Mitochondrial Transcription Factor A in Induction of Oxidative Phosphorylation and MyD88/TRIF...

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Mitochondrial Transcription Factor A in Induction of Oxidative Phosphorylation andMyD88/TRIF Dependent and Critical for TLR4-Mediated AKT Activation Is

SamavatiBirnbaum, Luigi Franchi, Gabriel Nuñez and LobeliaIcksoo Lee, Maik Hüttemann, Bobby Monks, Morris J. Christian P. Bauerfeld, Ruchi Rastogi, Gaila Pirockinaite,

http://www.jimmunol.org/content/188/6/2847doi: 10.4049/jimmunol.1102157February 2012;

2012; 188:2847-2857; Prepublished online 6J Immunol

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The Journal of Immunology

TLR4-Mediated AKT Activation Is MyD88/TRIF Dependentand Critical for Induction of Oxidative Phosphorylation andMitochondrial Transcription Factor A in MurineMacrophages

Christian P. Bauerfeld,*,1 Ruchi Rastogi,† Gaila Pirockinaite,† Icksoo Lee,‡

Maik Huttemann,‡ Bobby Monks,x Morris J. Birnbaum,x Luigi Franchi,{ Gabriel Nunez,{

and Lobelia Samavati†,‡

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is

associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis, and function requires coordinated expres-

sion of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam)

and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory

responses, but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase

that plays an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence we

investigated the role of LPS-mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macro-

phages (B6-MCL) and bone marrow-derived macrophages. We show that LPS challenge led to increased expression of T-fam and

COX subunits I and IV in a time-dependent manner through early phosphorylation of the PI3K/AKT pathway. PI3K/AKT

pathway inhibitors abrogated LPS-mediated T-fam and COX induction. Lack of induction was associated with decreased ATP

production, increased proinflammatory cytokines (TNF-a), NO production, and cell death. The TLR4-mediated AKT activation

and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll/IL-1R domain-containing adaptor-inducing

IFN-b. Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in

response to TLR4 agonist. The Journal of Immunology, 2012, 188: 2847–2857.

Mammalian immune cells evolved to recognize patho-gens through pattern recognition receptors, such asTLRs that recognize a wide range of microbial path-

ogens. Recognition of microbial components by TLRs triggers a

cascade of cellular signaling leading to inflammatory gene ex-pression and clearance of invading organisms. LPS, a majorbacteria-derived cell wall component recognized by TLR4, elicitsstrong host defenses in mammals with pronounced inflamma-tory responses (1). Such responses are characterized by activationof inflammatory cascades with release of soluble cytokines andchemokines such as TNF-a, IL-6, and generation of reactive ox-ygen species (ROS) and NO that subsequently facilitate theeradication of pathogens. Although inflammation is necessary toeradicate pathogens, excessive inflammation may lead to hosttissue injury and cell death. This might be, at least in part, becauseof the effect of inflammation on mitochondrial bioenergetics andmitochondrial injury.Mitochondria provide the majority of cellular energy in the form

of ATP and are the major source of ROS (2). Cellular immuneresponse to pathogens requires high energy in the form of ATP,and it appears that TLR-mediated mitochondrial ROS (mROS) areimportant for macrophage bactericidal activity (3). However, ex-cessive reactive oxygen and nitrogen species generation duringinflammation may damage mitochondria, causing bioenergeticfailure. This process is thought to be due to inhibition of theelectron transfer chain (ETC) (4) through secondary modificationof enzymes involved in oxidative phosphorylation (OxPhos) anduncoupling of electron transport from ATP synthesis. Using aninflammation model, we have shown that TNF-a inhibits the ac-tivity of cytochrome c oxidase (COX) in bovine liver and inmurine hepatocytes (5). This inhibition was associated with de-creased ATP production and defective electron transport to mo-lecular oxygen. Several reports suggest regeneration of mitochon-

*Department of Pediatrics, St. John Hospital and Medical Center, Detroit, MI 48236;†Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine,Wayne State University School of Medicine, Detroit, MI 48201; ‡Center for Molec-ular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI48201; xDepartment of Medicine, University of Pennsylvania School of Medicine,Philadelphia, PA 19104; and {Department of Pathology, University of MichiganMedical School, Ann Arbor, MI 48109

1Current address: Division of Critical Care, Department of Pediatrics, University ofTexas Health Science Center, San Antonio, TX.

