S-Adenosylmethionine Negatively Regulates the Mitochondrial Respiratory Chain Repressor MCJ in the Liver.

MCJ (Methylation-Controlled J protein), an endogenous repressor of the mitochondrial respiratory chain, is upregulated in multiple liver diseases but little is known about how it is regulated. S-adenosylmethionine (SAMe), the biological methyl donor, is frequently depleted in chronic liver diseases. Here, we show that SAMe negatively regulates MCJ in the liver. While deficiency in methionine adenosyltransferase alpha 1 (MATα1), enzyme that catalyzes SAMe biosynthesis, leads to hepatic MCJ upregulation, MAT1A overexpression and SAMe treatment reduced MCJ expression. We found that MCJ is methylated at lysine residues and that it interacts with MATα1 in liver mitochondria, likely to facilitate its methylation. Lastly, we observed that MCJ is upregulated in alcohol-associated liver disease, a condition characterized by reduced MAT1A expression and SAMe levels along with mitochondrial injury. MCJ silencing protected against alcohol-induced mitochondrial dysfunction and lipid accumulation. Our study demonstrates a new role of MATα1 and SAMe in reducing hepatic MCJ expression.

CSNK2A1-mediated MAX phosphorylation upregulates HMGB1 and IL-6 expression in cholangiocarcinoma progression.

BACKGROUND: We established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia. METHODS: BALB/c mice were administered DEN by oral gavage. Cells isolated from livers were analyzed for expression of CSNK2A1, MAX and MAX-interacting proteins. Human CCA cell lines (MzChA-1, HuCCT1), normal human cholangiocyte (H69), human hepatic stellate cells (LX-2), macrophages (RAW 264.7), and primary hepatic cells were used for cellular and molecular biology assays. RESULTS: Expression of MAX, CSNK2A1, C-MYC, ß-catenin, HMGB1, and IL-6 was upregulated in hepatic cells from CCA liver tissue. The half-life of MAX is higher in CCA cells, and this favors their proliferation. Overexpression of MAX increased growth, migration, and invasion of MzChA-1, whereas silencing of MAX had the opposite effect. MAX positively regulated IL-6 and HMGB1 through paracrine signaling in HepG2, LX2, and RAW cells and autocrine signaling in MzChA-1 cells. CSNK2A1-mediated MAX phosphorylation shifts MAX-MAX homodimer to C-MYC-MAX and ß-catenin-MAX heterodimers and increases the HMGB1 and IL-6 promoter activities. Increase of MAX phosphorylation promotes cell proliferation, migration, invasion, and cholangiocarcinogenesis. The casein kinase 2 inhibitor CX-4945 induces cell cycle arrest and inhibits cell proliferation, migration, invasion, and carcinogenesis in MzChA-1 cells through the downregulation of CSNK2A1, MAX, and MAX-interaction proteins. CONCLUSION: C-MYC-MAX and ß-catenin-MAX binding to E-box site or ß-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities, respectively. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. CX-4945 provides perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.

Hyperphosphorylation of hepatic proteome characterizes nonalcoholic fatty liver disease in S-adenosylmethionine deficiency.

Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a -/- mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a -/- mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a -/- mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a -/- mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a -/- mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.

Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models.

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12's scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12's scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12's site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12's phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47's collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.

Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid ß-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

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

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.

Star-related lipid transfer protein 10 (STARD10): a novel key player in alcohol-induced breast cancer progression.

BACKGROUND: Ethanol abuse promotes breast cancer development, metastasis and recurrence stimulating mammary tumorigenesis by mechanisms that remain unclear. Normally, 35% of breast cancer is Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)-positive that predisposes to poor prognosis and relapse, while ethanol drinking leads to invasion of their ERBB2 positive cells triggering the phosphorylation status of mitogen-activated protein kinase. StAR-related lipid transfer protein 10 (STARD10) is a lipid transporter of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); changes on membrane composition of PC and PE occur before the morphological tumorigenic events. Interestingly, STARD10 has been described to be highly expressed in 35-40% of ERBB2-positive breast cancers. In this study, we demonstrate that ethanol administration promotes STARD10 and ERBB2 expression that is significantly associated with increased cell malignancy and aggressiveness. MATERIAL AND METHODS: We investigated the effect of ethanol on STARD10-ERBB2 cross-talk in breast cancer cells, MMTV-neu transgenic mice and in clinical ERBB2-positive breast cancer specimens with Western Blotting and Real-time PCR. We also examined the effects of their knockdown and overexpression on transient transfected breast cancer cells using promoter activity, MTT, cell migration, calcium and membrane fluidity assays in vitro. RESULTS: Ethanol administration induces STARD10 and ERBB2 expression in vitro and in vivo. ERBB2 overexpression causes an increase in STARD10 expression, while overexpression of ERBB2's downstream targets, p65, c-MYC, c-FOS or c-JUN induces STARD10 promoter activity, correlative of enhanced ERBB2 function. Ethanol and STARD10-mediated cellular membrane fluidity and intracellular calcium concentration impact ERBB2 signaling pathway as evaluated by enhanced p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters. CONCLUSION: Our finding proved that STARD10 and ERBB2 positively regulate each other's expression and function. Taken together, our data demonstrate that ethanol can modulate ERBB2's function in breast cancer via a novel interplay with STARD10.

Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated-Beta-Catenin Signaling in Murine Liver and Human Liver Cancer Cells.

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.

SUMOylation and phosphorylation cross-talk in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and occurs predominantly in patients with underlying chronic liver disease and cirrhosis. The large spectrum of protein post-translational modification (PTM) includes numerous critical signaling events that occur during neoplastic transformation. PTMs occur to nearly all proteins and increase the functional diversity of proteins. We have reviewed the role of two major PTMs, SUMOylation and phosphorylation, in the altered signaling of key players in HCC. SUMOylation is a PTM that involves addition of a small ubiquitin-like modifiers (SUMO) group to proteins. It is known to regulate protein stability, protein-protein interactions, trafficking and transcriptional activity. The major pathways that are regulated by SUMOylation and may influence HCC are regulation of transcription, cell growth pathways associated with B-cell lymphoma 2 (Bcl-2) and methionine adenosyltransferases (MAT), oxidative stress pathways [nuclear erythroid 2-related factor 2 (Nrf2)], tumor suppressor pathways (p53), hypoxia-inducible signaling [hypoxia-inducible factor-1 (HIF-1)], glucose and lipid metabolism, nuclear factor kappa B (NF-κB) and ß-Catenin signaling. Phosphorylation is an extensively studied PTM in HCC. The mitogen-activated protein kinase (MAPK), phosphatidyl inositol/AK-strain transforming (PI3K/AKT), and C-SRC pathways have been extensively studied for deregulation of kinases and alteration in signaling of targets through phosphorylation of their substrates. Cross-talk between phosphorylation and SUMOylation is known to influence transcriptional activity of proteins and protein-protein interactions. In HCC, several SUMOylation-dependent phosphorylation events have been studied such as MAPK activation and c-SRC activity that have been reviewed in this work. The drastic effects of site-specific phosphorylation or SUMOylation on enzyme activity of signaling players and its effect on growth and tumorigenesis suggests that these PTMs are novel targets for therapeutic intervention in HCC.

SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Alcohol acts through numerous pathways leading to alcoholic liver disease (ALD). Cytochrome P450 (CYP2E1), an ethanol-inducible enzyme, metabolizes ethanol-producing toxic reactive oxygen species (ROS) and is regulated at the posttranslational level. Small ubiquitin-like modifier (SUMO)ylation is a posttranslational modification that involves the addition of SUMOs, which modulate protein stability, activity, and localization. We demonstrated that ubiquitin-conjugation enzyme 9, the SUMO-conjugating enzyme, is induced in the livers of an intragastric ethanol mouse model. Our aim is to examine whether SUMOylation could regulate ethanol-induced CYP2E1 expression in ALD and to elucidate the molecular mechanism(s). CYP2E1 and UBC9 expression in vitro and in vivo was detected by real-time PCR and immunoblotting/immunostaining. SUMOylation was assayed by mass spectrometry and coimmunoprecipitation. Ubc9 expression was induced in ethanol-fed mouse livers, and silencing inhibited ethanol-mediated CYP2E1 microsomal retention and enzymatic activity. CYP2E1 SUMOylation was found to be induced by ethanol in vitro and in vivo. Ubc9 silencing prevents ethanol-induced lipid accumulation and ROS production. UBC9 was highly expressed in human ALD livers. Finally, we found that lysine 410 is a key SUMOylated residue contributing to CYP2E1 protein stability and activity preventing CYP2E1 SUMOylation. Ethanol-mediated up-regulation of CYP2E1 via SUMOylation enhancing its protein stability and activity and may have important implications in ALD.-Tomasi, M. L., Ramani, K., Ryoo, M., Cossu, C., Floris, A., Murray, B. J., Iglesias-Ara, A., Spissu, Y., Mavila, N. SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease.

Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation.

