Sequential Serum-dependent Activation of CREB and NRF-1 Leads to Enhanced Mitochondrial Respiration through the Induction of Cytochrome c

Progression through the cell cycle requires ATP for protein synthesis, cytoskeletal rearrangement, chromatin remodeling, and protein degradation. The mechanisms by which mammalian cells increase respiratory capacity and ATP production in preparation for cell division are largely unexplored. Here, we demonstrate that serum induction of cytochrome c mRNA and processed protein in quiescent BALB/3T3 fibroblasts is associated with a marked increase in mitochondrial respiration. Cytochrome c was induced in the absence of any increase in citrate synthase activity or in subunit IV of the cytochrome c oxidase complex mRNA or protein, indicating that the enhanced respiratory rate did not require a general increase in mitochondrial biogenesis or respiratory chain expression. Transfections with a series of cytochromec promoter mutants showed that both nuclear respiratory factor 1 (NRF-1) and cAMP-response element-binding protein (CREB) binding sites contributed equally to induced expression by serum. Moreover, CREB and NRF-1 were phosphorylated sequentially in response to serum, and the NRF-1 phosphorylation was accompanied by an increase in its ability to trans-activate target gene expression. The results demonstrate that the differential transcriptional expression of cytochrome c, through sequential transcription factor phosphorylations, leads to enhanced mitochondrial respiratory capacity upon serum-induced entry to the cell cycle. Progression through the cell cycle requires ATP for protein synthesis, cytoskeletal rearrangement, chromatin remodeling, and protein degradation. The mechanisms by which mammalian cells increase respiratory capacity and ATP production in preparation for cell division are largely unexplored. Here, we demonstrate that serum induction of cytochrome c mRNA and processed protein in quiescent BALB/3T3 fibroblasts is associated with a marked increase in mitochondrial respiration. Cytochrome c was induced in the absence of any increase in citrate synthase activity or in subunit IV of the cytochrome c oxidase complex mRNA or protein, indicating that the enhanced respiratory rate did not require a general increase in mitochondrial biogenesis or respiratory chain expression. Transfections with a series of cytochromec promoter mutants showed that both nuclear respiratory factor 1 (NRF-1) and cAMP-response element-binding protein (CREB) binding sites contributed equally to induced expression by serum. Moreover, CREB and NRF-1 were phosphorylated sequentially in response to serum, and the NRF-1 phosphorylation was accompanied by an increase in its ability to trans-activate target gene expression. The results demonstrate that the differential transcriptional expression of cytochrome c, through sequential transcription factor phosphorylations, leads to enhanced mitochondrial respiratory capacity upon serum-induced entry to the cell cycle. nuclear respiratory factor 1 cAMP-response element-binding protein cAMP-response element phosphate-buffered saline carbonyl cyanide m-chlorophenylhydrazone tetramethyl-p-phenylenediamine di-HCl cytochromec oxidase subunit IV of the cytochrome coxidase complex 3-(cyclohexylamino)propanesulfonic acid casein kinase II rat cytochrome oxidase subunit IV gene Entry into the cell cycle requires energy in the form of ATP for the execution of a number of regulatory and metabolic events. Protein synthesis consumes large amounts of cellular energy and is required for entry into S phase (1.Brooks R.F. Cell. 1977; 12: 311-317Abstract Full Text PDF PubMed Scopus (234) Google Scholar). In quiescent 3T3 fibroblasts, cellular protein content increases within 4–5 h of serum stimulation and precedes DNA replication by several hours (2.Tiercy J.-M. Weil R. Eur. J. Biochem. 1983; 131: 47-55Crossref PubMed Scopus (6) Google Scholar). Nontoxic inhibition of mitochondrial respiratory function inhibits progression to G1 in parallel with a reduction in cellular ATP (3.Sweet S. Singh G. Cancer Res. 1995; 55: 5164-5167PubMed Google Scholar). Protein synthesis (1.Brooks R.F. Cell. 1977; 12: 311-317Abstract Full Text PDF PubMed Scopus (234) Google Scholar) and degradation (4.