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• W1980320422 abstract "Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. Mammals detect and respond to inadequacies in protein or amino acid (AA) 2The abbreviations used are: AAamino acidAARAA responseASNSasparagine synthetaseATFactivating transcription factorCAREC/EBP-ATF response elementsEGR1early growth response 1ELK1E-twenty-six (ETS)-like factor 1GCN2general control nonderepressible 2HisOHhistidinolMEKCAconstitutively active MEKqPCRreal time quantitative PCRSREserum response elementSRFserum response factorTSStranscription start siteMEFmouse embryonic fibroblastERendoplasmic reticulumTettetracyclinePolpolymerase. content via a collection of signaling cascades jointly referred to as the AA response (AAR) (reviewed in Refs. 1.Kilberg M.S. Shan J. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol. Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar and 2.Chaveroux C. Lambert-Langlais S. Cherasse Y. Averous J. Parry L. Carraro V. Jousse C. Maurin A.C. Bruhat A. Fafournoux P. Molecular mechanisms involved in the adaptation to amino acid limitation in mammals.Biochimie. 2010; 92: 736-745Crossref PubMed Scopus (57) Google Scholar). The best characterized among these networks involves activation of the general control nonderepressible 2 (GCN2) kinase, which senses increased intracellular uncharged tRNA levels. Once activated, GCN2 phosphorylates the eukaryotic translation initiation factor (eIF2α), triggering a global suppression of protein translation initiation, but an increase in the translation of selected mRNAs including that for activating transcription factor 4 (ATF4) (3.Vattem K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11269-11274Crossref PubMed Scopus (1125) Google Scholar). Endoplasmic reticulum (ER) stress also triggers ATF4 synthesis through activation of a separate eIF2 kinase called PKR-like endoplasmic reticulum kinase (PERK) (4.Harding H.P. Zhang Y. Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.Nature. 1999; 397: 271-274Crossref PubMed Scopus (2522) Google Scholar, 5.Harding H.P. Zhang Y. Bertolotti A. Zeng H. Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response.Mol. Cell. 2000; 5: 897-904Abstract Full Text Full Text PDF PubMed Scopus (1552) Google Scholar). ATF4 mediates an increase in transcription from a number of genes that contain an enhancer sequence composed of a half-site for C/EBP and a half-site for the ATF family of transcription factors (6.Wolfgang C.D. Chen B.P. Martindale J.L. Holbrook N.J. Hai T. gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (143) Google Scholar, 7.Fawcett T.W. Martindale J.L. Guyton K.Z. Hai T. Holbrook N.J. Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response.Biochem. J. 1999; 339: 135-141Crossref PubMed Scopus (368) Google Scholar); consequently, these sequences are referred to as a C/EBP-ATF response element (CARE). Most, but not all (8.Gjymishka A. Palii S.S. Shan J. Kilberg M.S. Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells.J. Biol. Chem. 2008; 283: 27736-27747Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), functional CARE sites respond to ATF4, regardless of which eIF2 kinase was activated, so CARE sites can exhibit ER stress element activity or AAR element activity (reviewed in Ref. 1.Kilberg M.S. Shan J. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol. Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The products of these CARE-containing genes control a wide range of physiological processes and microarray analyses of human HepG2-C3A (9.Lee J.I. Dominy Jr., J.E. Sikalidis A.K. Hirschberger L.L. Wang W. Stipanuk M.H. HepG2/C3A cells respond to cysteine-deprivation by induction of the amino acid deprivation/integrated stress response pathway.Physiol. Genomics. 