Lipopolysaccharide Stimulates Mitochondrial Biogenesis via Activation of Nuclear Respiratory Factor-1
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m304719200
ISSN1083-351X
AutoresHagir B. Suliman, Martha Sue Carraway, Karen E. Welty‐Wolf, A. Richard Whorton, Claude A. Piantadosi,
Tópico(s)Metabolism and Genetic Disorders
ResumoExposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase γ, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (ρo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage. Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase γ, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (ρo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; ROS, reactive oxygen species; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation; TNF, tumor necrosis factor; ETC, electron transport chain; mtTFA, mitochondrial transcription factor A; NRF, nuclear respiratory factor; γGCS, γ-glutamylcysteine synthetase; EB, ethidium bromide; t-BH, tertiary butyl hydroperoxide; PI3K, phosphatidylinositol 3-kinase; PCNA, proliferating cell nuclear antigen; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; GSH, glutathione; RT, reverse transcriptase; Polγ, polymerase γ; muNRF, mutant nuclear respiratory factor. produces deleterious effects on mitochondria in the liver through reactive oxygen species (ROS) generation (1Taylor D.E. Ghio A.J. Piantadosi C.A. Arch. Biochem. Biophys. 1995; 316: 70-76Crossref PubMed Scopus (141) Google Scholar, 2Simonson S.G. Welty-Wolf K. Huang Y.C. Griebel J.A. Caplan M.S. Fracica P.J. Piantadosi C.A. Circ. Shock. 1994; 43: 34-43PubMed Google Scholar), glutathione depletion (3Kantrow S.P. Taylor D.E. Carraway M.S. Piantadosi C.A. Arch. Biochem. Biophys. 1997; 345: 278-288Crossref PubMed Scopus (143) Google Scholar, 4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar), mtDNA damage (4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar), and impairment of OXPHOS (5Taylor D.E. Kantrow S.P. Piantadosi C.A. Am. J. Physiol. 1998; 275: L139-LL44Crossref PubMed Google Scholar). These effects are associated with early receptor-mediated cytokine responses, e.g. production of interleukin-1 and tumor necrosis factor-α (TNF-α), which initiate the early immune response (6Tracey K.J. Cerami A. Annu. Rev. Cell Biol. 1993; 9: 317-343Crossref PubMed Scopus (761) Google Scholar). The hepatic response to LPS also includes activation of genes of mitochondrial recovery (4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar). Of particular interest is that TNF-α accelerates ROS production by mitochondria (7Schulze-Osthoff K. Bakker A.C. Vanhaesebroeck B. Beyaert R. Jacob W.A. Fiers W. J. Biol. Chem. 1992; 267: 5317-5323Abstract Full Text PDF PubMed Google Scholar, 8Schulze-Osthoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (549) Google Scholar), and exogenous ROS mimic aspects of TNF-α activity (9Kinnula V.L. Aito H. Klefstrom J. Alitalo K. Raivio K.O. Cancer Lett. 1998; 125: 191-198Crossref PubMed Scopus (6) Google Scholar, 10Arai T. Kelly S.A. Brengman M.L. Takano M. Smith E.H. Goldschmidt-Clermont P.J. Bulkley G.B. J. Biol. Chem. 1998; 331: 853-861Google Scholar). Furthermore, low TNF-α levels typically induce hepatocytes proliferation not cell death (11Gottlieb E. Vander Heiden M.G. Thompson C.B. Mol. Cell. Biol. 2000; 20: 5680-5689Crossref PubMed Scopus (310) Google Scholar, 12Beyer H.S. Stanley M. Theologides A. Biochem. Int. 1990; 22: 405-410Crossref PubMed Scopus (7) Google Scholar), whereas intense TNF receptor-1 stimulation produces cytotoxicity (13Cunningham P.N. Dyanov H.M. Park P. Wang J. Newell K.A. Quigg R.J. J. Immunol. 