Expression Pattern, Regulation, and Functions of Methionine Adenosyltransferase 2β Splicing Variants in Hepatoma Cells
2007; Elsevier BV; Volume: 134; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.10.027
ISSN1528-0012
AutoresHeping Yang, Ainhoa Iglesias–Ara, Nathaniel Magilnick, Meng Xia, Komal Ramani, Hui Chen, Taunia D. Lee, José M. Mato, Shelly C. Lu,
Tópico(s)RNA regulation and disease
ResumoBackground & Aims: Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5′-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2. Methods: Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays. Results:MAT2β is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-α, which induces MAT2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-α induced the promoter activity of MAT2β V1, likely via nuclear factor κB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-α–induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types. Conclusions: Different variants of MAT2β regulate growth and death, which broadens their importance in biology. Background & Aims: Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5′-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2. Methods: Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays. Results:MAT2β is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-α, which induces MAT2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-α induced the promoter activity of MAT2β V1, likely via nuclear factor κB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-α–induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types. Conclusions: Different variants of MAT2β regulate growth and death, which broadens their importance in biology. Methionine adenosyltransferase (MAT) is a critical cellular enzyme responsible for the synthesis of S-adenosylmethionine using methionine and adenosine triphosphate.1Mato J.M. Corrales F.J. Lu S.C. et al.S-Adenosylmethionine: a control switch that regulates liver function.FASEB J. 2002; 16: 15-26Crossref PubMed Scopus (375) Google Scholar Its importance is due to the fact that S-adenosylmethionine is the principal biological methyl donor, the precursor of aminopropyl groups used in polyamine biosynthesis, and, in the liver, a precursor of glutathione.1Mato J.M. Corrales F.J. Lu S.C. et al.S-Adenosylmethionine: a control switch that regulates liver function.FASEB J. 2002; 16: 15-26Crossref PubMed Scopus (375) Google Scholar In mammals, 2 different genes, MAT1A and MAT2A, encode for 2 homologous MAT catalytic subunits, α1 and α2.2Kotb M. Mudd S.H. Mato J.M. et al.Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products.Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (189) Google ScholarMAT1A is expressed mostly in the liver and it encodes the α1 subunit found in 2 native MAT isozymes, which are either a dimer (MATIII) or tetramer (MATI) of this single subunit.2Kotb M. Mudd S.H. Mato J.M. et al.Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products.Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (189) Google ScholarMAT2A encodes for a catalytic subunit (α2) found in a native MAT isozyme (MATII), which is widely distributed.1Mato J.M. Corrales F.J. Lu S.C. et al.S-Adenosylmethionine: a control switch that regulates liver function.FASEB J. 2002; 16: 15-26Crossref PubMed Scopus (375) Google Scholar, 2Kotb M. Mudd S.H. Mato J.M. et al.Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products.Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (189) Google ScholarMAT2A also predominates in the fetal liver and is progressively replaced by MAT1A during liver development.3Gil B. Casado M. Pajares M. et al.Differential expression pattern of S-adenosylmethionine synthetase isoenzymes during rat liver development.Hepatology. 1996; 24: 876-881PubMed Google Scholar In adult liver, increased expression of MAT2A is associated with rapid growth or dedifferentiation of the liver.4Cai J. Sun W.M. Hwang J.J. et al.Changes in S-adenosylmethionine synthetase in human liver cancer: molecular characterization and significance.Hepatology. 1996; 24: 1090-1097Crossref PubMed Google Scholar, 5Huang Z.Z. Mao Z. Cai J. et al.Changes in methionine adenosyltransferase during liver regeneration in the rat.Am J Physiol. 1998; 275: G14-G21PubMed Google Scholar, 6Huang Z.Z. Mato J.M. Kanel G. et al.Differential effect of thioacetamide on hepatic methionine adenosyltransferase expression in the rat.Hepatology. 