2008; 33: 218-229Crossref PubMed Scopus (79) Google Scholar) or HepG2 hepatocellular carcinoma cells (10.Shan J. Lopez M.C. Baker H.V. Kilberg M.S. Expression profiling after activation of the amino acid deprivation response in HepG2 human hepatoma cells.Physiol. Genomics. 2010; 41: 315-327Crossref PubMed Scopus (41) Google Scholar), as well as mouse embryonic fibroblasts (MEF) (11.Deval C. Chaveroux C. Maurin A.C. Cherasse Y. Parry L. Carraro V. Milenkovic D. Ferrara M. Bruhat A. Jousse C. Fafournoux P. Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways.FEBS J. 2009; 276: 707-718Crossref PubMed Scopus (106) Google Scholar), has revealed that AA limitation activates hundreds of genes, a majority of which have functions other than within protein or AA homeostasis. Interestingly, the list of genes induced during the AAR in HepG2 cells included several immediate-early response genes such as selected members of the FOS-JUN and the early growth response (EGR) transcription factor families (10.Shan J. Lopez M.C. Baker H.V. Kilberg M.S. Expression profiling after activation of the amino acid deprivation response in HepG2 human hepatoma cells.Physiol. Genomics. 2010; 41: 315-327Crossref PubMed Scopus (41) Google Scholar). Follow-up studies established that cJUN is a novel AAR-inducible gene, for which the induction is ATF4-independent, and instead involves autoactivation in response to signaling from the JNK arm of MAPK signaling (12.Fu L. Balasubramanian M. Shan J. Dudenhausen E.E. Kilberg M.S. Auto-activation of c-JUN gene by amino acid deprivation of hepatocellular carcinoma cells reveals a novel c-JUN-mediated signaling pathway.J. Biol. Chem. 2011; 286: 36724-36738Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The elevated cJUN expression impacts the regulation of other AAR targets, including ATF3 (13.Fu L. Kilberg M.S. Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response.Physiol. Genomics. 2013; 45: 127-137Crossref PubMed Scopus (18) Google Scholar). amino acid AA response asparagine synthetase activating transcription factor C/EBP-ATF response elements early growth response 1 E-twenty-six (ETS)-like factor 1 general control nonderepressible 2 histidinol constitutively active MEK real time quantitative PCR serum response element serum response factor transcription start site mouse embryonic fibroblast endoplasmic reticulum tetracycline polymerase. EGR1, which encodes a zinc finger transcription factor, is an important immediate-early response gene activated by a broad range of extracellular stimuli. EGR1 impacts cell growth, proliferation, differentiation, and apoptosis (reviewed in Ref. 14.Pagel J.I. Deindl E. Early growth response 1: a transcription factor in the crossfire of signal transduction cascades.Indian J. Biochem. Biophys. 2011; 48: 226-235PubMed Google Scholar). The signaling pathways that control EGR1 transcription vary depending on the initial stimulus and target tissue, but many studies have established that phosphorylation of constitutively bound E twenty-six-like factor (ELK1) in response to MEK-ERK signaling is an important mechanism (14.Pagel J.I. Deindl E. Early growth response 1: a transcription factor in the crossfire of signal transduction cascades.Indian J. Biochem. Biophys. 2011; 48: 226-235PubMed Google Scholar, 15.Hodge C. Liao J. Stofega M. Guan K. Carter-Su C. Schwartz J. Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2.J. Biol. Chem. 1998; 273: 31327-31336Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). ELK1 belongs to the ternary complex factor subfamily of the ETS (E twenty-six) superfamily of transcription factors (16.Hollenhorst P.C. McIntosh L.P. Graves B.J. Genomic and biochemical insights into the specificity of ETS transcription factors.Annu. Rev. Biochem. 2011; 80: 437-471Crossref PubMed Scopus (330) Google Scholar, 17.Odrowaz Z. Sharrocks A.D. ELK1 uses different DNA binding modes to regulate functionally distinct classes of target genes.PLoS Genet. 2012; 8: e1002694Crossref PubMed Scopus (55) Google Scholar). Once increased in its expression, EGR1 regulates the transcription of target genes by binding to GC-rich sequences (14.Pagel J.I. Deindl E. Early growth response 1: a transcription factor in the crossfire of signal transduction cascades.Indian J. Biochem. Biophys. 2011; 48: 226-235PubMed Google Scholar, 18.Cao X.M. Koski R.A. Gashler A. McKiernan M. Morris C.F. Gaffney R. Hay R.V. Sukhatme V.P. Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals.Mol. Cell. Biol. 1990; 10: 1931-1939Crossref PubMed Scopus (298) Google Scholar). Egr1 knock-out mice, though viable, exhibit impaired liver regeneration following partial hepatectomy, and Egr1 has been proposed as a central regulator of cell cycle progression during hepatocellular regeneration following injury (19.Liao Y. Shikapwashya O.N. Shteyer E. Dieckgraefe B.K. Hruz P.W. Rudnick D.A. Delayed hepatocellular mitotic progression and impaired liver regeneration in early growth response-1-deficient mice.J. Biol. Chem. 2004; 279: 43107-43116Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Thus, control of hepatic EGR1 expression by AA limitation or ER stress may be a critical factor in liver physiology. The present study documents that the AAR-initiated induction of EGR1 transcription is not mediated by the well documented GCN2-eIF2-ATF4 signaling pathway, but instead by AA-responsive MEK-ERK signaling. ERK-dependent phosphorylation of ELK1, constitutively bound to the EGR1 gene is associated with increased transcription and a marked elevation of EGR1 expression. Therefore, these results provide evidence for the existence of an AA-controlled MEK signaling pathway that terminates with phosphorylation of ELK1. The AA-dependent transcription via p-ELK1 reveals a new family of transcription factors, the ETS family, within the AAR. Correspondingly, transcription is induced through ETS genomic enhancer sequences previously unknown to have AAR element activity. Furthermore, the induction of immediate-early response genes in AA-deprived tumor cells provides a possible link between protein/AA nutrition and cell growth in the transformed state. All of the cell lines used in these studies were cultured in DMEM (pH 7.4; Mediatech, Herndon, VA), supplemented with 1× nonessential AA, 2 mm glutamine, 100 μg/ml streptomycin sulfate, 100 units/ml penicillin G, 0.25 μg/ml amphotericin B, and 10% (v/v) fetal bovine serum. The HEK293T-ATF4 cell line was created by Ord et al. (20.Ord D. Meerits K. Ord T. TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4.Exp. Cell Res. 2007; 313: 3556-3567Crossref PubMed Scopus (72) Google Scholar) after virally transforming HEK293T cells with a tetracycline (Tet)-inducible construct that contains the ATF4 coding region. The HEK293T-ATF4 DMEM was the same as above but was also supplemented with 10% (v/v) tetracycline-free fetal bovine serum, 25 μg/ml Zeocin, and 2.5 μg/ml blasticidin. All cells were maintained at 37 °C in an atmosphere of 5% CO2 and 95% air and maintained in growth phase at 60–70% confluence. Approximately 12 h prior to treatments, cells were replenished with fresh DMEM to ensure more complete nutrition when experiments were initiated. For the HEK293T-ATF4 cells, overexpression of ATF4 in