2002; 168: 5817-5823Crossref PubMed Scopus (313) Google Scholar). Mitochondria play crucial roles both in cell survival and death, and mitochondrial damage may initiate apoptosis, necrosis, or both. Mitochondria rely on an intrinsic genome (mtDNA) that is replicated and transcribed semi-autonomously but whose maintenance requires nuclear factors. The 16-kbp circular mtDNA contains limited genetic information relative to nuclear DNA; it encodes two rRNAs, 22 tRNAs, and 13 polypeptides of the electron transport chain (ETC). Mitochondrial transcription is regulated by mitochondrial transcription factor A (mtTFA) that contains domains for high mobility group-box proteins critical for DNA promoter binding and transcriptional activation. Nuclear high mobility group-box proteins enhance transcription and structural organization of chromatin and exhibit dual DNA binding ability (14Grosschedl R. Giese K. Pagel J. Trends Genet. 1994; 10: 94-99Abstract Full Text PDF PubMed Scopus (734) Google Scholar, 15Wolffe A.P. Science. 1994; 264: 1100-1103Crossref PubMed Scopus (171) Google Scholar). mtTFA initiates transcription by binding to mtDNA upstream of control elements of both heavy and light strand promoters (16Moraes C.T. Kenyon L. Hao H. Mol. Biol. Cell. 1999; 10: 3345-3356Crossref PubMed Scopus (71) Google Scholar). The mtTFA knockout mouse shows mtDNA depletion and lack of OXPHOS, and dies early in embryogenesis, establishing the role of mtTFA in coordinating mitochondria-nuclear genomic activity and regulation of mtDNA copy number (17Larsson 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). Regulation of mtTFA transcription is not well understood, but loss of mitochondrial transcription inhibits cell growth because of lack of energy for cell replication. Two redox-responsive transcription factors, nuclear respiratory factors-1 and -2 (NRF-1 and NRF-2), have been implicated in its expression (18Scarpulla R.C. Bioenerg Biomembr. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar, 19Martin M.E. Chinenov Y. Yu M. Schmidt T.K. Yang X.Y. J. Biol. Chem. 1996; 271: 25617-25623Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), as well as that of several ETC proteins (18Scarpulla R.C. Bioenerg Biomembr. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar, 20Evans M.J. Scarpulla R.C. J. Biol. Chem. 1989; 264: 14361-14368Abstract Full Text PDF PubMed Google Scholar). NRF-1 is phosphorylated, translocates to the nucleus, and binds to DNA as a homodimer. Because NRF-1 is encoded in the nucleus but controls synthesis of ETC components, it is a candidate for linking nuclear and mitochondrial genome activity during repair of damage to both mitochondria and cells. A role for NRF-1 in the duality of mtTFA function in mtDNA replication and transcription has not been explored in the context of LPS damage, which led to the present investigation. The implication is that elucidating a role for NRF-1 in communication between nucleus and mitochondrion after LPS-induced mitochondrial damage could provide new insight into the molecular mechanisms underlying a major clinical problem, organ failure of severe sepsis. Animal protocols were approved by the Duke University Animal Care and Use Committee. Male Sprague-Dawley rats weighing 300–400 g were injected with a single dose of intraperitoneal LPS (Escherichia coli, 055 B5; Difco, Inc.) dissolved in 1 ml of sterile pyrogen-free 0.9% sodium chloride at a sub-lethal dose (1 mg/kg) chosen for significant but reversible hepatic injury (4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar). Control animals were injected with an equal volume of 0.9% sodium chloride. Animals were killed 6, 24, and 48 h after injection, the abdomen was opened, and the livers were excised quickly and placed in cold isolation buffer. Fresh liver (4 g) was used to isolate mitochondria, and the remainder was snap-frozen and stored at –80 °C. For microscopy livers were flushed with phosphate-buffered saline, pH 7.0, and perfusion-fixed with 4% paraformaldehyde. The livers were removed, immersed in fixative for 2 h, and stored in 2% paraformaldehyde at 4 °C until paraffin embedding, sectioning, and staining for light microscopy. Highly purified, tightly coupled liver mitochondria were prepared as reported by discontinuous gradient centrifugation (1Taylor D.E. Ghio A.J. Piantadosi C.A. Arch. Biochem. Biophys. 