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (515) Google Scholar) as well as the depolymerization of the microtubular network during interphase (5.Marcussen M. Larson P.J. Cell Motil. Cytoskeleton. 1996; 35: 94-99Crossref PubMed Scopus (35) Google Scholar) all depend upon the intracellular ATP concentration. ATP has also been implicated in the regulation of cyclin-dependent kinases, which in turn control cell proliferation (6.Sheaff R.J. Groudine M. Roberts J.M. Clurman B.E. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (794) Google Scholar). The bulk of cellular ATP comes from the mitochondrial electron transport chain and oxidative phosphorylation system (7.Hatefi Y. Eur. J. Biochem. 1993; 218: 759-767Crossref PubMed Scopus (61) Google Scholar). This system comprises of five integral membrane complexes along with the dissociable electron carriers, ubiquinone and cytochrome c. An electrochemical gradient of protons is established across the mitochondrial inner membrane as a result of electron transfer. The dissipation of this proton gradient through the ATP synthase (complex V) is coupled to the synthesis of ATP (Fig. 1) (8.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar). The respiratory apparatus is the product of ∼90 different genes localized in both the nuclear and mitochondrial genomes (9.Attardi G. Schatz G. Annu. Rev. Cell Biol. 1988; 4: 289-333Crossref PubMed Scopus (1064) Google Scholar, 10.Wallace D.C. Annu. Rev. Biochem. 1992; 61: 1175-1212Crossref PubMed Scopus (1194) Google Scholar). Although the mitochondrial genes are essential, they encode only 13 respiratory polypeptides along with the tRNAs and rRNAs required for their translation in the mitochondrial matrix. Thus, nuclear genes specify the majority of respiratory subunits, and nuclear gene products govern mitochondrial gene expression in part by controlling the transcription and replication of mitochondrial DNA (11.Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (816) Google Scholar, 12.Grossman L.I. Lomax M.I. Biochim. Biophys. Acta Gene Struct. Expression. 1997; 1352: 174-192Crossref PubMed Scopus (101) Google Scholar, 13.Scarpulla R.C. J. Bioenergetics and Biomembranes. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar, 14.Scarpulla R.C. Trends Cardiovasc. Med. 1996; 6: 39-45Crossref PubMed Scopus (60) Google Scholar). In mammalian cells, little is known of the signaling pathways that mediate changes in the expression and function of the respiratory apparatus in meeting cellular energy demands. One means for responding to changing requirements for ATP is to modulate the synthesis or activity of respiratory chain constituents in response to extracellular signals. For example, in yeast, metabolites in the environment regulate the expression of cytochrome c and other respiratory proteins through the action of specific transcriptional activators and repressors (15.Zitomer R.S. Lowry C.V. Microbiol. Rev. 1992; 56: 1-11Crossref PubMed Google Scholar). Cytochrome c participates in the reduction of oxygen by cytochrome oxidase, the terminal and putative rate-limiting step of electron transfer (16.Poyton R.O. Trueblood C.E. Wright R.M. Farrell L.E. Ann. NY Acad. Sci. 1988; 550: 289-307Crossref PubMed Scopus (89) Google Scholar). The induction of cytochrome c by oxygen and nonfermentable carbon sources is part of a shift to aerobic growth. It is conceivable that in mammalian cells, the rate of respiration under certain physiological conditions may also be governed by regulating levels of cytochrome c. Mechanisms governing cytochrome c expression in mammalian cells may also be important in controlling apoptosis and levels of reactive oxygen species (reviewed in Ref. 17.Skulachev V.P. FEBS Lett. 1998; 423: 275-280Crossref PubMed Scopus (445) Google Scholar). We have previously isolated the human and rodent cytochromec genes and investigated the determinants of cytochromec transcription in mammalian cells (13.Scarpulla R.C. J. Bioenergetics and Biomembranes. 