed bisulfite genomic sequencing because the less sensitive technique, based on the use of MspI and HpaII restriction endonucleases, used at 8 month of age, did not reveal differences between GNMT-knockout and WT livers at 3 month of age. Data represent means ± SDs from 3 animals per subgroup (*P < 0.05 versus WT). (D, E) To analyze the methylation state of the promoters of RASSF1 and SOCS2 at 8 month of age, Southern blot analyses of genomic DNA digested with the methylation-sensitive isoschizomer restriction enzymes MspI and HpaII were performed as described in the Materials and Methods sections. Two MspI fragments were detected in Southern blots when hepatic DNA was hybridized with a RASSF1 probe (D). All DNA samples from WT mice liver digested with HpaII showed the same pattern of restriction fragments as that produced by MspI digestion, indicating that the CCGG sites were unmethylated. However, all DNA samples from GNMT-KO mice liver tumors digested with HpaII showed additional higher-molecular-weight bands (indicated by an arrow), showing that the RASSF1 promoter was hypermethylated. When hepatic DNA was hybridized with a SOCS2 probe (E), an additional band (indicated by arrows) was also detected when DNA from GNMT-KO liver tumors was digested with HpaII compared with when digested with MspI, whereas liver samples from WT mice showed the same pattern after digestion with either enzyme (HpaII-WT, DNA samples from WT liver mice digested with HpaII; HpaII-KO, DNA samples from GNMT-KO liver tumors digested with HpaII; 4 animals from each subgroup were analyzed). (F, G) Analysis of H3K27me3 bound to RASSF1 and SOCS2. The amount of H3K27me3 bound to RASSF1 (F) and SOCS2 (G) was determined by chromatin immunoprecipitation, as described in the Materials and Methods section in liver samples of 3- and 8-month-old WT mice, in liver samples of 3-month-old GNMT-knockout mice, and in liver tumors from 8-month-old knockout animals. Relative values of H3K27me3 are shown. Data represent means ± SDs from 5 animals per subgroup (*P < 0.05 versus WT).