1999; 29: 1471-1478Crossref PubMed Scopus (47) Google Scholar There is a regulatory subunit (β) that is associated only with MATII.2Kotb M. Mudd S.H. Mato J.M. et al.Consensus nomenclature for the mammalian methionine adenosyltransferase genes and gene products.Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 7Halim A. LeGros L. Geller A. et al.Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.J Biol Chem. 1999; 274: 29720-29725Crossref PubMed Scopus (85) Google Scholar The β subunit is encoded by the gene MAT2β that is expressed in extrahepatic tissues but not in normal liver.7Halim A. LeGros L. Geller A. et al.Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.J Biol Chem. 1999; 274: 29720-29725Crossref PubMed Scopus (85) Google Scholar, 8LeGros Jr, H.L. Halim A.B. Geller A.M. et al.Cloning, expression, and functional characterization of the β regulatory subunit of human methionine adenosyltransferase (MAT II).J Biol Chem. 2000; 275: 2359-2366Crossref PubMed Scopus (67) Google Scholar, 9Martinez-Chantar M.L. Garcia-Trevijano E.R. Latasa M.U. et al.Methionine adenosyltransferase II β subunit gene expression provides a proliferative advantage in human hepatoma.Gastroenterology. 2003; 124: 940-948Abstract Full Text Full Text PDF PubMed Scopus (73) Google ScholarMAT2β is induced in cirrhosis and in hepatocellular carcinoma (HCC).9Martinez-Chantar M.L. Garcia-Trevijano E.R. Latasa M.U. et al.Methionine adenosyltransferase II β subunit gene expression provides a proliferative advantage in human hepatoma.Gastroenterology. 2003; 124: 940-948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Importantly, both increased MAT2A and MAT2β offer liver cancer cells a growth advantage.9Martinez-Chantar M.L. Garcia-Trevijano E.R. Latasa M.U. et al.Methionine adenosyltransferase II β subunit gene expression provides a proliferative advantage in human hepatoma.Gastroenterology. 2003; 124: 940-948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Cai J. Mao M. Hwang J.J. et al.Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.Cancer Res. 1998; 58: 1444-1450PubMed Google Scholar Given the importance of MAT genes, we have been interested in understanding their transcriptional regulation. Both nuclear factor κB (NF-κB) and activator protein 1 (AP-1) are positive regulators of MAT2A gene transcription.11Yang H.P. Sadda M.R. Yu V. et al.Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor α: role of NF-κB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar To better understand transcriptional regulation of MAT2β, we cloned and characterized the 5′-flanking region. During this process, we identified multiple alternate splicing variants of MAT2β not described previously. Here we report the first in-depth analysis of expression patterns of the 2 major MAT2β variants (V1 and V2) in normal human tissues and HCC, their differential regulation by tumor necrosis factor (TNF)-α, and their modulation of TNF-α–mediated apoptosis. All of the published work on MAT2β pertains only to V1.7Halim A. LeGros L. Geller A. et al.Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.J Biol Chem. 1999; 274: 29720-29725Crossref PubMed Scopus (85) Google Scholar, 9Martinez-Chantar M.L. Garcia-Trevijano E.R. Latasa M.U. et al.Methionine adenosyltransferase II β subunit gene expression provides a proliferative advantage in human hepatoma.Gastroenterology. 2003; 124: 940-948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12LeGros L. Halim A.B. Chamberlin M.E. et al.Regulation of the human MAT2β gene encoding the regulatory β subunit of methionine adenosyltransferase, MAT II.J Biol Chem. 2001; 276: 24918-24924Crossref PubMed Scopus (48) Google Scholar We uncovered the novel finding that in addition to regulating MATII, MAT2β V1 also regulate cell death through modulating c-Jun-N-terminal kinase (JNK) activity. All reagents were of analytical grade and obtained from commercial sources. Normal and cancerous liver tissues were obtained as described.13Yang H.P. Huang Z.Z. Zeng Z.H. et al.The role of c-Myb and Sp1 in the up-regulation of methionine adenosyltransferase 2A gene expression in human hepatocellular carcinoma.FASEB J. 2001; 15: 1507-1516Crossref PubMed Scopus (63) Google Scholar The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Keck School of Medicine, University of Southern California Human Research Review Committee. HepG2, HuH-7, and RKO cells were grown according to instructions provided by the American Type