1997; 29: 109-119Crossref PubMed Scopus (229) Google Scholar). The somatically expressed gene has a complex promoter comprised of recognition sites for NRF-1,1 Sp1, CAAT box factors, and CREB/activating transcription factor among others (18.Evans M.J. Scarpulla R.C. J. Biol. Chem. 1989; 264: 14361-14368Abstract Full Text PDF PubMed Google Scholar). Tandem cAMP-response elements (CREs) were trans-activated by CREB in the presence of protein kinase A, and these CREs accounted for the cAMP induction of cytochrome c mRNA (19.Gopalakrishnan L. Scarpulla R.C. J. Biol. Chem. 1994; 269: 105-113Abstract Full Text PDF PubMed Google Scholar). These findings represent the only established link between a signaling molecule (cAMP) and a specific transcriptional activator (CREB) in the expression of a respiratory chain gene in vertebrate systems. NRF-1 was originally characterized as an activator of cytochromec expression and was subsequently found to act on many nuclear genes required for mitochondrial respiratory function (18.Evans M.J. Scarpulla R.C. J. Biol. Chem. 1989; 264: 14361-14368Abstract Full Text PDF PubMed Google Scholar, 20.Evans M.J. Scarpulla R.C. Genes Dev. 1990; 4: 1023-1034Crossref PubMed Scopus (332) Google Scholar). These include genes encoding respiratory subunits (21.Virbasius C.A. Virbasius J.V. Scarpulla R.C. Genes Dev. 1993; 7: 2431-2445Crossref PubMed Scopus (279) Google Scholar), the rate-limiting heme biosynthetic enzyme (22.Braidotti G. Borthwick I.A. May B.K. J. Biol. Chem. 1993; 268: 1109-1117Abstract Full Text PDF PubMed Google Scholar), as well as mtDNA transcription and replication factors (20.Evans M.J. Scarpulla R.C. Genes Dev. 1990; 4: 1023-1034Crossref PubMed Scopus (332) Google Scholar, 23.Virbasius J.V. Scarpulla R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1309-1313Crossref PubMed Scopus (615) Google Scholar). NRF-1 is phosphorylated both in vivo and in vitro on serine residues within a concise amino-terminal domain (24.Gugneja S. Scarpulla R.C. J. Biol. Chem. 1997; 272: 18732-18739Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Phosphorylation of these sites in vitro enhances NRF-1 DNA binding activity, suggesting that these modifications may regulate NRF-1 function. Such a mechanism might allow the nuclear transcriptional machinery to respond both to extracellular signals and to intracellular ATP concentrations in controlling the expression of the respiratory chain. However, no in vivo regulatory function has yet been ascribed to NRF-1 phosphorylation. Here, we examine changes in respiratory activity and cytochromec gene expression associated with the serum-induced proliferation of quiescent 3T3 fibroblasts. We find that the transcriptional induction of cytochrome c is intimately associated with enhanced respiratory activity in preparation for entry to the cell cycle. The results demonstrate that a change in respiratory capacity can be implemented through the differential expression of a key respiratory protein rather than through coordinate synthesis of the entire chain. BALB/3T3 cells, clone A31, passage 64 from the American Type Culture Collection, were tested for contact inhibition and used between passages 67 and 69 because the respiratory response to serum was diminished or absent in transformed cells. Tissue culture reagents were from Sigma except for fetal bovine serum, which was from HyClone Laboratories, Inc. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and supplemented with 4 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 1 mm sodium pyruvate. For all experiments, cells were plated at a density of 5 × 103 cells/cm2 in growth medium and allowed to grow for two days until ∼40–50% confluent. The cells were washed twice with Dulbecco's phosphate-buffered saline (PBS) and then serum-starved in Dulbecco's modified Eagle's medium, 0.5% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin for 60 h. Following starvation, the cells were stimulated in Dulbecco's modified Eagle's medium, 20% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin for the indicated times. The viable cell number was found to be constant over 12 h of serum stimulation. Schneider's Drosophilaline-2 (SL2) cells were obtained from the American Type Culture Collection at passage 519. All experiments were carried out between passages 521 and 