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• W2096754987 abstract "The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondriarich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network. The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondriarich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network. Mitochondrial transcription factor A (Tfam 2The abbreviations used are: Tfamtranscription factor ANRFnuclear respiratory factorROSreactive oxygen speciesPI3Kphosphatidylinositol 3,4,5-triphosphate kinaseMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidemutmutantEMSAelectrophoretic mobility shift assaysiRNAsmall interfering RNARTreverse transcriptionChIPchromatin immunoprecipitationBRCA1breast cancer-associated gene 1.; mtTFA) is a 25-kDa nuclear-encoded protein with a 42-amino acid pro-sequence that is removed after mitochondrial importation (1Fisher R.P. Clayton D.A. J. Biol. Chem. 1985; 260: 11330-11338Abstract Full Text PDF PubMed Google Scholar). The functional protein has an amino-terminal high mobility group domain, a basic linker region, a second high mobility group domain, and a basic carboxyl-terminal tail (2Parisi M.A. Clayton D.A. Science. 1991; 252: 965-969Crossref PubMed Scopus (442) Google Scholar, 3Rantanen A. Jansson M. Oldfors A. Larsson N.G. Mamm. Genome. 2001; 12: 787-792Crossref PubMed Scopus (58) Google Scholar). Biochemical studies have demonstrated that human Tfam, like other high mobility group-domain proteins, binds, unwinds, and bends DNA without respect to sequence specificity (4Fisher R.P. Lisowsky T. Parisi M.A. Clayton D.A. J. Biol. Chem. 1992; 267: 3358-3367Abstract Full Text PDF PubMed Google Scholar). The mammalian Tfam yield after purification is ∼1 molecule/10–20 bp of mtDNA (5Alam T.I. Kanki T. Muta T. Ukaji K. Abe Y. Nakayama H. Takio K. Hamasaki N. Kang D. N ucleic Acids Res. 2003; 31: 1640-1645Crossref PubMed Scopus (277) Google Scholar, 6Takamatsu C. Umeda S. Ohsato T. Ohno T. Abe Y. Fukuoh A. Shinagawa H. Hamasaki N. Kang D. EMBO Rep. 2002; 3: 451-456Crossref PubMed Scopus (172) Google Scholar), as in yeast and avian cells (7Diffley J.F.X Stillman B. J. Biol. Chem. 1992; 267: 3368-3374Abstract Full Text PDF PubMed Google Scholar, 8Matsushima Y. Matsumura K. Ishii S. Inagaki H. Suzuki T. Matsuda Y. Beck K. Kitagawa Y. J. Biol. Chem. 2003; 278: 31149-31158Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 9Parisi M.A. Xu B. Clayton D.A. Mol. Cell. Biol. 1993; 13: 1951-1961Crossref PubMed Scopus (162) Google Scholar), and a high molar ratio may signify that Tfam protects mtDNA. Immunochemical studies of Tfam also support the idea that it is involved in nucleoid formation, i.e. packaging mtDNA into protein-DNA aggregates (5Alam T.I. Kanki T. Muta T. Ukaji K. Abe Y. Nakayama H. Takio K. Hamasaki N. Kang D. N ucleic Acids Res. 2003; 31: 1640-1645Crossref PubMed Scopus (277) Google Scholar, 10Garrido N. Griparic L. Jokitalo E. Wartiovaara J. van der Bliek A.M. Spelbrink J.N. Mol. Biol. Cell. 2003; 14: 1583-1596Crossref PubMed Scopus (272) Google Scholar). transcription factor A nuclear respiratory factor reactive oxygen species phosphatidylinositol 3,4,5-triphosphate kinase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide mutant electrophoretic mobility shift assay small interfering RNA reverse transcription chromatin immunoprecipitation breast cancer-associated gene 1. Tfam helps regulate mammalian mtDNA copy number, and in mice, Tfam disruption causes cytopathy, embryonic lethality, and diabetes due to mtDNA depletion and loss of oxidative phosphorylation (11Larsson N.G. Wang J. Wilhelmsson H. Oldfors A. Rustin P. Lewandoski M. Barsh G.S. Clayton D.A. Nat. Genet. 