Culture Collection (Rockville, MD). Before treatment of HepG2 cells with TNF-α, medium was changed to withhold serum overnight. Cells were then treated with TNF-α (25 ng/mL), insulin-like growth factor (IGF)-1 (100 ng/mL), or epidermal growth factor (EGF; 100 ng/mL) for 15 minutes to 8 hours for various assays described in the following text. pCMV-p50, pCMV-p65, and pRSV-cJun expression plasmids were kindly provided by Dr Richard Rippe (University of North Carolina at Chapel Hill, Chapel Hill, NC). Dominant negative (DN) JNK1 and JNK2 adenoviral vectors were kindly provided by Dr Mark Czaja (Albert Einstein School of Medicine, Bronx, NY). An oligonucleotide probe corresponding to −60 to +3 of the human MAT2β complementary DNA8LeGros Jr, H.L. Halim A.B. Geller A.M. et al.Cloning, expression, and functional characterization of the β regulatory subunit of human methionine adenosyltransferase (MAT II).J Biol Chem. 2000; 275: 2359-2366Crossref PubMed Scopus (67) Google Scholar (cDNA) was used to screen the human genomic library EMBL 3 (Clontech Laboratories, Inc, Mountain View, CA). Five positive plaques were selected; DNA was isolated and digested with EcoRI. The insert fragment was subcloned into pGL-3 basic vector (Promega, Madison, WI) and sequenced as described.14Yang H.P. Huang Z.Z. Wang J.H. et al.Cloning and characterization of the 5'-flanking region of the rat glutamate-cysteine ligase catalytic subunit.Biochem J. 2001; 357: 447-455Crossref PubMed Scopus (48) Google Scholar A 4.1-kilobase 5′-flanking region of the human MAT2β was cloned into the SmaI site of promoterless pGL-3 basic vector. Primer extension analysis was performed as described.14Yang H.P. Huang Z.Z. Wang J.H. et al.Cloning and characterization of the 5'-flanking region of the rat glutamate-cysteine ligase catalytic subunit.Biochem J. 2001; 357: 447-455Crossref PubMed Scopus (48) Google Scholar Two antisense oligonucleotide primers complementary to −1 to +17 and +4 to +21 nucleotides relative to the translational start site of the human MAT2β Cdna8LeGros Jr, H.L. Halim A.B. Geller A.M. et al.Cloning, expression, and functional characterization of the β regulatory subunit of human methionine adenosyltransferase (MAT II).J Biol Chem. 2000; 275: 2359-2366Crossref PubMed Scopus (67) Google Scholar were end-labeled with [γ-32P]adenosine triphosphate using T4 polynucleotide kinase. A total of 2.5 μg of poly (A+) RNA from HepG2 cells was annealed to 106 cpm of the primers and extended with 4 U of Vent DNA polymerase (New England Biolabs, Inc, Ipswich, MA). The positive insert fragment in the sense orientation upstream of the luciferase coding sequence of the pGL-3 basic vector was subjected to polymerase chain reaction to generate 5 deletion constructs. Forward primers were from −2073 to –2046, −1319 to –1294, −990 to –967, −713 to –688, and −250 to –225, and reverse primer was +3 to –20 to generate deletion constructs –2073/+3, −1319/+3, −990/+3, –713/+3, and –250/+3 MAT2β-LUC, respectively. All sequences are relative to the ATG start codon. Total RNA from human tissues and cells was isolated using TRIzol kit (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. First-strand cDNA was synthesized from 250 ng messenger RNA (mRNA) using RACE kit (Ambion, Austin, TX) according to the manufacturer's recommendations. To express recombinant human MAT2β (either V1 or V2), the full-length cDNA was cloned into the mammalian expression vector pcDNA3.1D/V5-His/TOPO (Invitrogen) using the pcDNA3.1 Directional TOPO Expression kit according to the manufacturer's instructions. Forward primers (V1, 5′-CACCATGGTGGGGCGGGAG-3′; V2, 5′-CACCATGCCTGAAATGCCAGAG-3′) were designed to incorporate the sequence CACC 5′ to the ATG translation start site. The reverse primer (5′-ATGAAAGACCGTTTGTCTCCATCT -3′) is suitable for amplification of both variants and was designed to begin one codon upstream of the stop codon, effectively eliminating the stop codon from the polymerase chain reaction amplicon, enabling fusion of the MAT2β protein to the viral V5 epitope and His tag at the carboxyl terminus. Clones obtaining the expression plasmid pcDNA3.1D/V5-His/MAT2βvar1 (V1 expression vector) or pcDNA3.1D/V5-His/MAT2βvar2 (V2 expression vector) were identified initially by restriction digest analysis to detect orientation and were confirmed to contain the MAT2β cDNA in frame with the V5 and His-tag by sequence analysis using BGH and T7 priming. To increase variant expression, HuH-7 cells were transfected with V1 or V2 expression vectors for up to 72 hours. Expression of variants was documented by Western blot analysis using anti-V5 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-MAT2β antibodies (Novus Biologicals, Inc, Littleton, CO). RNA (10 μg) from human tissues (total RNA Master Panel II; Clontech Laboratories, Inc) were subjected to Northern blot analysis using specific V1 (−202 to +3) or V2 (−2372 to −2261) and housekeeping β-actin cDNA probes as described.11Yang H.P. Sadda M.R. Yu V. et al.Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor α: role of NF-κB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar Results of Northern blot analysis were normalized to β-actin. Effects of TNF-α, IGF-1, and EGF on gene expression in HepG2 cells were examined using Northern blot analysis as described previously. The effect of TNF-α on MAT2β V1 promoter activity was examined by measuring luciferase activity driven by MAT2β V1 promoter luciferase gene constructs in transfected HepG2 cells treated with TNF-α (25 ng/mL) during the last 8 hours of the transfection. To confirm the role of NF-κB and AP-1 on MAT2β V1 promoter activity, HepG2 cells were cotransfected with expression vectors for p65, p50, or c-Jun for 24 hours before measuring luciferase activity. Controls were transfected with empty vectors for p65, p50, or c-Jun. The V1 coding sequence was analyzed for sites of small interfering RNA (siRNA) targeting using siRNA Target Designer (Promega), and 3 potential sequences for each variant were selected (Supplementary Table 1; see supplemental material online at www.gastrojournal.org). HepG2 and RKO cells were grown to 60%–70% confluency before transfection. Lipofectamine 2000 was used as the transfection reagent, and transfections were performed according to the manufacturer's protocol using Opti-MEM media (Invitrogen Life Sciences). The effect of siRNA on the MAT2β protein level was assessed by Western blot analysis. In some experiments, HepG2 cells treated with siRNA against V1 or scrambled siRNA for 24 hours were treated with adenoviral vectors expressing DNJNK1, DNJNK2, or empty vector alone for another 24 hours. The siRNA transfection efficiency of Lipofectamine RNAiMax in HepG2 cells was determined by the BLOCK-iT Alexa Fluor Red Fluorescent Oligo protocol (Invitrogen) and averaged 93.5%. DNA synthesis was measured by 3H-thymidine incorporation into DNA as described.10Cai J. Mao M. Hwang J.J. et al.Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.Cancer Res. 1998; 58: 1444-1450PubMed Google Scholar Apoptosis was measured using DNA fragmentation and Hoechst staining as described.11Yang H.P. Sadda M.R. Yu V. et al.Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor α: role of NF-κB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar Similar results were obtained using the Apo-direct kit (Pharmingen, San Diego, CA), which uses the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and flow cytometry as described.15Delatte S.J. Hazen-Martin D.J. Re G.G. et al.Restoration of p53 function in anaplastic Wilms' tumor.J Pediatr Surg. 2001; 36: 43-50Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Hoechst staining and TUNEL yielded comparable results and were pooled for analysis. All of the cells (whether or not they were transfected) were counted. JNK and extracellular signal–regulated kinase (ERK) activation was assessed by Western blot analysis using antibodies that recognize the phosphorylated (activated) versus total JNK and ERK forms as described.16Chen L. Zeng Y. Yang H.P. et al.Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A.FASEB J. 2004; 18: 914-916Crossref PubMed Scopus (187) Google Scholar NF-κB activation was assessed by Western blot analysis using nuclear protein extracts and anti-p50 and anti-p65 antibodies (Santa Cruz Biotechnology). For loading control, actin was used for cytosolic proteins and histone 3 was used for nuclear proteins. Measurement of intracellular reactive oxygen species (ROS) production in HepG2 cells treated with TNF-α (25 ng/mL), H2O2 (100 μmol/L) for 30 minutes, or V1 or V2 RNA interference (RNAi) for 24–48 hours was performed as described.17Balcerczyk A. Soszynski M. Rybaczek D. et al.Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium.Biochem Pharmacol. 