the absence of other possible AAR signals was induced by adding tetracycline at the concentrations and times indicated. For activation of the AAR, cells were incubated in either DMEM lacking histidine (catalog number D9801-02; United States Biological, Swampscott, MA) or complete DMEM containing 2 mm histidinol (HisOH), an amino alcohol that triggers the AAR. HisOH competitively inhibits histidinyl tRNA synthetase, causing an increase in uncharged tRNAHis and thereby inducing the AAR (21.Thiaville M.M. Dudenhausen E.E. Zhong C. Pan Y.X. Kilberg M.S. Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription.Biochem. J. 2008; 410: 473-484Crossref PubMed Scopus (40) Google Scholar). Replicating experiments with either DMEM-histidine or DMEM + HisOH yielded no qualitative differences. The MEK inhibitor PD98059 (Sigma-Aldrich) was diluted in DMSO. The initial concentrations tested were chosen based on previous studies (22.Thiaville M.M. Pan Y.X. Gjymishka A. Zhong C. Kaufman R.J. Kilberg M.S. MEK signaling is required for phosphorylation of eIF2α following amino acid limitation of HepG2 human hepatoma cells.J. Biol. Chem. 2008; 283: 10848-10857Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and then optimized as described in the text. All cell lines were pretreated with an equal volume of DMSO (control) or PD98059 for 1 h prior to activation of the AAR for the indicated times in the continued presence of inhibitor. HEK293T cells (0.5 × 106 cells/60-mm dish) were plated in DMEM 24 h before transfection to achieve 30–40% confluence. The cells were transiently transfected with plasmids expressing full-length ATF4 cDNA, a constitutively active MEK1 (MEKCA, kindly provided by Dr. Xingming Deng), or as a control, green fluorescent protein (GFP-pcDNA3.1) at 5 μg/60-mm dish, using a calcium phosphate protocol (23.Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Production of high-titer helper-free retroviruses by transient transfection.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8392-8396Crossref PubMed Scopus (2298) Google Scholar). The constitutively active MEKCA was created by the mutations S218E and S222D, two phospho-serine residues in the activation loop of MEK1 (24.Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Transformation of mammalian cells by constitutively active MAP kinase kinase.Science. 1994; 265: 966-970Crossref PubMed Scopus (1259) Google Scholar). The cells to be transfected were incubated with the plasmids overnight, washed twice with PBS, replenished with complete DMEM, and incubated for another 36 h prior to activation of the AAR. HC-04 human hepatocytes (MRA-156, MR4; ATCC, Manassas, VA) were transiently transfected using calcium phosphate. The GIPZ-shRNA plasmid constructs against GCN2 (catalog number RHS4430-101133792) or a nonsilencing Control (catalog number RHS4346) were purchased from Open Biosystems (Huntsville, AL). The shRNA constructs were packaged in HEK293T cells with the Trans-Lentiviral shRNA packaging kit (catalog number TLP5912) following the manufacturer's protocol. HepG2 cells were incubated at 37 °C for 6 h with lentiviral particles containing the shRNA construct. The infected cells were cultured with fresh culture medium for 48 h before puromycin selection (2.5 μg/ml) for at least 14 days. After the initial puromycin selection, individual clones were isolated by serial dilution and screened for the reduction in ATF4 induction following activation of the AAR. The siRNA si-ERK1 (L-003592-00-0005), siERK2 (L-003555-00-0005), siJNK1 (L-03514-00-0005), and siJNK2 (L-003505-00-0005) were purchased from Dharmacon/Thermoscientific and are unmodified siGENOME SMARTpool constructs. Transient