1995; 316: 70-76Crossref PubMed Scopus (141) Google Scholar, 2Simonson S.G. Welty-Wolf K. Huang Y.C. Griebel J.A. Caplan M.S. Fracica P.J. Piantadosi C.A. Circ. Shock. 1994; 43: 34-43PubMed Google Scholar, 3Kantrow S.P. Taylor D.E. Carraway M.S. Piantadosi C.A. Arch. Biochem. Biophys. 1997; 345: 278-288Crossref PubMed Scopus (143) Google Scholar, 4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar). Nuclei were obtained by centrifugation of liver homogenate at 1000 × g for 20 min and further purified by centrifugation through 1.75 m sucrose. Protein content was determined by BCA method (Pierce, Rockford, IL) using bovine serum albumin as a standard. RNA was extracted with TRIzol reagent (Invitrogen), separated on 1.2% agarose gels, stained, and photographed. RNA was transferred to nylon membranes (Nytran Super Charge; Schleicher & Schuell) and UV cross-linked. PCR products were radiolabeled with [α-32P]dCTP using random primers and Ready-to-Go DNA labeling beads (Amersham Biosciences). Northern hybridization was done using QuickHyb (Stratagene). A γGCS (catalytic subunit) cDNA probe of 390 bp was used corresponding to nucleotides 79 to 468 of the rat sequence (21Yan N. Meister A. J. Biol. Chem. 1990; 265: 1588-1593Abstract Full Text PDF PubMed Google Scholar). After hybridization, membranes were washed twice in 0.2% SSC (0.15 m NaCl/0.015 m sodium citrate) containing 0.1% SDS at room temperature for 15 min then in 0.1% SSC/0.1% SDS at 60 °C for 45 min. The membranes were exposed to x-ray film (Eastman Kodak Co.) to develop autoradiograms. Genomic DNA was extracted with a DNA extraction kit (DNA Isolator PS Kit; Wako Pure Chemical Industries, Ltd., Tokyo, Japan), and DNA concentration was measured by optical density. mtDNA was prepared from rat liver mitochondria by NaI isolation (mtDNA Extractor WB kit; Wako Pure Chemical Industries, Ltd.) and stored at –20 °C. Synthesis of mtDNA Mutant Fragments—A DNA fragment of 590 bp of cytochrome b was amplified by PCR using 14314 FW primer, 5′-CATCAGTCACCCACATCTGC-3′, and 14904RV, 5′-GGTTAGCGGGTGTATAATTG-3′, and cloned into pGEM-T vector (Promega, Madison, WI). The cloned DNA fragment was truncated by 175 bp using BsmFI to generate an internal standard competitor of 415 bp. The competitor DNA was amplified in E. coli, extracted with phenol/chloroform followed by alcohol precipitation, separated into aliquots, and stored at –20 °C until use. Copy Number Determination by Competitive PCR—mtDNA copy number was quantified by competitive PCR based on co-amplification of target template with a known amount of competitor to account for variables affecting PCR amplification and assure a constant target/competitor ratio during amplification (22Suliman H.B. Majiwa P.A. Feldman B.F. Mertens B. Logan-Henfrey L. Gene (Amst.). 1996; 171: 275-280Crossref PubMed Scopus (15) Google Scholar). The rat cytochrome b-specific primers amplify 590 bp of the target mtDNA and 415 bp of the competitor DNA. Triplicate samples containing 2 × 103 copies (unless otherwise indicated) of competitor and 50 ng of target mtDNA were co-amplified in a thermal cycler (PE Applied Biosystems, Foster City, CA) at different cycle numbers optimized for visible product on ethidium bromide (EB)-stained gels in the PCR exponential phase. PCR products were separated by electrophoresis in 2% agarose. The intensities of the EB-stained bands were measured by densitometry, and the ratio