541. Cells were cultured at 25 °C in Schneider's insect medium (Sigma) containing 10% fetal bovine serum as described (25.Cherbas L. Cherbas P. Roberts D.B. Drosophila: A Practical Approach. Oxford University Press, New York1998Google Scholar). Cells assayed for oxygen utilization were harvested using a 1× trypsin/EDTA solution, collected by centrifugation at 1000 × g, and resuspended in PBS. Viable cells were counted as determined by dye exclusion using 1× trypan blue solution. The cell suspension was pelleted and resuspended to 5 × 104 cells/μl in respiration buffer containing 30 mm Tris-HCl (pH 7.4), 75 mmsucrose, 50 mm KCl, 0.5 mm EDTA, 0.5 mm MgCl2, and 2 mm potassium phosphate. Oxygen utilization by ∼1.5 × 106 cells was measured in 650 μl of air-saturated respiration buffer at 37 °C with constant mixing using an oxygen-sensitive electrode and dual oxygen electrode amplifier (INSTECH Model 203). After the rate of oxygen uptake driven by endogenous cellular substrates was recorded, rates of oxygen utilization were determined upon sequential addition of the following substrates and inhibitors to the indicated final concentrations: 10 mm each glutamate plus malate (pH 7.4), 0.5 μm CCCP, 0.02 μm rotenone, 0.02 μm antimycin A, 2 mm ascorbate plus 0.2 mm TMPD, pH 6.0, 0.3 mm KCN, pH 8.0 (26.Boffoli D. Scacco S. Vergari R. Solarino G. Santacroce G. Papa S. Biochim. Biophys. Acta. 1994; 1226: 73-82Crossref PubMed Scopus (285) Google Scholar). The ascorbate/TMPD respiration rate was measured as that which was KCN sensitive. No increase in oxygen uptake was observed in the presence of digitonin indicating that the substrates are permeable. Cells were lysed in 20 mmphosphate, pH 7.4, 0.1% laurylmaltoside at 5 × 103cells/μl and incubated for 15 min on ice. Citrate synthase activity was assayed using extract from 2.5 × 104 cells in a 300-μl reaction containing 100 mm Tris, pH 8.0, 0.5 mm acetyl-CoA, 0.5 mm5,5′-dithio-bis(2-nitrobenzoic acid). The reaction was started by the addition of oxaloacetate to a final concentration of 0.5 mm. The change in absorbance at 419 nm was followed for 5 min (27.Srere P.A. Methods Enzymol. 1969; 13: 3-11Crossref Scopus (2035) Google Scholar). For the cytochrome oxidase (COX) assay, a 2-mmsolution of ferricytochrome c was reduced with several grains of sodium dithionite and chromatographed on a G50 Sephadex column (0.7 × 10 cm), and the ferricytochrome c eluate was assayed at 550 nm. COX activity using extract derived from 2 × 104 cells was assayed in 300 μl of 10 mmphosphate, pH 7.4, 10 μm ferricytochrome c by the change in absorbance at 550 nm (28.Cooperstein S.J. Lazarow A. J. Biol. Chem. 1951; 189: 665-670Abstract Full Text PDF PubMed Google Scholar). For spectrophotometric determination of cellular cytochrome contents, ∼3 × 106 cells were suspended in 20 mmTris, pH 7.5, 1% Triton X-100, 0.1 mm ferricyanide and incubated on ice for 10 min. The suspension was centrifuged at 10,000 × g; the supernatant was divided between reference and sample cuvettes (80-μl quartz). A Cary 300 Bio UV-visible spectrophotometer was zeroed across a spectral range of 420 to 640 nm with both cuvettes in place. Reduction of the sample with ascorbate plus TMPD was carried out in 0.4 mm KCN, 4 mm ascorbate, and 0.4 mm TMPD, and the absorbance spectra taken. The sample was subsequently reduced with 0.4 mm sodium dithionite, and a second absorbance spectra determined. Spectral data were expressed as reduced minus oxidized cytochrome spectral absorbance (29.Bookelman H. Trijbels J.M.F. Sengers R.C.A. Janssen A.J.M. Biochem. Med. 1978; 19: 366-373Crossref PubMed Scopus (133) Google Scholar). Nuclear extracts were prepared for immunoblotting as described (30.Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). For preparation of BALB/3T3 whole cell extracts, cells from a 150-mm plate at 60% confluence were lysed in 150 μl of RIPA buffer (50 mm Tris-HCL, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate) containing 5 mm NaF, 500 nmokadaic acid, 1 mm sodium orthovanadate as phosphatase inhibitors, 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin and allowed to incubate on ice for 30 min. The lysate was cleared by centrifugation at 20,000 ×g for 5 min, and the supernatant was collected. SL2 whole cell extracts were prepared from 10% of the transfected cells by lysis in 40 μl of RIPA. The remaining cells were used for reporter enzyme assays as described below. Extracts were subjected to SDS-polyacrylamide gel electrophoresis (36.5 acrylamide:1 bis-acrylamide) at 200 V for detection of cytochromec and COXIV (15% polyacrylamide) and CREB and phosphoCREB (10% polyacrylamide). The gels were transferred to either polyvinylidene difluoride (cytochrome c and COXIV) or to nitrocellulose (CREB and NRF-1). COXIV and cytochrome c were transferred in 10 mm caps, 10% methanol, pH 11. CREB was transferred in Towbin buffer (25 mm Tris, 192 mm glycine, 20% methanol, pH 8.3). Immunodetection was carried out with ECL reagents according to the manufacturer (Amersham Pharmacia Biotech). Plasmid p7ZfmCyt.c for the synthesis of a mouse cytochrome c riboprobe was made by cloning a 261-base pair HincII-BamHI fragment from the mouse gene (31.Limbach K.J. Wu R. Nucleic Acids Res. 1985; 13: 617-630Crossref PubMed Scopus (30) Google Scholar) into SmaI-BamHI-digested pGEM7Zf. A plasmid for the production of a rat COXIV riboprobe was constructed by cloning the 281-base pairBglII-PvuII fragment containing exon 4 (32.Virbasius J.V. Scarpulla R.C. Nucleic Acids Res. 1990; 18: 6581-6586Crossref PubMed Scopus (53) Google Scholar) into the BamHI and HincII sites of pGEM4Blue. The rat cytochrome c promoter luciferase plasmid, pGL3RC4/-326, was constructed by excising the promoter, 5′-untranslated region, and first intron fragment from pRC4CAT/-326 (33.Evans M.J. Scarpulla R.C. Mol. Cell. Biol. 1988; 8: 35-41Crossref PubMed Scopus (54) Google Scholar) with BglII, blunting with Klenow DNA polymerase, and digesting with XhoI. The resultant fragment was cloned into pGL3 basic vector (Promega) that had been cut with HindIII, blunt-ended with Klenow, and digested with XhoI. pGL3RC4/-66, pGL3RC4/-326, and pGL3RC4/-326;CRE DM1, were all derived from their respective CAT plasmids (19.Gopalakrishnan L. Scarpulla R.C. J. Biol. Chem. 1994; 269: 105-113Abstract Full Text PDF PubMed Google Scholar, 33.Evans M.J. Scarpulla R.C. Mol. Cell. Biol. 1988; 8: 35-41Crossref PubMed Scopus (54) Google Scholar) by substituting a XhoI-HindIII fragment. pGL3RC4/-326 NRF-1Mut was constructed from the parent plasmid pGL3RC4/-326 using oligonucleotide RC4NRF-1MutF 5′-ACCATGCTAGCCCTCATTAGCGCGCACCTTGCT-3′ and the QuikChangeTM site-directed mutagenesis kit (Promega). Similarly, pGL3RC4/-326;CRE DM1/NRF-1Mut was constructed from pGL3RC4/-326;CRE DM1 using RC4NRF-1MutR 5′-AGCAAGGTGCGCGCTAATGAGGGCTAGCATGGT-3′. TheHindIII-XhoI fragments from the amplified reaction products were subcloned back into the parent pGL3RC4/-326 and sequenced. The construction of pSG5NRF-1/3xHA has been described (24.Gugneja S. Scarpulla R.C. J. Biol. Chem. 1997; 272: 18732-18739Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In pPac5c, the Drosophila actin 5c promoter drives expression of cloned coding regions (34.Urness L.D. Functional Studies of the E74A Protein during Drosophila Metamorphosis, Ph.D. thesis/dissertation. University of Utah, 1995Google Scholar). To make pPac5cNRF-1, pSG5NRF-1/119-1662 was digested with EcoRI andPstI, and the 230-base pair fragment was cloned into pBluescript. The resulting plasmid was digested with EcoRV and PstI to release the NRF-1 fragment. Next, pET3dNRF-1/6His was cut with PstI and Acc65I and, together with the EcoRV/PstI NRF-1 fragment, cloned into pPac5c linearized with EcoRV andAcc65I. pPac5cNRF-1Mut8xA was made from pSG5NRF-1Mut 8xA (24.Gugneja S. Scarpulla R.C. J. Biol. Chem. 1997; 272: 18732-18739Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) as described for pPac5cNRF-1. p4xNRF-1Luc has been described (35.Gugneja S. Virbasius C.A. Scarpulla R.C. Mol. Cell. Biol. 1996; 16: 5708-5716Crossref PubMed Scopus (65) Google Scholar). pSG5CREB was made by subcloning the EcoRI/BglII fragment from pRSVCREB into the EcoRI/BamHI sites of pSG5. Total RNA was prepared using TRIzol reagent (Life Technologies, Inc.), and RNase protections were performed as described (36.Gugneja S. Virbasius J.V. Scarpulla R.C. Mol. Cell. Biol. 1995; 15: 