siRNA transfections with 50 nm for each member to yield a total of 100 nm for siERK1+siERK2 or siJNK1+siJNK2 were performed in 12-well plates according to the manufacturer's protocol using DharmaFECT4 transfection reagent (T-2004-02) 72 h prior to activating the AAR. The same amount of siRNA with a scrambled sequence (D-001810-02-05) was used as the siControl. Total RNA was isolated with the TRIzol reagent (Invitrogen) following the manufacturer's directions. Transcription activity (hnRNA) and steady state mRNA levels were assayed by qPCR, as described previously (25.Shan J. Fu L. Balasubramanian M.N. Anthony T. Kilberg M.S. ATF4-dependent regulation of the JMJD3 gene during amino acid deprivation can be rescued in Atf4-deficient cells by inhibition of deacetylation.J. Biol. Chem. 2012; 287: 36393-36403Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). A 1-μg aliquot of total RNA was used to synthesize first strand cDNA with the qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). For qPCR, each cDNA sample was diluted 10× with TE buffer, 2 μl of this diluted solution was mixed with 10 μl of SYBR Green master mixture, and 5 pmol of forward and reverse primers were added in a total volume of 20 μl. The mixture was subjected to 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The primers used are listed in Table 1. After qPCR, melting curves were acquired by stepwise increase from 55 to 95 °C to ensure that only a single product was amplified in the reaction. GAPDH was used as an internal control, and all calculations were based on the difference of threshold cycle number of the analyzed gene relative to the GAPDH mRNA content in the same sample.TABLE 1PCR primersPrimer specificityPrimer sequences (human)aFP, forward primer; RP, reverse primer.ASNS, mRNAFP 5′-GCAGCTGAAAGAAGCCCAAGT-3′RP 5′-TGTCTTCCATGCCAATTGCA-3′GCN2, mRNAFP 5′-GAAATGGTAAACATCGGGCAAACTC-3′RP 5′-TTCACAAGAGCCAGGAGAATCTTCAC-3′GAPDH, mRNAFP 5′-TTGGTATCGTGGAAGGACTC-3′RP 5′-ACAGTCTTCTGGGTGGCAGT-3′VEGF-A, mRNAFP 5′-ACTGAGGAGTCCAACATCAC-3′RP 5′-CTTGTCTTGCTCTATCTTTC-3′EGR1, mRNAFP 5′-AGAAGGACAAGAAAGCAGACAAAAGTGT-3′RP 5′-GGGGACGGGTAGGAAGAGAG-3′EGR1, transcription activityFP 5′-CTACGAGCACCTGACCGCAGG-3′RP 5′-ACAGGACGCCAGGATGGTGG-3′EGR1 P1, ChIP assayFP 5′-CCCCGTCTCAGAAAGAATAAAAACATTA-3′RP 5′-CCTTGTGTCTGAATGTCCATTTTGC-3′EGR1 P2, ChIP assayFP 5′-CCTCTTTCGGATTCCCGCAG-3′RP 5′-GGTCCTTGTGGTGAGGGGTCA-3′EGR1 P3, ChIP assayFP 5′-GAGGGAGCGAGGGAGCAACC-3′RP 5′-CTCCAAATAAGGTGCTGCCCAAA-3′EGR1 P4, ChIP assayFP 5′-CATATTAGGGCTTCCTGCTTCCCATA-3′RP 5′-CCGCCTCTATTTGAAGGGTCTGG-3′EGR1 P5, ChIP assayFP 5′-GTCACGACGGAGGCGGACC-3′RP 5′-CGGCGGCTCCCCAAGTTC-3′EGR1 P6, ChIP assayFP 5′-CGCAGAGGACCGAGCTTTTGT-3′RP 5′-GCAGCCCCGCTCATCAAAA-3′EGR1 P7, ChIP assayFP 5′-GGGGATTCTCCGTATTTGCGTC-3′RP 5′-GGCTACCATTGACTCCCGAGGT-3′EGR1 P8, ChIP assayFP 5′-GTCCCAGCTCATCAAACCCAGC-3′RP 5′-AGAAGCGGCGATCACAGGACTC-3′VEGF-A upstream, ChIP assayFP 5′-ACTTTCCTGCTCCCTCCTCGC-3′RP 5′-CCACCAAGGTTCACAGCCTG-3′VEGF-A promoter, ChIP assayFP 5′-CGCTCGGTGCTGGAATTTGATA-3′RP 5′-TGGGGAATGGCAAGCAAAAA-3′VEGF-A intron 1, ChIP assayFP 5′-GCTGTCACTGCCACTCGGTCTC-3′RP 5′-GCAGCAATCCACCCCAAAAC-3′VEGF-A intron 4, ChIP assayFP 5′-GTGAGGATGTAGTCACGGATTC-3′RP 5′-CCAAAGGTCACATAGCGGGA-3′JNK1, mRNAFP 5′-CCATTTCAGAATCAGACTCATGCCA-3′RP 5′-TGTGGTGTGAAAACATTCAAAAGGC-3′JNK2, mRNAFP 5′-GGGATTGTTGTGCTGCATTTGATAC-3′RP 5′-TGGTTCTGAAAAGGACGGCTTAGTTT-3′ERK1, mRNAFP 5′-CGCTTCCGCCATGAGAATGTC-3′RP 5′-CAGGTCAGTCTCCATCAGGTCCTG-3′ERK2, mRNAFP 5′-CGTGTTGCAGATCCAGACCATGAT-3′RP 5′-TGGACTTGGTGTAGCCCTTGGAA-3′ATF3, mRNAFP 5′-GAGCGGAGCCTGGAGCAAAA-3′RP 5′-GGGGACGATGGCAGAAGCACT-3′a FP, forward primer; RP, reverse primer. Open table in a new tab Total RNA was isolated