Received for publication July 26, 2011. Accepted for publication January 3, 2012.

This work was supported by the Department of Medicine, Wayne State UniversitySchool of Medicine (to L.S.); the Center for Molecular Medicine and Genetics,Wayne State University School of Medicine (to L.S. and M.H.); and National In-stitutes of Health Grants R01 DK61707 (to G.N.) and R01 DK56886 (to M.J.B.).

Address correspondence and reprint requests to Dr. Lobelia Samavati, Division ofPulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Wayne StateUniversity School of Medicine, 3 Hudson, 3990 John R Street, Detroit, MI 48201.E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: BMDM, bone marrow-derived macrophage; COX,cytochrome c oxidase; ETC, electron transfer chain; FCCP, carbonyl cyanide 4-(tri-fluoromethoxy) phenylhydrazone; MnSOD, manganese superoxide dismutase;mROS, mitochondrial reactive oxygen species; NRF-1, nuclear respiratory factor1; OxPhos, oxidative phosphorylation; PGC-1a, peroxisome proliferator-activatedreceptor g coactivator-1-a; ROS, reactive oxygen species; T-fam, mitochondrialtranscription factor A; TMRM, tetramethylrhodamine-methylester; TRIF, Toll/IL-1R domain-containing adapter-inducing IFN-b; WT, wild-type.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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dria, upregulation of ETC capacity, and generation of new mito-chondria later in the disease process after acute insults such ashypoxemia or sepsis are important for survival (6–8). The exactmechanism underlying the recovery of bioenergetic failure afterinflammatory insult is not well studied.LPS stimulation under specific conditions leads to activation of

the prosurvival cascade followed by growth and differentiation. Forexample, it has been shown that LPS may serve as a signal topromote preconditioning and to protect against reperfusion in-jury (9). This bifunctionality of LPS is neither well understoodnor studied. Among numerous pathways activated through TLR-mediated signaling, the PI3K/AKT pathway plays a central role inthe regulation of cell survival and proliferation (9, 10). Evidencesuggests an important negative regulatory role for the PI3K/AKTpathway in innate immune responses to TLR agonists (11). Al-though growth factor-mediated PI3K activation and the subse-quent AKT activation is well characterized, TLR-mediated AKTactivation and its role in inflammation and mitochondrial recoveryin response to TLR4 agonists is not well defined. It was shown thatcertain TLRs, such as TLR4, require the adaptor protein MyD88and Toll/IL-1R domain-containing adapter-inducing IFN-b (TRIF)to interact and activate the PI3K/AKT pathway (12, 13). ThePI3K/AKT pathway plays both proinflammatory and anti-inflam-matory roles in TLR signaling (11, 14). The PI3K/AKT pathwaymay be a link between immune response through TLRs and re-storing energy metabolism through mitochondrial biogenesis.Recovery of energy capacity by biogenesis requires coordinated

expression of genes encoded by two independent genomes (nuclearand mitochondrial), including transcription factor activation andtheir binding to promoter consensus sequences in nuclear genesencoding mitochondrial proteins (7, 15). Among the transcrip-tional regulators, the mitochondrial transcription factor A (T-fam),a nuclear-encoded protein, governs mitochondrial gene expression(16, 17). In addition, the peroxisome proliferator-activated re-ceptor g coactivator-1-a (PGC-1a) and nuclear respiratory factors1 and 2 (NRF-1 and -2) are involved in nuclear respiratory geneexpression and serve to integrate mitochondrial gene expressionwith a wide range of cellular functions (17, 18).The exact molecular mechanism of LPS-mediated bioenergetic