102-111Crossref PubMed Scopus (100) Google Scholar). Reactions contained 5 μg of total RNA from BALB/3T3 cells and 15 μg of yeast tRNA. Antisense probes for hybridization were prepared by linearizing plasmids withMluI for mouse cytochrome c or EcoRI for RCO4 and transcribing with T7 polymerase. The 345-nucleotide cytochrome c probe gave a protected fragment of 175 nucleotides, whereas the 350-nucleotide RCO4 probe gave a protected fragment of 132 nucleotides. The protected products were run on a 6% acrylamide (29:1), 8 m urea gel and were visualized by autoradiography. BALB/3T3 cells were plated at 5 × 103 cells/cm2 in 6-well plates and incubated for 24–36 h. For transfections, 200 ng of pGL3RC4 reporter plasmid, 100 ng of pCMV β-gal (CLONTECH), and 3.7 μg of pGEM7Zf were added to 100 μl of Dulbecco's modified Eagle's medium. SuperFect (Qiagen)(5 μl) was added and vortexed for 10 s, and the mixture was incubated for 10 min. After the addition of 600 μl of growth medium, the suspension was added to a PBS-washed well and incubated for 6–8 h. The cells were washed three times with PBS and then starved with 2 ml of starvation medium for 48 h. The medium was changed to either stimulation or starvation medium, and the cells were incubated for an additional 13.5 h. COS cells were transfected with calcium/phosphate-precipitated DNA (33.Evans M.J. Scarpulla R.C. Mol. Cell. Biol. 1988; 8: 35-41Crossref PubMed Scopus (54) Google Scholar). SL2 cells were plated at 106 cells/ml in 100-mm dishes and transfected with calcium/phosphate-precipitated DNA (25.Cherbas L. Cherbas P. Roberts D.B. Drosophila: A Practical Approach. Oxford University Press, New York1998Google Scholar). Cell extracts were prepared, and luciferase assays were performed using reagents from Pharmingen. Spectrophotometric β-galactosidase assays were performed using the β-galactosidase enzyme assay system (Promega). Phosphoamino acid analysis was performed as described (24.Gugneja S. Scarpulla R.C. J. Biol. Chem. 1997; 272: 18732-18739Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). BALB/3T3 cells were labeled with [32P]orthophosphate after 60 h in starvation medium. The medium was changed to phosphate-free stimulation media, and 200 μCi of [32P]orthophosphate was added. Cells were incubated for 12 h before harvesting with RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS). Transfected SL2 cells were labeled with [32P]orthophosphate 24 h after removal of the DNA precipitate. The cells were replated in 4 ml of medium containing 1.5 mCi of [32P]orthophosphate. After 4 h they were washed with PBS and lysed in RIPA buffer. For in vivo 35S-labeling, cells were plated and after 16 h washed with PBS and labeled with 5 ml of methionine-free growth medium containing 250 μCi of [35S]methionine at 1000 Ci/mmol. After 16 h cells were washed with PBS, and 5 ml of methionine-free starvation medium containing 250 μCi of [35S]methionine was added. After 60 h the cells were again washed with PBS, and 5 ml of methionine-free stimulation medium containing 250 μCi of [35S]methionine was added. The cells were incubated for 12 h, washed with PBS, and lysed in RIPA buffer. In vivo labeled NRF-1 was immunoprecipitated from BALB/3T3 extracts as described (24.Gugneja S. Scarpulla R.C. J. Biol. Chem. 1997; 272: 18732-18739Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Extracts were precleared by incubation with normal rabbit IgG for 3 h followed by precipitation with protein A-agarose. We observed a marked increase in the endogenous rate of respiration in quiescent BALB/3T3 cells that were serum-stimulated (TableI). The increased respiration began within 3 h of stimulation and extended through at least 12 h. The elevated endogenous respiration rate, measured on a per cell basis, coincided with a similar increase in glutamate/malate-dependent respiration in the presence of an uncoupler. This is consistent with the enhancement of electron flow from NADH through the entire electron transport chain (Fig.1). An uncoupler (CCCP) was included to dissipate the proton gradient and thus alleviate respiratory control through complex V (Fig. 1). The increase in electron transport did not result from a general increase in