Alcoholic liver injury is associated with hepatic stellate cell (HSC) activation. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinase C and cyclin-D1, which is regulated by its phosphorylation, and spatiotemporally controls cell proliferation, invasiveness, and chemotaxis. HSC activation induces AKAP12 expression, but the role of AKAP12's scaffolding activity in liver function is unknown. Because AKAP12 phosphorylation is enhanced in ethanol-treated HSCs, we examined AKAP12's scaffolding functions in alcohol-mediated HSC activation and liver injury. AKAP12 expression, interaction, and phosphorylation were assayed in in vitro and in vivo ethanol models and human subjects by real-time PCR, coimmunoprecipitation, immunoblotting, and phosphorylated proteomics/Phos-tag. Ethanol induced AKAP12 phosphorylation in the liver and in primary HSCs, but not in hepatocytes. AKAP12's scaffolding activity for protein kinase C/cyclin-D1 decreased in ethanol-treated HSCs but not hepatocytes. AKAP12 negatively regulated HSC activation, which was reversed by ethanol-mediated AKAP12 phosphorylation. AKAP12 interacted with heat shock protein 47 (HSP47), which chaperones collagen and induces its secretion. Ethanol inhibited AKAP12-HSP47 and induced HSP47-collagen interaction. Ethanol-induced phosphorylated AKAP12 was unable to bind to HSP47 compared with its unphosphorylated counterpart, thereby proving that ethanol-mediated phosphorylation of AKAP12 inhibited the HSP47-AKAP12 scaffold. Silencing AKAP12 facilitated the chaperoning of collagen by HSP47. Hence, AKAP12 scaffolds HSP47 and regulates collagen-HSP47 interaction. Ethanol quenches AKAP12's scaffolding activity through phosphorylation and facilitates HSC activation.

Methionine adenosyltransferases in liver health and diseases.

Methionine adenosyltransferases (MATs) are essential for cell survival because they catalyze the biosynthesis of the biological methyl donor S-adenosylmethionine (SAMe) from methionine and adenosine triphosphate (ATP). Mammalian cells express two genes, MAT1A and MAT2A, which encode two MAT catalytic subunits, α1 and α2, respectively. The α1 subunit organizes into dimers (MATIII) or tetramers (MATI). The α2 subunit is found in the MATII isoform. A third gene MAT2B, encodes a regulatory subunit ß, that regulates the activity of MATII by lowering the inhibition constant (Ki) for SAMe and the Michaelis constant (Km) for methionine. MAT1A expressed mainly in hepatocytes maintains the differentiated state of these cells whereas MAT2A and MAT2B are expressed in non-parenchymal cells of the liver (hepatic stellate cells [HSCs] and Kupffer cells) and extrahepatic tissues. A switch from the liver-specific MAT1A to MAT2A has been observed during conditions of active liver growth and de-differentiation. Liver injury, fibrosis, and cancer are associated with MAT1A silencing and MAT2A/MAT2B induction. Even though both MAT1A and MAT2A are involved in SAMe biosynthesis, they exhibit distinct molecular interactions in liver cells. This review provides an update on MAT genes and their roles in liver pathologies.

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes.

Prohibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulin-like growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knock-out mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8-20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ∼30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and co-localized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40-50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growth-suppressive effect of PHB1 in the liver.

Ubiquitin-Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of the Inflammatory Response.

Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-κB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-κB inhibitor α (Ikbα) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikbα stability by SUMO-1 conjugation that further regulates NF-κB signaling. The role of UBC9 in liver inflammation is unknown. We reported that CDK1-mediated phosphorylation of UBC9 enhanced its stability. Herein, we describe an anti-inflammatory role of UBC9 that is lost when it is phosphorylated during inflammation. LPS exposure caused induction in UBC9 phosphorylation and CDK1 activation specifically in Kupffer cells in vivo and in RAW264.7 macrophages in vitro. Silencing or overexpression experiments in vitro and in vivo showed that UBC9 was required to blunt the proinflammatory response elicited by LPS. LPS stimulation raised the binding of phospho-UBC9 but not the unphosphorylated counterpart, to Ikbα in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-κB signaling by regulating Ikbα and this may be a novel mechanism that deregulates liver inflammatory signaling.

Methionine adenosyltransferase α2 sumoylation positively regulate Bcl-2 expression in human colon and liver cancer cells.

