MAT1A Knockout Mice Research Articles

S-adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells.

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

S -adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol-fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real-time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein-protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF-7 with SAMe or its metabolite 5'-methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)-Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho-Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho-Ubc9 levels increased in human liver, breast, and colon cancers. Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.

Methionine adenosyltransferase 1A gene deletion disrupts hepatic very low-density lipoprotein assembly in mice

Very low-density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S-adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3-month-old MAT1A-knockout mice (3-KO), with no signs of liver injury, and in 8-month-old MAT1A-knockout mice (8-KO), harboring nonalcoholic steatohepatitis. In 3-KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N-methyltransferase. MAT1A deletion also increased VLDL-apolipoprotein B secretion, leading to small, lipid-poor VLDL particles. Administration of SAMe to 3-KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8-KO mouse liver, in which TG accumulated and the phosphatidylcholine-to-phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3-KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low-density lipoprotein subclasses and decrease in high-density lipoprotein subclasses. MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A-KO mice.

S-Adenosylmethionine regulates connexins sub-types expressed by hepatocytes

Intercellular communication via GAP Junctions plays an important role in tissue homeostasis, apoptosis, carcinogenesis, cell proliferation and differentiation. Hepatocyte connexins (Cx) 26 and 32 levels are decreased during the de-differentiation process of primary hepatocytes in culture, a situation that is also characterized by a decrease in S-Adenosylmethionine (SAMe) levels. In this current study, we show that SAMe supplementation in cultured hepatocytes every 12 h, leads to an up-regulation of Cx26 and 32 mRNA and protein levels and blocks culture-induced Cx43 expression, although it failed to increase Cx26 and 32 membrane localization and GAP junction intracellular communication. SAMe reduced nuclear β-catenin accumulation, which is known to stimulate the TCF/LEF-dependent gene transcription of Cx43. Moreover SAMe-induced reduction in Cx43 and β-catenin was prevented by the proteasome inhibitor MG132, and was not mediated by GSK3 activity. SAMe, and its metabolite 5′-methylthioadenosine (MTA) increased Cx26 mRNA in a process partially mediated by Adenosine A 2A receptors but independent of PKA. Finally livers from MAT1A knockout mice, characterized by low hepatic SAMe levels, express higher Cx43 and lower Cx26 and 32 protein levels than control mice. These results suggest that SAMe maintains a characteristic expression pattern of the different Cxs in hepatocytes by differentially regulating their levels.

Activation of LKB1-Akt Pathway Independent of Phosphoinositide 3-Kinase Plays a Critical Role in the Proliferation of Hepatocellular Carcinoma From Nonalcoholic Steatohepatitis

LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient [SAMe-D]) from MAT1A knockout (MAT1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein-activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. The SAMe-D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies.

S -Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes

Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration.

S‐Adenosylmethionine in cell growth, apoptosis and liver cancer

S-Adenosylmethionine (SAMe), the principal biological methyl donor, is synthesized from methionine and ATP in a reaction catalyzed by methionine adenosyltransferase (MAT). In mammals, two genes (MAT1A and MAT2A), encode for two homologous MAT catalytic subunits, while a third gene MAT2beta, encodes for the beta-subunit that regulates MAT2A-encoded isoenzyme. Normal liver expresses MAT1A, whereas extrahepatic tissues express MAT2A. MAT2A and MAT2 beta are induced in human hepatocellular carcinoma (HCC), which facilitate cancer cell growth. Patients with cirrhosis of various etiologies, including alcohol, have decreased hepatic MAT activity and SAMe biosynthesis. Consequences of hepatic SAMe deficiency as illustrated by the Mat1a knock-out mouse model include increased susceptibility to steatosis and oxidative liver injury, spontaneous development of steatohepatitis and HCC. Predisposition to HCC can be partly explained by the effect of SAMe on growth. Thus, SAMe inhibits the mitogenic effect of growth factors such as hepatocyte growth factor and, following partial hepatectomy, a fall in SAMe level is required for the liver to regenerate. During liver regeneration, the fall in hepatic SAMe is transient. If the fall were to persist, it would favor a proliferative phenotype and, ultimately, development of HCC. Not only does SAMe control liver growth, it also regulates apoptosis. Interestingly, SAMe is anti-apoptotic in normal hepatocytes but pro-apoptotic in liver cancer cells. In liver cancer cells but not in normal human hepatocytes, SAMe can selectively induce Bcl-x(S), an alternatively spliced isoform of Bcl-x(L) that promotes apoptosis. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

Role of methionine adenosyltransferase and S-adenosylmethionine in alcohol-associated liver cancer

Two genes ( MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and, in the liver, a precursor of glutathione. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. MAT2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC) MAT1A is replaced by MAT2A. This is important pathogenetically because MAT2A expression is associated with lower SAMe levels and faster growth, whereas exogenous SAMe treatment inhibits growth. Rats fed ethanol intragastrically for 9 weeks also exhibit a relative switch in hepatic MAT expression, decreased SAMe levels, hypomethylation of c- myc, increased c- myc expression, and increased DNA strand break accumulation. Patients with alcoholic liver disease have decreased hepatic MAT activity owing to both decreased MAT1A expression and inactivation of the MAT1A-encoded isoenzymes, culminating in decreased SAMe biosynthesis. Consequences of chronic hepatic SAMe depletion have been examined in the MAT1A knockout mouse model. In this model, the liver is more susceptible to injury. In addition, spontaneous steatohepatitis develops by 8 months, and HCC develops by 18 months. Accumulating evidence shows that, in addition to being a methyl donor, SAMe controls hepatocyte growth response and death response. Whereas transient SAMe depletion is necessary for the liver to regenerate, chronic hepatic SAMe depletion may lead to malignant transformation. It is interesting that SAMe is antiapoptotic in normal hepatocytes, but proapoptotic in liver cancer cells. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.