recovery is not known. In this study, using a murine macrophagecell line and macrophages lacking MyD88/TRIF, we investigatedthe effect of LPS on mitochondrial bioenergetics. We show thatrecovery of cellular energy perturbation after LPS treatment isclosely related to PI3K/AKT activation, and inhibition of thispathway is directly related to bioenergetic failure and cell death.Using a genetic approach, we show that the AKT1 isoform playsa critical role in mitochondrial bioenergetics in response to TLR4.We present data that indicate that TLR4-mediated AKT activationrequires adaptor protein MyD88 and TRIF. Such activation iscritical for T-fam upregulation, and increased expression and ac-tivity of both COX subunit I (mitochondrially encoded) and COXsubunit IV (nuclear encoded).

Materials and MethodsChemicals

All chemicals were purchased from Sigma Chemical (St. Louis, MO) unlessspecified otherwise. Inhibitors (LY294002, wortmannin, okadaic acid) werepurchased from Calbiochem (San Diego, CA). Ultrapure LPS was pur-chased from Invivogen (San Diego, CA). Anti–phospho-AKT and PI3K,total AKT, AKT1, AKT2, and b-actin Abs were purchased from CellSignaling Technology (Beverly, CA). Abs against PI3K and T-fam werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Abs against mito-chondrial complexes, COX subunits I and IV (COX I and COX IV), werepurchased from Mitosciences (Eugene, OR). Ab against manganese su-peroxide dismutase (MnSOD) was purchased from New England Biolab

(Ipswich, MA). Anti-mouse IgG and anti-rabbit IgG HRP-linked Abs werepurchased from Cell Signaling Technology (Beverly, CA), and anti-goatHRP was purchased from Bio-Rad (Hercules, CA).

Cell culture

The mouse macrophage cell line (B6-MCL) and MyD88/TRIF2/2 cell linewere generous gifts of Dr. Eicke Latz (University of MassachusettsMedical School, Worcester, MA). Both cell lines were generated fromwild-type (WT) C57BL6 mice as previously described (19), and MyD88/TRIF2/2 macrophages were generated from the same background mice aspreviously described (20). B6-MCL and cells deficient in MyD88/TRIFwere grown in IMDM supplemented with 10% heat-inactivated FCS,antibiotics, sodium pyruvate, nonessential amino acids, and 2-ME.

RAW 264.7 cells were obtained from American Type Culture Collection(Manassas, VA). Cells were maintained in a 95% air, 5% CO2 humidifiedatmosphere at 37˚C in RPMI medium supplemented with L-glutamine,penicillin-streptomycin, and 10% FCS (Invitrogen, Carlsbad, CA).

Mice and macrophages

MyD882/2 and TRIF2/2 mice were generated as previously described(21). AKT12/2 and AKT22/2 mice were generated as previously de-scribed (22, 23). All mice were backcrossed onto the C57BL/6 backgroundat least 10 times. WT C57BL/6 mice were maintained at animal facilitiesat the University of Michigan (Ann Arbor, MI) and University of Penn-sylvania (Philadelphia, PA). Animal studies were approved by the Uni-versity of Michigan and University of Pennsylvania Committee on Use andCare of Animals.

Isolation of bone marrow-derived macrophages

Bone marrow-derived macrophages (BMDMs) from AKT12/2, AKT22/2,MyD882/2, and TRIF2/2 mice were prepared as described previously(24). In brief, femurs and tibias from 6- to 12-wk-old mice were dis-sected, and the bone marrow was flushed out. Bone marrow cells werecultured with IMDM supplemented with 30% L929 supernatant containingmacrophage-stimulating factor, glutamine, sodium pyruvate, 10% heat-inactivated FBS (Life Technologies-BRL), and antibiotics for 5–7 d. Mac-rophages were replated at a density of 2 3 106 cells/well the day beforethe experiment.