1998; 18: 231-236Crossref PubMed Scopus (1202) Google Scholar, 12Silva J.P. Kohler M. Graff C. Oldfors A. Magnuson M.A. Berggren P.O. Larsson N.G. Nat. Genet. 2000; 26: 336-340Crossref PubMed Scopus (366) Google Scholar). Tfam levels reflect cellular mtDNA content and diminish in cells depleted of mtDNA (13Davis A.F. Ropp P.A. Clayton D.A. Copeland W.C. Nucleic Acids Res. 1996; 24: 2753-2759Crossref PubMed Scopus (85) Google Scholar, 14Larsson N.G. Oldfors A. Holme E. Clayton D.A. Biochem. Biophys. Res. Commun. 1994; 200: 1374-1381Crossref PubMed Scopus (200) Google Scholar, 15Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Tfam expression is coordinated and regulated by a highly specific set of transcription factors, most notably two major trans-acting proteins, the nuclear respiratory factors (NRF-1 and -2) (16Virbasius J.V. Scarpulla R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1309-1313Crossref PubMed Scopus (615) Google Scholar). Also, the NRF-1 transcription function on Tfam promoter is stimulated by peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1) (17Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3192) Google Scholar). Promoter alignment studies indicate that mouse and rat Tfam promoters contain conserved Sp1 and NRF-2 recognition sites, but a standard NRF-1 consensus binding site has not been reported for either species (3Rantanen A. Jansson M. Oldfors A. Larsson N.G. Mamm. Genome. 2001; 12: 787-792Crossref PubMed Scopus (58) Google Scholar). Genomic footprinting of tumor cells has shown that high levels of Sp1 binding to the Tfam promoter up-regulate the mRNA expression (18Dong X. Ghoshal K. Majumder S. Yadav S.P. Jacob S.T. J. Biol. Chem. 2002; 277: 43309-43318Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Although these controls on Tfam activity have been identified, much information is lacking about retrograde signaling from mitochondria to nucleus and about factors that regulate the protein response to various cellular stressors. Communication between mitochondria and the nucleus is essential to energy homeostasis, and close coordination is especially important because the electron transport chain produces reactive oxygen species (ROS) (19Shigenaga M.K. Hagen T.M. Ames B.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 71: 10771-10778Crossref Scopus (1837) Google Scholar), which are damaging to mtDNA and loss of oxidative phosphorylation proteins. ROS leakage increases from damaged mitochondria, and low levels of ROS have been proposed as possible retrograde mediators of signaling from mitochondria to nuclei (20Scarpulla R.C. J. Bioenerg. Biomembr. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar). Specific pathways have not been elucidated, but we found previously in rat liver cells that the lipophilic oxidant, tertiary butyl hydroperoxide (t-BOOH), at low concentrations up-regulates NRF-1 and Tfam (15Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). This response was interpreted in the context of inflammation and recovery from mitochondrial genomic damage by an apparent redox signal for nuclear-encoded mitochondrial gene expression. To investigate whether ROS participate in retrograde signaling from mitochondria to nucleus, we tested the hypothesis in rat cells that peroxide regulates Tfam expression through NRF-1 phosphorylation and Tfam promoter binding to service mtDNA maintenance. Materials—All reagents were purchased commercially unless indicated otherwise. [γ-32P]ATP (3000 Ci/mmol) was from Amersham Biosciences, and phospho-Akt (Ser473) and Akt substrate antibodies were from Cell Signaling Technology (Beverly, MA). Rabbit αAkt/protein kinase B and NRF-1 antibody and αPTEN monoclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). MitoTracker® Green FM and