respiratory chain synthesis or mitochondrial biogenesis. The activity of the mitochondrial matrix enzyme, citrate synthase, which is correlated with mitochondrial content (37.LaNoue K.F. Strzelecki T. Finch F. J. Biol. Chem. 1984; 259: 4116-4121Abstract Full Text PDF PubMed Google Scholar), remained unchanged in this time interval (Table I). In addition, there was no increase in cytochrome c oxidase activity as measured spectrophotometrically using reduced cytochromec as a substrate. Thus, the increased respiration did not coincide with a general increase in mitochondrial proliferation or respiratory chain expression.Table IEnzyme activities over a time course of serum stimulationSerum stimulation (h)Enzyme/substrate0312Activityfmol/min/cellEndogenousafmol of O2 consumed/min/cell using 1.5 × 106 intact cells in a 650-μl reaction volume.,bActivity values are the average of three independent experiments ± the standard error of the mean.0.88 ± 0.09 (1)cRelative activities normalized to citrate synthase.2.20 ± 0.28 (2.0)3.96 ± 0.12 (4.3)Glutamate + malateafmol of O2 consumed/min/cell using 1.5 × 106 intact cells in a 650-μl reaction volume.,bActivity values are the average of three independent experiments ± the standard error of the mean.1.65 ± 0.35 (1)3.75 ± 0.36 (1.8)5.13 ± 0.32 (2.9)Ascorbate + TMPD a,b,d,e3.49 ± 0.67 (1)4.86 ± 0.35 (1.1)8.02 ± 0.19 (2.2)Cytochrome oxidaseefmol of ferrocytochrome c oxidized/min/cell using a total cell extract from 2 × 104 cells in a 300-μl reaction volume.,ffmol of citric acid synthesized/min/cell using a total cell extract from 2 × 104 cells in a 300-μl reaction volume.18.33 ± 0.97 (1)18.01 ± 0.47 (0.8)10.64 ± 0.19 (0.6)Citrate synthasebActivity values are the average of three independent experiments ± the standard error of the mean.,ffmol of citric acid synthesized/min/cell using a total cell extract from 2 × 104 cells in a 300-μl reaction volume.3.16 ± 0.284.10 ± 0.263.38 ± 0.48d Assay performed in the presence of CCCP.a fmol of O2 consumed/min/cell using 1.5 × 106 intact cells in a 650-μl reaction volume.b Activity values are the average of three independent experiments ± the standard error of the mean.c Relative activities normalized to citrate synthase.e fmol of ferrocytochrome c oxidized/min/cell using a total cell extract from 2 × 104 cells in a 300-μl reaction volume.f fmol of citric acid synthesized/min/cell using a total cell extract from 2 × 104 cells in a 300-μl reaction volume. Open table in a new tab d Assay performed in the presence of CCCP. Although cytochrome oxidase activity was unchanged, increased respiration was observed using ascorbate plus TMPD (Table I), substrates that enhance respiration by reducing endogenous cytochromec (Fig. 1). This reaction was performed in the presence of antimycin A, which blocks the reduction of cytochrome c by NADH, and respiration was measured as the cyanide-sensitive component of the activity. In this manner, it was possible to dissect out the contribution of cytochrome c to the reduction of oxygen. The stimulation of oxygen consumption by ascorbate plus TMPD in response to serum stimulation demonstrated that cytochromec-dependent respiration contributed to the increased oxygen utilization. The observed increase in cytochromec-dependent respiration may be explained by the serum induction of cytochrome c relative to other respiratory components. Because cytochrome oxidase activity was not increased, the level of cytochrome c relative to cytochrome oxidase subunit IV (COXIV) was measured by immunoblotting. The results show a time-dependent elevation of cytochrome cprotein in total cell extracts, under conditions where COXIV protein levels remained unchanged (Fig. 2). When densitometric values were normalized to COXIV, cytochrome cincreased ∼1.5-fold after 3 h of serum stimulation and about 4-fold after 12 h. These values correlate well with the overall increase in respiration observed in these time intervals (TableI). To determine whether the induction of cytochrome c protein leads to higher levels of heme-containing holocytochrome, difference spectra obtained after 0 and 12 h of serum induction were compared. A clear increase in cytochrome c +c 1 absorbance at 55