of mtDNA-specific product was plotted against competitor density on a log scale. Copy number was determined from the x intercept or the point at which the ratio of the 590- and 415-bp products was 1 (log 0). A correction factor of 1.4 was used to account for differences in molecular weights. Cytoplasmic RNA was extracted with TRIzol (Invitrogen), and 1 μg from each sample was reverse-transcribed (in a total volume of 20 μl) using Moloney murine leukemia virus reverse transcriptase (180 units; Promega) in a reaction buffer containing random hexamer primers, dNTPs, and ribonuclease inhibitor RNasin (Promega). Gene transcripts were amplified in triplicate using gene specific primers (Table I). 18 S rRNA was used to control for variation in efficiency of RNA extraction, reverse transcription, and PCR for nuclear and mitochondrial RNA gene expression. Quantification of amplified mRNA was done by densitometry normalized to the 18 S rRNA mRNA signal density for each sample using image analysis software (Bio-Rad).Table IRT-PCR primer sequencesGeneSequence 5′-3′Product sizeAnnealing temperaturebp°CATPase 6SenseCCTCTTTCATTACCCCCACA32555AntisenseGAATTACGGCTCCTGCTCACytochrome cSenseGGAGGCAAGCATAAGACTG21355AntisenseGTCTGCCCTTTCTCCCTTCTCOX VSenseGTCACACGAGACAGATGA33958AntisenseCATCGAAGGGAGTTTACAmtSSBSenseAGCCAGCAGTTTGGTTCTTG28460AntisenseCCACTTTGCCTTCCACAAATmtTFASenseGCTTCCAGGAGGCTAAGGAT26455AntisenseCCCAATCCCAATGACAACTCNRF-1SenseCCACGTTGGATGAGTACACG26362AntisenseCTGAGCCTGGGTCATTTTGTNRF-2SenseCCGCTACACCGACTACGATT20660AntisenseACCTTCATCACCAACCCAAGPolγSenseGCCCATATCAGGGAGCAGTA25556AntisenseGTTGTGCACGTCTGCAAGAT Open table in a new tab Rat hepatoma cells obtained from American Type Culture Collection (H4IIE; Manassas, VA) were characterized in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C with 5% CO2. To deplete cells of mtDNA (ρo cells) normal H4IIE cells were cultured for 60 or more passages in the presence of 20 ng/ml EB in Dulbecco's modified Eagle's medium supplemented with 4.5 mg/ml glucose, 50 mg/ml uridine, and 100 mg/ml pyruvate to compensate for loss of respiration. Experiments were performed on cells 24 h post-confluence. One h before adding test agents cells were switched to phenol red-free Dulbecco's modified Eagle's medium with 0.5% fetal bovine serum (Invitrogen). Oxidant exposure of H4IIE (wild type) and mtDNA-deficient (ΔH4IIE) cells was produced with tertiary butyl hydroperoxide (t-BH). The t-BH (10–100 μm) diluted in serum-free medium was added to confluent monolayers in 6-well plates. Viability was assessed after trypsin release, staining with 0.4% trypan blue dye, and counting non-viable and total cells on a hemocytometer. PI3K inhibitor LY294002 was used at 50 μm. All measurements were performed in duplicate, and the experiments were repeated three times. Total DNA from H4IIE and respiration-deficient H4IIE clones was isolated, and 10-μg samples were digested to completion and separated by agarose electrophoresis. DNA was transferred to a nylon membrane as described (24Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5974) Google Scholar) and hybridized to the cytochrome b cloned probe labeled using Ready-to-Go DNA labeling beads (Amersham Biosciences) and [α-32P]dCTP (Amersham Biosciences). Mitochondrial membrane potential was compared in wild type and ΔH4IIE cells using a cell permeant cationic fluorescent indicator, rhodamine-123 (5 μm; Molecular Probes), which is sequestered by active mitochondria in proportion to membrane potential (23Royall J.A. Ischiropoulos H. Arch. Biochem. Biophys. 1993; 302: 348-355Crossref PubMed Scopus (1049) Google Scholar). ROS production was estimated in cells immediately after 3 h of exposure to t-BH using dihydrorhodamine-123 (Molecular Probes, Eugene, OR). Dihydrorhodamine-123 accumulates primarily in mitochondria and is oxidized by ROS to rhodamine-123 (23Royall J.A. Ischiropoulos H. Arch. Biochem. Biophys. 