Protein extraction and immunoblotting

Cells were harvested after the appropriate treatment and washed with PBS.Total cellular proteins were extracted after addition of a protease inhibitormixture and antiphosphatase I and II, by adding RIPA buffer (SigmaAldrich). Protein concentrations of samples were measured with the BCAassay (Bio-Rad). Equal amounts of protein (5–30 mg) were mixed withsame volume of sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05%bromophenol blue, and 1.25 M Tris-HCl, pH 6.8), loaded on to a 10%SDS-polyacrylamide gel, and run at 40 mA for 3 h. Proteins were trans-ferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA)for 30 min at 20 V using a SemiDry Transfer Cell (Bio-Rad). The poly-vinylidene difluoride membrane was blocked with 5% dry milk in TBST(TBS with 0.1% Tween 20) for 1 h, washed, and incubated with primaryAb (diluted between 1/500 and 1/1000 in 5% dry milk in TBST) overnightat 4˚C. The blots were washed four times with TBST and then incubatedfor 1 h with HRP-conjugated secondary anti-IgG Ab using a dilution be-tween 1/5000 and 1/10,000 in 5% dry milk in TBST. Membranes werewashed four times in TBST. Immunoreactive bands were visualized usinga chemiluminescent substrate (ECL-Plus; GE Healthcare, Pittsburgh, PA)for 5 min. Images were captured on Hyblot CL film (Denville Scientific,Metuchen, NJ). OD analysis of signals was performed using ImageQuantsoftware from Molecular Dynamics (version 5). Equal loading of the blotswas shown by either total AKT, PI3K, or b-actin.

Cell viability

Cell viability was measured using the MTT assay as described previously(25). Cells equivalent to 1 3 105 ml21 were seeded in 96-well cell cultureplates and incubated for 24 h before treatment, as indicated in the Results.Absorbance was measured at 550 nm. Relative cell viability was calculatedaccording to the formula: cell viability (%) = absorbance experimental/absorbance control 3 100.

ATP assay

B6-MCL were cultured to 80% confluency and treated as indicated in eachexperiment. After completion of experiments, cells were collected byscraping and immediately stored in aliquots at 280˚C until measurement.

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ATP was released using the boiling method, by addition of 300 ml boilingbuffer (100 mM Tris-Cl [pH 7.75], 4 mM EDTA) and immediate transferto a boiling water bath for 2 min (5). Samples were put on ice and soni-cated for 10 s. Samples were diluted 300-fold, and 50 ml was used todetermine the ATP concentration using the ATP bioluminescence assay kitHS II (Roche, Indianapolis, IN), according to the manufacturer’s protocol.Experiments were performed in triplicate, and data were standardized tothe protein concentration using the DC protein assay kit (Bio-Rad).

Mitochondrial membrane potential measurements

The mitochondrial membrane potential (DCm) of intact cells was mea-sured as described previously (26) with modifications. Cultured cells werewashed with PBS and trypsinized. The protein concentration of cells wasadjusted to 0.2 mg/ml in DMEM without phenol red (Life Technologies-Invitrogen, Carlsbad, CA) and not supplemented with FBS and antibiotics.Tetramethylrhodamine-methylester (20 nM; TMRM; T-668; MolecularProbes-Invitrogen) was added to the cell suspension. As a control, theDCm was dissipated using 1 mM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 500 nM oligomycin. Cells were incubated at37˚C for 30 min in the dark under slow rotation. Yellow fluorescence(excitation, 532-nm laser; emission, 585 nm; band pass, 42 nm) wasmeasured using a BD FACS Array (BD Biosciences, San Jose, CA), anddata were analyzed with WinMDI version 2.9 software.

Measurement of nitrite

The nitrite concentration in the culture media was used as a measure of NOproduction (27). After stimulation/incubation, the generation of NO in thecell culture supernatants was determined by measuring nitrite accumula-tion in the medium using Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]-ethylenediamine dihydrochloride in 5% H3PO4; Sigma). Onehundred microliters of culture supernatant and 100 ml Griess reagent weremixed and incubated for 5 min. The absorption was measured in an au-tomated plate reader at 540 nm. Sodium nitrite (NaNO2; Sigma) was usedto generate a standard curve for quantification. Background nitrite wassubtracted from the experimental values. Results for each condition wereobtained from three separate measurements of identically treated wells,and the data are derived from four independent experiments.