using the Qiagen RNeasy kit (Qiagen), including DNase I treatment before the final elution to eliminate any DNA contamination. The integrity of total RNA was monitored with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). A 100-ng aliquot of total RNA from four independent incubations for each of four conditions (DMEM versus DMEM lacking histidine, each with or without 2.5 μm PD98059) was amplified using the GeneChip® WT PLUS reagent kit (Affymetrix, Santa Clara, CA) following the manufacturer's instructions, and then 5.5 μg of cDNA was fragmented and terminally labeled. Labeled targets were hybridized to Affymetrix GeneChip® Human Transcriptome Array 2.0 for 16 h at 45 °C and washed according to Affymetrix standard protocols. For gene expression analysis, arrays were normalized using RMA as implemented in Partek Genomics Suite 6.6 (Partek Incorporated, St. Louis, MO). Analysis of variance was used to detect differentially expressed genes. The microarray data have been deposited in the NCBI Gene Expression Omnibus (accession number GSE58869). Whole cell protein was extracted with a RIPA buffer (50 mm Tris-HCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 2 mm EDTA, containing Pierce protease and phosphatase inhibitor mini-tablets (Thermo Scientific, Waltham, MA). Immunoblotting was performed as described previously (26.Chen H. Pan Y.X. Dudenhausen E.E. Kilberg M.S. Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrient-responsive bZIP transcription factors as well as localized histone acetylation.J. Biol. Chem. 2004; 279: 50829-50839Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The membrane was then incubated with one of the following antibodies: rabbit anti-ATF4 polyclonal antibody (27.Su N. Kilberg M.S. C/EBP homology protein (CHOP) interacts with activating transcription factor 4 (ATF4) and negatively regulates the stress-dependent induction of the asparagine synthetase gene.J. Biol. Chem. 2008; 283: 35106-35117Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), mouse anti-phospho ERK mouse monoclonal antibody (sc-7383), rabbit anti-total ERK polyclonal antibody (sc-94), rabbit anti-EGR1 polyclonal antibody (sc-189), and mouse anti-GAPDH monoclonal antibody (sc-32233) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The rabbit anti-β-actin polyclonal antibody (A2066) was from Sigma-Aldrich. The rabbit anti-JNK polyclonal antibody (9252s) was from Cell Signaling Technology (Danvers, MA). The bound secondary antibody was detected using an enhanced chemiluminescence kit (32106; Thermo Scientific) and then exposing the blot to Classic Blue Autoradiography Film BX (MIDSCI, St. Louis, MO). ChIP analysis was performed according to a previously published protocol (26.Chen H. Pan Y.X. Dudenhausen E.E. Kilberg M.S. Amino acid deprivation induces the transcription rate of the human asparagine synthetase gene through a timed program of expression and promoter binding of nutrient-responsive bZIP transcription factors as well as localized histone acetylation.J. Biol. Chem. 2004; 279: 50829-50839Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). HepG2 cells were seeded at a density of 1.5 × 107/150-mm dish with DMEM and cultured for ∼36 h, which includes a transfer to fresh DMEM during the final 12 h prior to AAR induction. Immunoprecipitation was performed with one of the following antibodies: rabbit anti-ATF4 polyclonal antibody described previously (27.Su N. Kilberg M.S. C/EBP homology protein (CHOP) interacts with activating transcription factor 4 (ATF4) and negatively regulates the stress-dependent induction of the asparagine synthetase gene.J. Biol. Chem. 2008; 283: 35106-35117Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), rabbit