1993; 302: 348-355Crossref PubMed Scopus (1049) Google Scholar). Rhodamine-123 fluorescence (excitation wavelength, 490 nm; emission, 535 nm) was measured on a microplate reader (Safire; Tecan US, Research Triangle Park, NC), and signal intensity was expressed qualitatively in relative units. Protein samples (nuclear or cytoplasmic) were separated by SDS-PAGE and prepared for immunoblot analysis (1Taylor D.E. Ghio A.J. Piantadosi C.A. Arch. Biochem. Biophys. 1995; 316: 70-76Crossref PubMed Scopus (141) Google Scholar). Nonspecific binding sites were blocked with TBST (Tris-buffered saline/Tween) containing 5% nonfat dry milk for 12 h at 4 °C. Membranes were incubated with antibody as follows: polyclonal rabbit anti-PCNA (1:1000; Pharmingen, San Diego, CA), polyclonal rabbit anti-cyclin D1 and Cdk4 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal mouse anti-β-actin (1:5000; Sigma), polyclonal rabbit anti-NRF-1 (1:8000; see Ref. 20Evans M.J. Scarpulla R.C. J. Biol. Chem. 1989; 264: 14361-14368Abstract Full Text PDF PubMed Google Scholar), anti-tubulin (1:1000; Sigma), rabbit polyclonal anti-P-Akt (1:1000; Cell Signaling Technology, Beverly, MA), and rabbit polyclonal anti-Akt (1:1000; Cell Signaling Technology). After five washes in TBST, membranes were incubated in a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (Amersham Biosciences). The membranes were developed by enhanced chemiluminescence (Amersham Biosciences). Protein expression was quantitated on digitized images from the center of the dynamic range and normalized to β-actin or tubulin in the same sample. At least four samples were used for each densitometry measurement. EMSA for NRF-1 was performed with [γ-32P]ATP polynucleotide kinase-labeled oligonucleotides annealed to double-strand oligonucleotides. Binding reactions were carried out on 20 μg of nuclear extract from liver or hepatoma cell nuclei. Binding assays were performed in a volume of 20 μl with 5 μg of bovine serum albumin and 1 μg of sonicated salmon sperm DNA. Preliminary experiments were performed to determine the range of nuclear protein that generated a linear signal response for the gel shift on autoradiographs. Control binding experiments were performed under conditions of mutant NRF-1 probe. For supershifts, the reactions included 1 μl of undiluted goat anti-NRF-1 antiserum. For competition, excess unlabeled oligonucleotide was incubated with nuclear extract before adding labeled oligonucleotide. After 20 min at room temperature, reaction mixtures were loaded on prerun 5% native polyacrylamide for 2.5 h at 10 V/cm, and the gels were dried and exposed to x-ray film. The DNA oligonucleotide sequences used for EMSA were as follows (NRF-1 recognition sites are underlined): human mtTFA NRF-1A, 5′-CGCTCTCCCGCGCCTGCGCCAATT-3′; NRF-1B, 5′-GGGCGGAATTGGCGCAGGCGCGGG-3′. Control oligonucleotides were synthesized as follows (mutated regions underlined): muNRF-1C, 5′-CGCTCTCCGAAAATGAAAACAATT-3′; muNRF-1D, 5′-GGGCGGAATTGTTTTCATTTTCGG. Data were expressed as mean ± S.E. Statistics were performed by ANOVA followed by Tukey's post hoc comparison using computer software. A p ≤ 0.05 was considered significant. n values refer to independent replicates. Regression analysis was performed using Statview (SAS, Version 5.0.1). Oxidative Stress and Hepatic Cell and Mitochondrial Injury after LPS—LPS depletes hepatic glutathione (GSH) and increases glutathione disulfide (GSSG/GSH) even in highly coupled mitochondria (4Suliman H.B. Carraway M S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar); this stress occurred within 6 h and recovered by 48 h (not shown). GSH recovery was associated with increased expression of the regulatory heavy