2005; 69: 1263-1273Crossref PubMed Scopus (29) Google Scholar Data are given as mean ± SEM. Statistical analysis was performed using analysis of variance and Fisher exact test. For mRNA and protein levels, ratios of genes and proteins to respective housekeeping densitometric values were compared. Significance was defined by P < .05. The sequence of the 4.0-kilobase product is shown in Figure 1 (GenBank accession no. AY223864). Analysis of the transcription factor binding site was performed using Transcription Factor Search and MatInspector. The 5′-flanking region of the human MAT2β contains multiple consensus binding sites for NF-κB and AP-1. Two transcriptional start sites were identified, as indicated by forward arrows (Figure 1). Multiple alternative splicing variants were identified in both HepG2 cells and HCC (Supplementary Figure 1; see supplemental material online at www.gastrojournal.org). V1 and V2 were reported previously to the GenBank (NM_013283 and NM_182796, respectively), and they differ in the N-terminus by 20 amino acids. V2 uses a different first exon lying 2.29 kilobases upstream of the translational start site of V1 (Figure 2A). V2a is missing the first 51 base pairs of exon 7, while V2b is missing exons 3–6 (Supplementary Figure 1A; see supplemental material online at www.gastrojournal.org). All 4 variants are expressed in both HCC and HepG2 cells, but V2a and V2b are expressed at very low levels as compared with V2 (Supplementary Figure 1B; see supplemental material online at www.gastrojournal.org).Figure 2MAT2β V1 and V2 genomic structure and expression in normal human tissues. (A) Genomic structure and N-terminus amino acid sequences of V1 and V2. (B) Expression patterns of the 2 major variants in different normal human tissues by Northern blot analysis. (C) Expression of MAT genes in normal liver and HCC. Northern blot analysis was performed in 4 normal livers and 4 HCC specimens. Each lane contains 15 μg of total RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 2 main variants (V1 and V2) are differentially expressed in normal human tissues (Figure 2B). Some tissues express mainly V1 (prostate, brain, lung, thyroid, adrenal gland, fetal liver, and spinal cord), others express mainly V2 (skeletal muscle, testis, and heart), some express both forms (thymus and kidney), and some express almost no MAT2β V1 or V2 (bone marrow, trachea, salivary gland, uterus, adult liver, and placenta). In human HCC, both MAT2β V1 and V2 mRNA levels are increased like MAT2A, while MAT1A is silenced (Figure 2C). TNF-α is a pleiotropic cytokine that we have previously shown to induce MAT2A expression at the transcriptional level via AP-1 and NF-κB.11Yang H.P. Sadda M.R. Yu V. et al.Induction of human methionine adenosyltransferase 2A expression by tumor necrosis factor α: role of NF-κB and AP-1.J Biol Chem. 2003; 278: 50887-50896Crossref PubMed Scopus (54) Google Scholar TNF-α also induced MAT2β V1 but had no influence on V2 mRNA level (Figure 3). TNF-α treatment also increased the mRNA levels of p50, p65, c-Fos, and c-Jun in a time-dependent manner (Figure 3). Similar to TNF-α, IGF-1 and EGF also predominantly induced the expression of MAT2β V1 (Supplementary Figure 2; see supplemental material online at www.gastrojournal.org). Next we investigated whether AP-1 and NF-κB regulate basal MAT2β V1 expression in response to TNF-α treatment. Figure 4A shows that TNF-α maximally induced the promoter construct –713/+3, which contains several AP-1 consensus binding sites (Figure 1). Overexpression of c-Jun also induced MAT2β V1 promoter constructs (Figure 4A) and endogenous V1 (but not V2) expression (Figure 4B). Overexpression of either p50 or p65 induced the MAT2β V1 promoter construct –1319/+3 but not –713/+3 (Figure 4A) and increased mRNA level of V1 but not V2 (Figure 4B). Three consensus NF-κB binding sites are located in the region of –1319 to −990 (Figure 1). There is cross-talk between NF-κB and AP-1 as overexpression of c-Jun increased p65 (but not p50) mRNA levels and overexpression of either p65 or p50 increased both c-Jun and c-Fos mRNA levels (Figure 4B). We next examined whether TNF-α treatment led to an increase in NF-κB and AP-1 binding to the MAT2β V1 promoter in the endogenous chromatin configuration using the chromatin immunoprecipitation assay (Supplementary Figure 3; see supplemental material online at www.gastrojournal.org). As expected, nuclear binding to the NF-κB (p65 and p50) and AP-1 sites (c-Jun, Jun D, and c-Fos) increased following TNF-α treatment. To address the influence of MAT2β on cell growth, V1 and V2 expression was reduced by RNAi in HepG2 cells and increased by overexpression vectors in HuH-7 cells, which express minimal MAT2β.9Martinez-Chantar M.L. Garcia-Trevijano E.R. Latasa M.U. et al.Methionine adenosyltransferase II β subunit gene expression provides a proliferative advantage in human hepatoma.Gastroenterology. 