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and inhibits apoptosis via Bcl-2 by unknown mechanism. Methionine adenosyltransferase 2A (MAT2A) encodes for MATα2, the catalytic subunit of the MATII isoenzyme that synthesizes S-adenosylmethionine (SAMe). Ubc9, Bcl-2 and MAT2A expression are up-regulated in several malignancies. Exogenous SAMe decreases Ubc9 and MAT2A expression and is pro-apoptotic in liver and colon cancer cells. Here we investigated whether there is interplay between Ubc9, MAT2A and Bcl-2. We used human colon and liver cancer cell lines RKO and HepG2, respectively, and confirmed key finding in colon cancer specimens. We found MATα2 can regulate Bcl-2 expression at multiple levels. MATα2 binds to Bcl-2 promoter to activate its transcription. This effect is independent of SAMe as MATα2 catalytic mutant was also effective. MATα2 also directly interacts with Bcl-2 to enhance its protein stability. MATα2's effect on Bcl-2 requires Ubc9 as MATα2's stability is influenced by sumoylation at K340, K372 and K394. Overexpressing wild type (but not less stable MATα2 sumoylation mutants) protected from 5-fluorouracil-induced apoptosis in both colon and liver cancer cells. Colon cancer have higher levels of sumoylated MATα2, total MATα2, Ubc9 and Bcl-2 and higher MATα2 binding to the Bcl-2 P2 promoter. Taken together, Ubc9's protective effect on apoptosis may be mediated at least in part by sumoylating and stabilizing MATα2 protein, which in turn positively maintains Bcl-2 expression. These interactions feed forward to further enhance growth and survival of the cancer cell.

Role of methionine adenosyltransferase α2 and ß phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.

Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATß proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATß proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATß proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATß predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATß but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATß phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.

Mechanism and significance of changes in glutamate-cysteine ligase expression during hepatic fibrogenesis.

GSH is synthesized sequentially by glutamate-cysteine ligase (GCL) and GSH synthase and defends against oxidative stress, which promotes hepatic stellate cell (HSC) activation. Changes in GSH synthesis during HSC activation are poorly characterized. Here, we examined the expression of GSH synthetic enzymes in rat HSC activation and reversion to quiescence. Expression of the GCL catalytic subunit (GCLC) fell during HSC activation and increased when activated HSCs revert back to quiescence. Blocking the increase in GCLC expression kept HSCs in an activated state. Activated HSCs have higher nuclear levels and binding activity of MafG to the antioxidant response element (ARE) of GCLC but lower Nrf2/MafG heterodimer binding to the ARE. Quiescent HSCs have a lower nuclear MafG level but higher Nrf2/MafG heterodimer binding to ARE. This occurred because of enhanced sumoylation of Nrf2 and MafG by SUMO-1, which promoted Nrf2 binding to ARE and heterodimerization with MafG. In vivo, knockdown of GCLC exacerbated bile duct ligation-induced liver injury and fibrosis. Ursodeoxycholic acid and S-adenosylmethionine are anti-fibrotic in bile duct ligation, but this effect was nearly lost if GCLC induction was blocked. In conclusion, sumoylation of Nrf2 and MafG enhances heterodimerization and increases GCLC expression, which keeps HSCs in a quiescent state. Antifibrotic agents require activation of GCLC to fully exert their protective effect.

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

UNLABELLED: 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. CONCLUSION: 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.

Transcriptional regulation of methionine adenosyltransferase 2A by peroxisome proliferator-activated receptors in rat hepatic stellate cells.

UNLABELLED: Methionine adenosyltransferases (MATs) are critical enzymes that catalyze the formation of the methyl donor S-adenosyl methionine (SAM). The MAT2A gene, which encodes the catalytic subunit α2, is induced in dedifferentiated liver. We previously demonstrated that MAT2A expression is enhanced in activated hepatic stellate cells (HSCs) and that silencing this gene reduces HSC activation. In this study, we examined the molecular mechanisms responsible for the transcriptional regulation of the MAT2A gene in HSCs. We identified peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the rat MAT2A promoter. The PPARγ agonist rosiglitazone (RSG) promoted quiescence in the activated rat HSC cell line (BSC) or culture-activated primary rat HSCs, decreased MAT2A expression and promoter activity, and enhanced PPARγ binding to MAT2A PPREs. In vivo HSC activation in bile duct-ligated rats lowered PPARγ interaction with MAT2A PPREs. Silencing PPARγ increased MAT2A transcription, whereas overexpressing it had the opposite effect, demonstrating that PPARγ negatively controls this gene. Site-directed mutagenesis of PPREs abolished PPARγ recruitment to the MAT2A promoter and its inhibitory effect on MAT2A transcription in quiescent HSCs. PPRE mutations decreased the basal promoter activity of MAT2A in activated HSCs independent of PPARγ, indicating that other factors might be involved in PPRE interaction. We identified PPARß binding to wild-type but not to mutated PPREs in activated cells. Furthermore, silencing PPARß inhibited MAT2A expression and promoter activity. Forced expression of MAT2A in RSG-treated HSCs lowered PPARγ and enhanced PPARß expression, thereby promoting an activated phenotype. CONCLUSION: We identified PPARγ as a negative regulator of MAT2A in quiescent HSCs. A switch from quiescence to activation abolishes this control and allows PPARß to up-regulate MAT2A transcription.