MitoTracker® Red CM-H2XRos were from Invitrogen. LY294002 was from Alexis Corp. (San Diego, CA). pcDNA 3.1/V5-His TOPO TA expression kit was purchased from Invitrogen, and the Chip It kit was from Active Motif (Carlsbad, CA). All other chemicals and reagents, including RPMI and Dulbecco's-modified Eagle's medium with 25 mm Hepes and 4.5 g/liter glucose, were from Sigma. Cell Culture—H4IIE rat hepatoma cells (ATTCC, Manassas, VA) were grown in stationary culture in Dulbecco's-modified Eagle's medium supplemented with 10% fetal calf serum in 5% CO2 at 37 °C. Control hepatocytes were obtained from primary cultures of healthy liver of SD rats. HCC1937 cells (ATTCC) were grown and maintained in RPMI 1640 medium with 10% fetal bovine serum, 2.5 g/liter glucose, and 1 mm sodium pyruvate. Cell growth and viability were measured using the Cell Titer-Blue Assay (Promega, Madison WI). Where indicated, cells were preincubated with t-BOOH at 0–135 mm for 1, 3, or 5 h and without or with 50 μm LY294002 (phosphatidylinositol 3,4,5-triphosphate kinase (PI3K) inhibitor) 30 min before oxidant exposure. For specific inhibition of Akt isoenzymes, cells were preincubated with Akt inhibitor VIII (50 μm, Calbiochem) for 30 min before oxidant exposure. Mitochondrial Integrity—Mitochondrial function was assessed by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye to formazan in situ by the “succinate-tetrazolium reductase” system of the mitochondrial respiratory chain, which is active only in metabolically intact cells (21Berridge M.V. Tan A.S. Arch. Biochem. Biophys. 1993; 303: 474-482Crossref PubMed Scopus (1111) Google Scholar). H4IIE cells were plated at a density of 5 × 104 cells/well in a 96-well plate and exposed to 10 μm t-BOOH at 37 °C under 5% CO2. Cells were stained with MTT (0.5 mg/ml) for 4 h after incubation with t-BOOH. The resultant formazan crystals were dissolved in 200 μl of Me2SO, and absorbance was measured at 540 nm on an automatic microplate assay reader (Genius, Tecan Systems, Inc. San Jose, CA). MTT reduction was defined relative to untreated control cells, i.e. 100 × (absorbance of treated/absorbance of control sample). Western Blot Analysis—Protein samples (nuclear or cytoplasm) were separated by standard SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Nonspecific binding sites were blocked with Tris-buffered saline/Tween containing 5% non-fat dry milk for 12 h at 4 °C. Polyvinylidene difluoride membranes were incubated with antibodies to NRF-1 (1:2000), NRF-2 (1:5000), actin (1:5000; Sigma), tubulin (1:1000; Sigma), phosphorylated-Akt (1:1000) Akt (1:1000), or PTEN (1:2000). After five washes in Tris-buffered saline/Tween, polyvinylidene difluoride membranes were incubated in horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (1:10,000; Amersham Biosciences). The membranes were developed by enhanced chemiluminescence (Amersham Biosciences), and protein expression was quantified on digitized images from the mid-dynamic range and normalized to β-actin or tubulin in the same sample. At least four samples were used for densitometry measurements. Rat Tfam mRNA Expression—RNA was extracted with TRIzol reagent (Invitrogen), and 1 μg of each sample was reverse-transcribed (20-μl volume) using Moloney murine leukemia virus reverse transcriptase (Promega) in a buffer of random hexamer primers, dNTPs, and ribonuclease inhibitor (RNasin, Promega). Gene transcripts were amplified in triplicate by RT-PCR using specific primers Tfam sense, 5′-GCTTCCAGGAGGCTAAGGAT-3′, and Tfam antisense, 5′-CCCAATCCCAATGACAACTC-3′. 