catalytic subunit of γGCS (24Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5974) Google Scholar). The mRNA for γGCS heavy chain increased 4-fold by Northern analysis at 24 h after LPS (0.46 ± 0.10 at 0 h versus 1.87 ± 0.12 at 24 h; see Fig. 1A, p < 0.05). Competitive PCR demonstrated transient decreases in mtDNA copy number coincident with GSH depletion/recovery (Fig. 1B). mtDNA copy number decreased significantly 6 h after LPS, stabilized at 24 h, and had recovered by 48 h. This coincided with an increased hepatocyte mitotic index (Fig. 1C). Nuclear and Mitochondrial mRNA—Mitochondrial transcription was checked using gene-specific primers for mitochondrial mRNA for four mitochondrial proteins (Fig. 2A), COX I, ND1, and ATPase 6 referenced to nuclear 18 S rRNA. At 24 h significant decreases were found in ATPase 6, COX I, and ND1 transcripts (22 to 27%), which recovered at 48 h. Because mitochondrial mRNA is transcribed as single polycistronic molecules from mtDNA heavy chain, similar COX I, ND1, and ATPase 6 mRNA contents indicate near steady-state conditions. Message for nuclear-encoded Complex IV subunits (cytochrome c oxidase, COX IV, COX V), and cytochrome c were analyzed by RT-PCR normalized to the stable nuclear-encoded 18 S rRNA (Fig. 2B). COX IV and cytochrome c mRNA doubled 24 h after LPS whereas COX V mRNA did not change relative to 18 S rRNA. Mitochondrial DNA Replication—Mitochondrial biogenesis requires mtDNA replication under nuclear regulation. To investigate mtDNA regulation, we examined three nuclear genes encoding proteins for mtDNA replication (Fig. 3). DNA polymerase γ (Polγ) replicates mtDNA, mitochondrial single-stranded DNA-binding protein (mtSSB) serves DNA replication, repair, and recombination, and mtTFA is the only currently known activator of mammalian mitochondrial transcription. Polγ, mtSSB, and mtTFA transcripts increased significantly by 6 h after LPS, further at 24 h, and by 48 h had declined toward pre-injury baseline. LPS Induced Protein Binding of NRF-1—The transcription factors NRF-1 and NRF-2 are involved in regulating expression of nuclear genes encoding major mitochondrial proteins that regulate mtDNA transcription and replication (18Scarpulla R.C. Bioenerg Biomembr. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar). We investigated the effect of LPS on NRF-1 activation by EMSA. An increase in NRF-1 binding was evident at 6 h after LPS exposure. Approximately 70% of the increase in NRF-1 protein binding was lost 24 h after LPS exposure (Fig. 4A). To confirm specificity of NRF-1 binding, we performed supershift assays with anti-NRF-1 antibody, and a mutated oligonucleotide sequence for NRF-1 interfered with protein binding (Fig. 4B). The murine NRF-1 peptide sequence revealed an Akt phosphorylation motif (RXRXX(S/T)) at position 104–109. Therefore, we assessed Akt activation by Western blot using anti-Akt and anti-phospho-Akt antibodies. To activate Akt fully, phosphorylation of two sites is necessary, one in the activation domain, and the other in the COOH-terminal hydrophobic motif (25Neri L.M. Borgatti P. Capitani S. Martelli A.M. Biochim. Biophys. Acta. 2002; 1584: 73-80Crossref PubMed Scopus (166) Google Scholar). Phospho-Akt increased significantly 6 h after LPS treatment and declined by 24 h but remained above baseline (Fig. 4C). LPS Induced Expression of NRF-2—We analyzed changes in NRF-2 mRNA by RT-PCR in liver after LPS. NRF-2 is also a nuclear regulatory protein for mitochondria; its mRNA was expressed at low levels in control liver (Fig. 4D), but transient NRF-2 mRNA induction was found within 6 h of LPS injection followed by a decline over the next 24 h (Fig. 4D). Up-regulation of NRF-2 expression is novel, and probable transcriptional regulation of this gene has not been demonstrated