2003; 124: 940-948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar RNAi treatment typically reduced V1 and V2 mRNA levels to 24% and 20% of control, respectively (Figure 5A, top). Each RNAi is specific and had no influence on the expression of the other variant; scrambled RNAi had no influence on the expression of either variant (not shown). Western blot analysis shows that HepG2 cells express more V1, because more MAT2β protein remained after V2 RNAi treatment (Figure 5A, bottom). HuH-7 cells express very little MAT2β and overexpression with either V1 or V2 increased MAT2β by 800% and 500%, respectively (Figure 5B). Reduced expression of either V1 or V2 reduced DNA synthesis in HepG2 cells (Figure 5C), while overexpression increased DNA synthesis in HuH-7 cells, with V1 exerting a stronger influence than V2 (Figure 5D). TNF-α is known to induce apoptosis by a JNK-mediated mechanism.18Papa S. Zazzeroni F. Pham C.G. et al.Linking JNK signaling to NF-κB: a key to survival.J Cell Sci. 2004; 117: 5197-5208Crossref PubMed Scopus (249) Google Scholar, 19Schwabe R.F. Brenner D.A. Mechanisms of liver injury. I. TNF-α-induced liver injury: role of IKK, JNK, and ROS pathways.Am J Physiol. 2006; 290: G583-G589Google Scholar In normal hepatocytes, TNF-α has no cytotoxic effect unless NF-κB induction is blocked.19Schwabe R.F. Brenner D.A. Mechanisms of liver injury. I. TNF-α-induced liver injury: role of IKK, JNK, and ROS pathways.Am J Physiol. 2006; 290: G583-G589Google Scholar NF-κB is believed to induce expression of protective genes against apoptosis. This prompted us to examine whether MAT2β V1 may be one of these protective genes. Figure 6A shows that knockdown of V1 to 24% of control level induced apoptosis by itself. TNF-α treatment for 24 hours also induced apoptosis in HepG2 cells. However, cells pretreated with V1 RNAi (but not V2 RNAi) were sensitized to TNF-α–induced apoptosis. Figure 6B shows that the opposite occurs, namely, HepG2 cells overexpressing V1 (but not V2) were protected from TNF-α–induced apoptosis. Sustained JNK activation is believed to be the cause of TNF-α–induced apoptosis in many cell types, although there is controversy with regard to whether it is JNK1 or JNK2 that plays the dominant role.20Liu J. Minemoto Y. Lin A. c-Jun N-Terminal Protein Kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis.Mol Cell Biol. 2004; 24: 10844-10856Crossref PubMed Scopus (176) Google Scholar, 21Wang Y. Singh R. Lefkowitch J.H. et al.Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway.J Biol Chem. 2006; 281: 15258-15267Crossref PubMed Scopus (193) Google Scholar We next investigated the effect of MAT2β V1 knockdown on JNK activation and signaling. Figure 7A shows that knockdown of MAT2β V1 (but not V2) and TNF-α both increased JNK activation (both JNK forms), but the magnitude of activation was higher with combined V1 knockdown and TNF-α treatment. One of the downstream targets of JNK1 is c-Jun, and c-Jun phosphorylation increased in parallel with changes in JNK1 phosphorylation (not shown). A key inducer of JNK activation is ROS.18Papa S. Zazzeroni F. Pham C.G. et al.Linking JNK signaling to NF-κB: a key to survival.J Cell Sci. 2004; 117: 5197-5208Crossref PubMed Scopus (249) Google Scholar, 19Schwabe R.F. Brenner D.A. Mechanisms of liver injury. I. TNF-α-induced liver injury: role of IKK, JNK, and ROS pathways.Am J Physiol. 2006; 290: G583-G589Google Scholar We next measured ROS generation in cells treated with V1 or V2 RNAi for 48 hours or TNF-α (25 ng/mL) or H2O2 (100 μmol/L) for 30 minutes. Cells treated with TNF-α or H2O2 had higher ROS generation (200% of untreated control), but V1 or V2 RNAi had no influence on the level of ROS (data not shown). Another regulator of JNK activity is NF-κB, which has been shown to inhibit JNK activation.18Papa S. Zazzeroni F. Pham C.G. et al.Linking JNK signaling to NF-κB: a key to survival.J Cell Sci. 2004; 117: 5197-5208Crossref PubMed Scopus (249) Google Scholar, 19Schwabe R.F. Brenner D.A. Mechanisms of liv
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