Elena R. García-Trevijano

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(MAT1A and 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 Sadenosylmethionine 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 18month‐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.

S-Adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S-adenosylmethionine in the maintenance of the differentiated status of the liver

Methionine metabolism starts with the formation of S‐adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (MAT). MAT is the product of two different genes: MAT1A, which is expressed only in the adult liver, and MAT2A, which is widely distributed, expressed in the fetal liver, and replaces MAT1A in hepatocarcinoma. In the liver, preservation of high expression of MAT1A and low expression of MAT2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes MAT1A expression progressively decreased, as described for other liver‐specific genes, and MAT2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased MAT1A expression AdoMet addition markedly increased MAT1A transcription in a dose‐dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3‐deazaadenosine and L‐ethionine, but not D‐ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates MAT1A and MAT2A expression and helps to maintain the differentiated status of the hepatocyte.—García‐Trevijano, E. R., Ujue Latasa, M., Victoria Carretero, M., Bera‐sain, C., Mato, J. M., and Avila, M. A. S‐Adenosylmethionine regulates MAT1A and MAT2A gene expression in cultured rat hepatocytes: a new role for S‐adenosylme‐thionine in the maintenance of the differentiated status of the liver. The FASEB J. 14, 2511–2518 (2000)

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 κB (NFκB), 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 NFκB 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.

5′‐methylthioadenosine modulates the inflammatory response to endotoxin in mice and in rat hepatocytes

5′‐methylthioadenosine (MTA) is a nucleoside generated from S‐adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti‐inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro‐inflammatory cytokines. MTA administration completely prevented LPS‐induced lethality. The life‐sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor‐α (TNF‐α), inducible NO synthase (iNOS) expression, and by the stimulation of IL‐10 synthesis. These responses to MTA were also observed in LPS‐treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro‐inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen‐activated protein kinase (MAPK), c‐jun phosphorylation, inhibitor kappa B alpha (IκBα) degradation, and nuclear factor κB (NFκB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti‐inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases. (HEPATOLOGY 2004;39:1088–1098.)

INDUCTION OF TIMP-1 EXPRESSION IN RAT HEPATIC STELLATE CELLS AND HEPATOCYTES : A NEW ROLE FOR HOMOCYSTEINE IN LIVER FIBROSIS

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.

Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF

Arginine methylation of RAF proteins limits ERK activation after growth factor stimulation, thereby determining the biological response. Minimized by Methylation Many growth factors signal through the RAS to RAF to extracellular signal–regulated kinase (RAS-ERK) cascade, a signaling pathway that involves the sequential phosphorylation and activation of a series of protein kinases. Despite their common activation of RAS-ERK signaling, however, different growth factors elicit distinct biological responses. For instance, nerve growth factor (NGF) stimulates differentiation of PC12 cells, whereas epidermal growth factor (EGF) stimulates PC12 cell proliferation. Andreu-Pérez et al. found that the amplitude and duration of ERK phosphorylation in response to certain growth factors are kept in check by methylation of activated RAF proteins, a modification that enhanced RAF protein degradation and thereby limited downstream signaling in the RAS-ERK pathway. When RAF methylation was prevented experimentally, the amplitude and duration of the ERK signal elicited by EGF in PC12 cells (normally smaller and briefer than that elicited by NGF) increased, and the biological response switched from proliferation to differentiation. The RAS to extracellular signal–regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.

Hepatocyte growth factor induces MAT2A expression and histone acetylation in rat hepatocytes: role in liver regeneration

SPECIFIC AIMSWe have studied the molecular mechanisms and mediators behind the induction of methionine adenosyltransferase 2 A (MAT2A) gene expression in the regenerating rat liver after partial he...

S-Adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes

BACKGROUND/AIMS Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines. METHODS The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively. RESULTS S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter. CONCLUSIONS Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.

Methylthioadenosine reverses brain autoimmune disease

To assess the immunomodulatory activity of methylthioadenosine (MTA) in rodent experimental autoimmune encephalomyelitis (EAE) and in patients with multiple sclerosis.

Transformed but not normal hepatocytes express UCP2.

Uncoupling protein 2 (UCP2) expression in liver is restricted to non‐parenchymal cells. By means of differential display screening between normal rat liver and H4IIE hepatoma cells we have isolated a cDNA clone encompassing part of UCP2 cDNA. Northern blot analysis revealed that UCP2 is expressed in some hepatocarcinoma cell lines, while it is absent in adult hepatocytes. UCP2 mRNA in H4IIE cells was downregulated when cells were cultured for 36 h in 0.1% serum and its expression was restored upon addition of 10% serum or phorbol esters. Hypomethylation of UCP2 was observed in transformed UCP2 expressing cells. Our results indicate that UCP2 is expressed in some hepatocarcinoma cell lines and that serum components may participate in maintaining elevated UCP2 levels.