before. Cell Growth Regulatory Proteins—Cell cycle activation evaluated with a PCNA marker was increased significantly (p < 0.05) by LPS at 24 and 48 h (Fig. 5A). To determine whether G1 cyclin expression accompanied recovery from LPS injury, cyclin D1 and cyclin-dependent kinase-4 (Cdk4) expression were checked by Western analysis in liver nuclei and cytoplasm (Fig. 5B). At 24 h, cytoplasm and nuclear cyclin D1 protein levels were stable and nearly undetectable, but G1 cyclin was found after 48 h in both cytoplasm and nuclei (Fig. 5B). In comparison, Cdk4 constitutively present in control cytoplasm was equivocal in the nucleus. Cytoplasmic Cdk4 was unaffected by LPS, but the nucleoprotein was expressed significantly by 48 h (p < 0.05) (Fig. 5B). Nuclear translocation of D1 and Cdk4 indicate cell cycle progression. Respiration-deficient Cells—To assess the relationship between mitochondria and hepatic cell proliferation we selected a rat hepatoma (H4IIE) cell line that constitutively expresses NRF-1 and mtTFA and generated mtDNA depleted clones. Southern analysis of DNA from parental (H4IIE) and EB derivative cells identified two clones (ΔH4IIE and 1ΔH4IIE) as mtDNA-deficient cells (>98% reduction of the mtDNA content) (Fig. 6A). These cells grew slowly, depended on glycolysis, and required pyruvate and uridine (not shown). Both clones responded similarly, but the reported data were obtained from clone ΔH4IIE. To assess nuclear genes that control mitochondrial biogenesis, we examined mRNA for transcription factors NRF-1 and mtTFA in H4IIE and ΔH4IIE cells. Fig. 6B shows expression of NRF-1 and mtTFA relative to 18 S rRNA mRNA by RT-PCR in control and mtDNA-depleted cells. Depletion of mtDNA significantly down-regulated biogenesis gene expression in ΔH4IIE cells compared with H4IIE cells (Fig. 6C). Deletion of mtDNA decreased mitochondrial transmembrane potential and ROS generation relative to control cells as determined using fluorescence techniques (Fig. 7). The ΔH4IIE cells, like H4IIE cells, took up the fluorescent dye rhodamine-123 initially but lost it rapidly, whereas H4IIE cells retained the dye (Fig. 7A). The effect of mtDNA depletion on ROS generation was assessed by oxidation of dihydrorhodamine (Fig. 7B). In comparing parent (H4IIE) with mtDNA-deficient (ΔH4IIE) cells, significantly less fluorescence was detected in ΔH4IIE cells, but t-BH exposure caused both cell types to accumulate fluorescence signal in the mitochondria. Strong oxidants such as t-BH affect cell growth and viability by both direct oxidation and through secondary ROS generation (26Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (795) Google Scholar). In H4IIE and ΔH4IIE cells, viability was decreased significantly in a concentration-dependent manner after exposure to t-BH at concentrations of 50 μm or above for 24 h (not shown). However, lower concentrations of t-BH had either no effect or stimulated growth. To avoid cytotoxicity, proliferation studies were performed using 15 μm t-BH, which enhanced ΔH4IIE cell growth (and survival) significantly, restoring it almost to the level of control cells. H4IIE cells exhibited slight increases in growth rate at 24 to 48 h with t-BH (Fig. 7C). Translocation of mtDNA Regulatory Proteins—Translocation and binding of transcription factors NRF-1 and -2 induce mt-TFA expression in response to oxidative damage of mtDNA (27Paraidathathu T. de Groot H. Kehrer J.P. Free Radic. Biol. Med. 1992; 13: 289-297Crossref PubMed Scopus (83) Google Scholar, 28Asuncion J.G. Millan A. Pla R. Bruseghini L. Esteras A. Pallardo F.V. Sastre J. Vina J. FASEB J. 1996; 10: 333-338Crossref PubMed Scopus (272) Google Scholar). Therefore, we probed nuclear extracts of H4IIE and ΔH4IIE cells with anti-NRF-1 by Weste
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