18 S rRNA was used to control for variation in efficiency of RNA extraction, reverse transcription, and PCR for nuclear RNA expression. Plasmids Expressing Rat NRF-1—NRF-1 expression vectors were constructed by RT-PCR with Pfu polymerase (Stratagene). The rat NRF-1 coding sequence (GenBank™ accession number XM231566) was amplified with the NRF-1 primers sense, 5′-ATGGAGGAACACGGAGTGAC-3′, and antisense, 5′-GCTTTTTGGGACAGTGAAAT-3′. The product was ligated into pcDNA 3.1/V5-His TOPO TA expression vector to express wild type NRF-1 (WT-NRF1, pc-NRF-1) fusion protein from a cytomegalovirus promoter. Mutant NRF-1 (mut-NRF-1, pcΔNRF-1) was created in which threonine was replaced with alanine in an Akt motif by site-directed mutagenesis using the QuikChange II kit (Stratagene) and the mut-NRF-1 primers sense (5′-AAAGAGACAGCAGACgcggttgcttcg aaac-3′) and antisense (5′-GTTTCCGAAGCAACCGcgtctgctgtctcttt-3′). H4IIE cells at 50–70% confluence were transfected with these or empty vector using manufacturer's protocols (Invitrogen). Cells were grown to 80–90% confluence, and WT and mutant NRF-1 recombinant proteins were purified using the ProBond purification system with anti-V5-horseradish peroxidase (Invitrogen). Immunoprecipitation—WT or mutant NRF-1 recombinant vectors were transfected transiently into H4IIE cells. Non-transfected and transfected cells were treated with t-BOOH (10 μm) without or with LY294002 (50 μm) 30 min before oxidant exposure. After incubation for 3 h, the cells were washed with ice-cold phosphate-buffered saline and lysed for 20 min in lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). Cell lysates or nuclear extracts (100 μg) were incubated with phospho-(Ser/Thr) Akt substrate antibody or with αNRF-1 antibody overnight at 4 °C and protein A-agarose beads (Santa Cruz Biotechnology) for 2 h. Beads were washed twice in lysis buffer and twice in phosphate-buffered saline, and proteins were eluted with SDS sample buffer for Western analysis using anti-phospho-Ser/Thr monoclonal antibody (BD Biosciences Pharmingen) or NRF-1 primary antibody (1:2000). Cloning of Rat Hepatoma Tfam—The 5′ region encompassing the promoter and 5′ region of Tfam was cloned by PCR with Pfu polymerase (Stratagene). Primers were designed from the published Tfam sequence (GenBank™ accession number AF264733), and rat genomic DNA was used as a template. The amplified DNA was cloned into TOPO vector (Invitrogen), and both strands were sequenced using a PerkinElmer Life Sciences Dye Terminator Cycle Sequencing system with AmpliTaq DNA Polymerase combined with ABI 3730 and 3100 PRISM DNA-sequencing instruments. Real-time DNA PCR—DNA primers were designed to detect COII and β-actin at a maximum amplicon length of 150 bp: β-actin forward, 5′-TGTTCCCTTCCACAGGGTGT-3′, and reverse, 5′-TCCCAGTTGGTAACAATGCCA-3′; COII forward, 5′-TGAGCCATCCCTTCACTAGG-3′, and reverse, 5′-TGAGCCGCAAATTTCAGAG-3′. The PCR reaction mixture contained 1× platinum SYBR green qPCR SuperMix UDG (Invitrogen), 500 nm each primer, and ∼10 ng of total genomic DNA or mtDNA. Real-time PCR conditions were 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Fluorescence intensities during PCR were recorded and analyzed in an ABI Prism 7000 sequence detector system (Applied Biosystems 7000 SDS software). The threshold cycle (CT) is the cycle at which the first significant increase in the fluorescent signal is detected. CT values in the linear exponential phase were used to measure the original DNA template copy numbers from a standard curve generated from five 10-fold dilutions of either pure mtDNA (COII) or pure nuclear DNA (β-actin). Relative values for COII and β-actin within samples were used to obtain a ratio of mtDNA to nuclear DNA in that sample. Electrophoretic Mobility Shift Assay (EMSA)—NRF-1 EMSA was performed with [γ-32P]ATP polynucleotide kinase-labeled oligonucleotides annealed to double-stranded oligonucleotides