anti-RNA polymerase II polyclonal antibody (sc-899), rabbit anti-EGR1 polyclonal antibody (sc-189), rabbit anti-serum response factor (SRF, sc-335), and, as a nonspecific negative control, a normal rabbit IgG (sc-2027) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The total ELK1 (9182) and S103 phospho-SRF (4261) antibodies were purchased from Cell Signaling, whereas the S383 phospho-ELK1 antibody was obtained from Abcam (32799; Boston, MA). Immunoprecipitated DNA was analyzed with qPCR as described above, using primers listed in Table 1. The ChIP results are presented as the ratio to input DNA. Each experiment contained three or more individual samples to detect experimental variation, and each experiment was repeated one or more times with separate batches of cells to ensure reproducibility between experiments. The data are expressed as the averages ± standard deviations within an individual experiment containing three or four replicates, and the results, analyzed using Student's t test, with p ≤ 0.05 were considered statistically significant. A recent microarray expression analysis performed by our laboratory revealed the EGR1 gene to be the most highly induced gene following activation of the AAR in HepG2 hepatoma cells (10.Shan J. Lopez M.C. Baker H.V. Kilberg M.S. Expression profiling after activation of the amino acid deprivation response in HepG2 human hepatoma cells.Physiol. Genomics. 2010; 41: 315-327Crossref PubMed Scopus (41) Google Scholar). This observation is consistent with an earlier demonstration of EGR1 induction in MEF (11.Deval C. Chaveroux C. Maurin A.C. Cherasse Y. Parry L. Carraro V. Milenkovic D. Ferrara M. Bruhat A. Jousse C. Fafournoux P. Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways.FEBS J. 2009; 276: 707-718Crossref PubMed Scopus (106) Google Scholar). To investigate the mechanism of the EGR1 induction in HepG2 cells, both transcription activity and steady state mRNA content was assayed from 1 to 24 h (Fig. 1A). The results showed that transcription from the EGR1 gene was increased within 2 h of AAR activation and was enhanced by more than 30-fold at 8 h. Steady state mRNA content paralleled the transcription activity (Fig. 1A), and cellular EGR1 protein content was also increased (Fig. 1B). The half-life of the EGR1 mRNA was modestly stabilized by AAR activation (Fig. 1C), but transcription appeared to account for most of the increased mRNA. Although the EGR1 induction was also evident in nontransformed, immortalized HC-04 human hepatocytes (Fig. 1D), compared with the HepG2 cells, it was considerably smaller in magnitude (compare values in Fig. 1A “steady state mRNA” to Fig. 1D). To determine whether this difference was linked to cellular transformation, additional human hepatoma cell lines were examined. The induction of EGR1 mRNA in Hep3B cells was less than 2-fold, and in LH86 cells it was ∼3.5-fold (data not shown). Interestingly, among the human hepatoma cells that did not exhibit significant EGR1 induction was the HepG2-derived subclone, C3A cells. The C3A subclone was selected from a parental HepG2 culture based on more “hepatocyte-like” properties (American Type Cell Culture). Although striking, this contrast between HepG2 and C3A cells is consistent with the expression array data of Lee et al. (9.Lee J.I. Dominy Jr., J.E. Sikalidis A.K. Hirschberger L.L. Wang W. Stipanuk M.H. HepG2/C3A cells respond to cysteine-deprivation by induction of the amino acid deprivation/integrated stress response pathway.Physiol. Genomics. 2008; 33: 218-229Crossref PubMed Scopus (79) Google Scholar)," @default.
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