This is consistent
with the observation that NAM administration induces a marked reduction of hepatic SAM content as well as an increase in SAH content. Next, we examined the Ras and JAK/STAT signaling pathways. We have shown the persistent activation Vadimezan of the Ras and JAK/STAT signaling pathways through suppression of Ras and JAK/STAT inhibitors such as RASSF1A and SOCS1 in GNMT-KO mouse liver.6 In the present study, we observed that NAM administration to GNMT-KO mice prevented the hepatic suppression of RASSF1A and SOCS1 protein expression (Fig. 5C,D). Concomitant with this normalization of RASSF1A protein expression, we observed that the livers of NAM-treated GNMT-KO mice exhibited markedly lower expression of Ras mTOR inhibitor and downstream effectors of Ras involved in cell proliferation and survival (including pRAF1 and pERK1/2) than untreated knockout animals (Fig. 5C). Ras activity, assessed by immunoprecipitation with anti-pan Ras
antibody and probed with anti-RAF1 antibody, was markedly increased in GNMT-KO mice liver but much less elevated in NAM-treated GNMT-KO mice (Fig. 5C). Similarly, pERK1/2 content increased more than 15-fold in GNMT-KO livers and only seven-fold in NAM-treated KO livers (Fig. 5C). The levels of pRAF1 were elevated in GNMT-KO mice compared with WT animals but were similar in WT and NAM-treated GNMT-KO mice (Fig. 5C). Similarly, concurrent with the normalization of SOCS1 protein expression, we observed that whereas the liver protein levels of pSTAT3 and of the downstream mitotic markers effectors pHistone 3 and Ki-67 were significantly elevated in both GNMT-KO mice groups compared with WT animals, induction in the NAM-treated group was significantly lower than in the untreated group (Fig. 5D). The protein levels of activated JAK2 tyrosine kinase (pJAK2) and cyclin D1 were elevated in GNMT-KO mice compared with WT animals but were similar in WT and NAM-treated GNMT-KO mice (Fig. 5D). SAM is synthesized
by methionine adenosyltransferase (MAT). In mammals, there are three isoforms of MAT (MATI, Thalidomide MATII, and MATIII) that are encoded by two genes (MAT1A and MAT2A). MATI and MATIII are tetrameric and dimeric forms, respectively, of the same subunit (α1) encoded by MAT1A, whereas the MATII isoform is a tetramer of a different subunit (α2) encoded by MAT2A. Adult differentiated liver expresses predominantly MAT1A, whereas extrahepatic tissues and fetal liver express MAT2A.2, 18 The prevalent liver form, MATIII, has lower affinity for its substrates, is activated by methionine, and has higher Vmax, contrasting with the other two enzymes.2, 18 Based on the differential properties of hepatic MAT isoforms, it has been postulated that MATIII is the truly liver-specific isoform.2 Under normal conditions, MATI synthesizes most SAM required by the hepatic cells (as MATII does outside the liver).