or PCR fragments. Binding assays were carried out on 10 μg of nuclear extract from hepatocytes or hepatoma cells (15Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). For supershifts, the reactions included 0.5–1 μg of undiluted goat anti-NRF-1 antiserum. For competition, excess unlabeled oligonucleotide was incubated with nuclear extract before adding labeled or unspecific oligonucleotide. After 20 min at room temperature, samples were loaded on pre-run 5% native polyacrylamide for 2.5 h at 10 V/cm, and the gels were dried and exposed to x-ray film. DNA oligonucleotide sequences of human Tfam were used for EMSA (NRF-1 recognition sites are underlined): Tfam NRF-1A, 5′-CGCTCTCCCGCGCCTGCGCCAATT; Tfam NRF-1B, 5′-GGGCGGAATTGGCGCAGGCGCGGG. The DNA oligonucleotide sequences of human Tfam at which NRF-1 recognition sites is mutated have been reported (15Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Rat Tfam promoter oligonucleotides were Tfam-205, 5′-TCAGAGGGGCCTGCGGCT-3′, and Tfam-185, 5′-AGCCGCAGGCCCCTCTGA-3′. Oligonucleotides for Sp1 and AP1 were purchased from Promega, and a rat Tfam promoter PCR fragment was generated using oligonucleotide pairs Tfam forward, 5′-GATACATTTCATGACCACTC-3′, and Tfam reverse, 5′-ACGGATGATGGACGAACGA-3′. NRF-1 Phosphorylation—To determine the effect of mutation of the Akt motif on NRF-1 phosphorylation, hepatoma cells were transfected with either WT-NRF-1 vector or mutant NRF-1 vector using FuGENE 6 transfection reagent (Roche Applied Science). Cells were collected in lysis buffer containing 1% Nonidet P-40, 0.05% SDS, 0.5% deoxycholate, 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm sodium orthovanadate, 25 mm NaF, 2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mg/ml leupeptin, and 2 mg/ml aprotinin. Cell lysate was collected and immunoprecipitated using anti-V5 antibody to bind the carboxyl-terminal V5 epitope tag of recombinant NRF-1 protein. Immunocomplex proteins were incubated with 100 ng/50 μl reaction of Akt/protein kinase B (Cell Signaling Technology) for 30 min with trace amounts of [γ-32P]ATP. The mixture containing phosphorylated recombinant protein was electrophoresed on 12% SDS-polyacrylamide. The gels were dried and exposed to x-ray film. NRF-1 Gene Silencing—Silencing experiments were performed using small interfering NRF-1 (siNRF-1) duplexes to target sequences in the open reading frame of NRF-1 mRNA. Multiple nucleotide sense and antisense siRNAs were synthesized and obtained in annealed form from Ambion. The siRNA target sequences were submitted to BLAST searches against other rat genome sequences to ensure specificity. After preliminary studies, one pair of siRNA sequences was selected and transfected at a concentration of 80 nm into H4IIE cells using Oligofectamine™ (Invitrogen) sense, 5′-GGAGGUUAAUUCAGAGCUG-3′, and antisense, 5′-CAGCUCUGAAUUAACCUCC-3′. A scrambled negative control siRNA from Ambion was also used. The effect of siRNA on NRF-1 and Tfam mRNA expression was established by both conventional and quantitative real time RT-PCR. For real-time PCR expression analysis, cells transfected with siNRF-1 or scrambled siRNA were washed in phosphate-buffered saline, and total RNA was extracted using Trizol reagent (Invitrogen). RNA samples were then DNase-treated with a DNA-free kit (Ambion). Treated RNA (1 μg) was reverse-transcribed in 20-μl reaction mixtures with random hexamer primers and the TaqMan reverse transcription reagents kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Two microliters of undiluted cDNA were used per reaction; a standard curve for NRF-1 and glyceraldehyde-3-phosphate dehydrogenase was generated from serial dilutions of positively expressed cDNA. The probe and primer sets were “pre-designed assay on demand” probes (Applied Biosystems) tested and standardized for reproducible expression analysis. Primer and cDNA were added to TaqMan universal master mix (Applied Biosystems) containing all the reagents for PCR. Relative quantification of the PCR products was carried out using the ABI Prism 7000 (Applied Biosystems) and a method that compares the amount of target gene amplification normalized to an endogenous reference (glyceraldehyde-3-phosphate dehydrogenase). The formula was 2–ΔΔCT, representing the n-fold differential gene expression in a treated sample compared with the control, where CT is the mean threshold cycle (cycle at which the product is detected initially). ΔCt was the difference in the CTvalues for the target gene and the reference gene, glyceraldehyde-3-phosphate dehydrogenase (in each sample assayed), and ΔΔCt represents the difference between CT of control and sample. ChIP Assay—Cells were transfected with NRF-1, WT, or mutant vectors and 48 h later were treated with 50 μm LY294002 30 min before 10 μm t-BOOH. After 3 h, 37% formaldehyde was placed directly into the medium of 4.5 × 107 cells to achieve a 1% final concentration. After 30 min at 37 °C, formaldehyde was quenched with 0.125 m glycine. Cells were washed with phosphate-buffered saline, harvested, and processed for immunoprecipitation using a kit (ChIP It assay kit, Active Motif) and NRF-1 antibody or V5 antibody following the manufacturer's protocol. After ethanol precipitation, DNA was resuspended in 200 μl/107 cells, and 2–5 μl were used as the template for each PCR. Input samples represent 1% of total DNA and were diluted 1:5, and immunoprecipitated fractions were diluted 1:2. PCR was carried out on 1 μl of each sample using sense primer, 5′-GGC AGTTTGCTGCTGGGT-3′, and antisense primer, 5′-GGCACTGTGGGAGGCCCA-3′, to amplify a 331-bp segment of the rat Tfam gene at –359 to –28 from the transcription start site. PCR products were analyzed on 2% ethidium bromide-stained agarose gels. Immunocytochemistry and Laser Scanning Confocal Microscopy— Rat H4IIE cells or human HCC1937 cells were grown in one- or two-well LabTek chamber slides and treated with t-BOOH (10 μm). The cells were then washed with Hanks' solution and incubated in normal media with MitoTracker® green (200 nm for H4IIE cells) or MitoTracker® red (100 nm for HCC1937 cells), incubated for 20 min, and rinsed with Hanks' balanced salt solution. H4IIE cells were fixed with cold 70:30 acetone/ethanol (v/v) for 10 min and blocked for 1 h with 10% fetal bovine serum in Hanks' solution at room temperature. The cells were incubated for 1 h with rabbit ant-NRF-1 primary antibodies followed by FluoroLink Cy3-labeled goat anti-rabbit IgG for 1 h. After extensive washing to remove unbound antibodies, the cells were mounted using the Slow Fade kit (Invitrogen). HCC1937 cells were mounted with Slow Fade immediately after fixation. Specific immunostaining was assessed by using one or both secondary antibodies without primary antibody. Multiple dilutions of each primary and secondary antibody were tested and optimized to minimize nonspecific adsorption of fluorescent antibodies, ensure separation of fluorescent signals, and optimize fluorophore concentration. Laser scanning confocal microscopy was performed on an LSM 410 microscope (Carl Zeiss MicroImaging, Inc.). Fluorescence images were collected using appropriate band-pass filters at excitation wavelengths of 488 and 568 and emission wavelengths of 520 and 590 nm. Statistics—Group values were expressed as the means ± S.D. Significance was tested by Student's unpaired t test, and a p value of <0.05 was accepted as significant. The n values provided refer to independent samples. Tfam and