Role of S-adenosylmethionine in two experimental models of pancreatitis.

Severe necrotizing pancreatitis occurs in young female mice fed a choline-deficient and ethionine-supplemented (CDE) diet. Although the mechanism of the pancreatitis is unknown, one consequence of this diet is depletion of hepatic S-adenosylmethionine (SAM). SAM formation is catalyzed by methionine adenosyltransferases (MATs), which are encoded by liver-specific (MAT1A) and non-liver-specific (MAT2A) genes. In this work, we examined changes in pancreatic SAM homeostasis in mice receiving the CDE diet and the effect of SAM treatment. We found that both MAT forms are expressed in normal pancreas and pancreatic acini. After 48 h of the CDE diet, SAM levels decreased 50% and MAT1A-encoded protein disappeared via post-translational mechanisms, whereas MAT2A-encoded protein increased via pretranslational mechanisms. CDE-fed mice exhibited extensive necrosis, edema, and acute pancreatic inflammatory infiltration, which were prevented by SAM treatment. However, old female mice consuming the CDE diet that do not develop pancreatitis showed a similar fall in pancreatic SAM level. SAM was also protective in cerulein-induced pancreatitis in the rat, but the protection was limited. Although the pancreatic SAM level fell by more than 80% in the MAT1A knockout mice, no pancreatitis developed. This study thus provides several novel findings. First, the so-called liver-specific MAT1A is highly expressed in the normal pancreas and pancreatic acini. Second, the CDE diet causes dramatic changes in the expression of MAT isozymes by different mechanisms. Third, in contrast to the situation in the liver, where absence of MAT1A and decreased hepatic SAM level can lead to spontaneous tissue injury, in the pancreas the roles of SAM and MAT1A appear more complex and remain to be defined.

Role of abnormal methionine metabolism in alcoholic liver injury

Methionine catabolism occurs mostly in the liver through the formation of S-adenosylmethionine (SAM) in a reaction catalyzed by methionine adenosyltransferase (MAT). S-adenosylmethionine is the principal biologic methyl donor, a precursor for polyamines, and in liver, it is also a precursor for reduced glutathione (GSH). Liver-specific and non-liver-specific MAT are products of two different genes, MAT1A and MAT2A, respectively. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and induced during liver growth and de-differentiation. The type of MAT expressed by the cell affects the steady-state SAM level, DNA methylation, and growth rate. This has been demonstrated further by using the MAT1A knockout mouse model in which hepatic SAM and GSH levels decrease, the liver becomes larger and more susceptible to injury, and steatohepatitis develops spontaneously. Altered methionine metabolism in alcoholic liver disease results in decreased transmethylation and transsulfuration, changes that may play important pathogenic roles. Major changes include a relative switch in MAT expression; decreased hepatic SAM, GSH, and DNA methylation levels; decreased homocysteine metabolism; and hyperhomocysteinemia. Consequences of hepatic DNA hypomethylation include increased expression of c- myc and DNA strand break accumulation. One possible consequence of hyperhomocysteinemia is increased fibrogenesis. Abnormal methionine metabolism may also occur in Kupffer cells, which express both forms of MAT. If SAM levels also decrease in these cells, this may contribute to the induction of tumor necrosis factor (TNF) expression and release. In summary, altered hepatic methionine metabolism can have serious consequences that affect not only hepatocytes, but also hepatic stellate and Kupffer cells. These changes can lead to impaired antioxidant defense, altered gene expression, promotion of fibrogenesis, and even hepatocarcinogenesis.

Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A.

In mammals, methionine metabolism occurs mainly in the liver via methionine adenosyltransferase-catalyzed conversion to S-adenosylmethionine. Of the two genes that encode methionine adenosyltransferase(MAT1Aand MAT2A), MAT1A is mainly expressed in adult liver whereas MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic S-adenosylmethionine content and hyperplasia and spontaneously develop nonalcoholic steatohepatitis. In this study, we examined whether chronic hepatic S-adenosylmethionine deficiency generates oxidative stress and predisposes to injury and malignant transformation. Differential gene expression in MAT1A knockout mice was analyzed following the criteria of the Gene Ontology Consortium. Susceptibility of MAT1A knockout mice to CCl4-induced hepatotoxicity and malignant transformation was determined in 3- and 18-month-old mice, respectively. Analysis of gene expression profiles revealed an abnormal expression of genes involved in the metabolism of lipids and carbohydrates in MAT1A knockout mice, a situation that is reminiscent of that found in diabetes, obesity, and other conditions associated with nonalcoholic steatohepatitis. This aberrant expression of metabolic genes in the knockout mice was associated with hyperglycemia, increased hepatic CYP2E1 and UCP2 expression and triglyceride levels, and reduced hepatic glutathione content. The knockout animals have increased lipid peroxidation and enhanced sensitivity to CCl4-induced liver damage, which was largely due to increased CYP2E1 expression because diallyl sulfide, an inhibitor of CYP2E1, prevented CCl4-induced liver injury. Hepatocellular carcinoma developed in more than half of the knockout mice by 18 months of age. Taken together, our findings define a critical role for S-adenosylmethionine in maintaining normal hepatic function and tumorigenesis of the liver.