mtDNA Expression in H4IIE Rat Hepatoma Cells—Tfam mRNA levels were compared by RT-PCR in H411E rat hepatoma cells and normal rat hepatocytes. Tfam transcript analysis indicated a 5-fold higher expression level in hepatoma relative to normal hepatocytes (Fig. 1A). On this basis it was anticipated that transcription factors involved in Tfam gene expression would be up-regulated in rat hepatoma cells. Comparison of steady-state expression levels of NRF-1 and NRF-2 protein by Western blot analysis in normal rat hepatocytes and hepatoma cells (Fig. 1B) indicated that NRF-1 and NRF-2 protein levels were 4- and 3-fold higher, respectively, in rat hepatoma cells compared with normal hepatocytes (p < 0.05 for n = 4, graph not shown). For unknown reasons, these differences were only associated with 25% more mtDNA per H4IIE cell by real time PCR compared with normal rat hepatocytes (not shown). Because of the uncertainty that NRF-1 regulated Tfam gene expression in rat hepatoma cells, the 5′ upstream region of Tfam was cloned first. The nucleotide sequences for the 5′-upstream region and exon one of rat hepatoma Tfam are shown in Fig. 2A. The cloned region showed 99.4% sequence homology with the normal upstream region of rat Tfam (GenBank™ accession number AF264733). The transcription start site is numbered +1 according to the rat cDNA sequence (22Inagaki H. Hayashi T. Matsushima T. Lin K.H. Maeda S. Ichihara S. Kitagawa Y. Saito T. DNA Sequencing. 2000; 11: 131-135Crossref PubMed Scopus (25) Google Scholar). A computer-assisted sequence analysis of the 5′ promoter region identified a putative NRF-1 binding site in the Tfam promoter region (Fig. 2A). This motif is a near-perfect match for the NRF-1 consensus and contains all the invariant nucleotides in functional NRF-1 recognition sites (23FitzGerald P.C. Shlyakhtenko A. Mir A.A. Vinson C. Genome Res. 2004; 8: 1562-1574Crossref Scopus (174) Google Scholar, 24Scarpulla R.C. Biochim. Biophys. Acta. 2002; 1576: 1-14Crossref PubMed Scopus (515) Google Scholar, 25Kelly D.P. Scarpulla R.C. Genes Dev. 2004; 18: 357-368Crossref PubMed Scopus (983) Google Scholar). The binding site showed high homology with the human Tfam sequence (Fig. 2B) but overlapped with the Sp1 site, indicating the complex organization of the rat promoter. Effect of Exogenous Oxidant on Tfam Expression—To determine whether oxidants mediated Tfam gene expression, hepatoma cells were exposed to low concentrations of t-BOOH for 1 to 5 h. These exposures led to ∼10-fold increases in cellular Tfam gene expression (Fig. 3A) and were associated with stimulation of cell growth by 24h (Fig. 3B) and comparable increases in MTT reduction and mtDNA content by 48 h (Fig. 3C). Functional Characterization of the 5′-Flanking Region of Rat Tfam— To determine whether the putative NRF-1 consensus in rat Tfam could act as a cis element and bind NRF-1, EMSAs were performed in rat hepatoma cell nuclear extracts with oligonucleotides containing the NRF-1 recognition site of human Tfam. NRF-1 produced a slow-migrating band that was lost after the addition of a 100-fold excess of unlabeled oligonucleotide or PCR products containing the full rat Tfam promoter (–462/+79) or the GC-rich oligonucleotides –205 to –185 (Fig. 4A). Binding was checked and confirmed for recombinant NRF-1 protein alone. The results indicated that the cloned rat Tfam pr" @default.
• W2096754987 created "2016-06-24" @default.
• W2096754987 creator A5032021083 @default.
• W2096754987 creator A5070009351 @default.
• W2096754987 date "2006-01-01" @default.
• W2096754987 modified "2023-10-14" @default.
• W2096754987 title "Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1" @default.
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