ELISA

Murine TNF-a and IL-6 cytokine levels were measured in cell culturesupernatants according to the manufacturer’s instructions (ELISA DuoKits;R&D Systems) as previously described (25).

Measurement of COX activity

After appropriate treatments of cell homogenates in incubation buffer (250mM sucrose, 20 mM K-HEPES [pH 7.4], 10 mM MgSO4, 2 mM KH2PO4,1 mM PMSF, 10 mM KF, 2 mM EGTA, 2 mM oligomycin), COX activitywas analyzed in a closed 200-ml chamber equipped with a micro Clark-type oxygen electrode (Oxygraph system; Hansatech) as previously de-scribed (5). Measurements were performed in the absence of nucleotides.Measurements were carried out with 2 mM COX at 25˚C in the presence of20 mM ascorbate and increasing amounts of cow heart cytochrome c from0 to 40 mM. Oxygen consumption was recorded and analyzed with theOxygraph plus software. Turnover number is defined as consumed O2

(mM)/min/protein (mg) (5). Protein concentration was determined with theDC protein assay kit (Bio-Rad).

mROS measurements

Cells were stimulated with LPS (100 ng/ml) in the presence or absence ofwortmannin for different time points. As positive control, cells were treatedwith antimycin A (5 mM) for 30 min. To measure mROS, we incubatedcells with MitoSOX (Invitrogen) at a final concentration of 1 mM in thedark for 20 min at 37˚C. Cells were collected by centrifugation, washedthree times in PBS, and immediately resuspended in cold PBS and sub-jected to FACS analysis. MitoSOX Red was excited by laser at 488 nm,and the data were collected at forward scatter, side scatter, 585/42 nm(FL2), and 670LP (FL3) channel. Experiments were performed at least intriplicate. Data are presented as fold change in mean intensity of MitoSOXfluorescence when compared with PBS alone.

RNA extraction and quantitative reversetranscriptase/real-time PCR

Total RNAwas extracted using Stat 60 (Iso-Tex Diagnostics, Friendswood,TX) and reverse transcribed using the Reverse Transcription System(Promega, Madison, WI). The primers for amplification of T-fam, AKT,

PGC-1, NRF-1, COX IV, and a reference gene (b-actin) were used toamplify the corresponding cDNAs. Using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), we performed quantitative analysis of mRNA ex-pression with a MX3000p instrument (Stratagene, Santa Clara, CA). PCRamplification was performed in a total volume of 20 ml containing 2 mltotal cDNA and 20 pg primers (Invitrogen). The PCR amplification pro-tocol consisted of an initial incubation step for 10 min at 95˚C, followed by45 cycles of denaturation at 95˚C for 10 s, annealing for 20 s at 60˚C, andextension at 72˚C for 20 s. Relative mRNA levels were calculated afternormalizing to b-actin. Data were analyzed using the unpaired, two-tailedStudent t test, and the results were expressed as relative fold change.

Primer sequences were: b-actin, forward 59-GATTACTGCTCTGGC-TCCTAGC-39 and reverse 59-GACTCATCGTACTCCTGCTTGC-39; COXIV, forward 59-AGTTCAGTTGTACCGCATCCAG-39 and reverse 59-GG-GCCATACACATAGCTCTTCT-39; NRF-1, forward 59-CCATCTATCCG-AAAGAGACAGC-39 and reverse 59-GGGTGAGATGCAGAGTACAA-TC-39; PGC-1, forward 59-GAA GGC CGT GTG GTATAC ATT C-39 andreverse 59-CTG GCC TCT TTTACT TCT CGT C-39; and T-fam, forward59-CCTGAGGAAAAGCAGGCATA-39 and reverse 59-TCACTTCGTCC-AACTTCAGC-39.

Statistical analysis

Statistical analyses were performed using SPSS software, version 18.0(SPSS, Chicago, IL). One-way ANOVA test and post hoc repeated-measurecomparisons (least significant difference) were performed on all obtaineddata. ELISA, MTT, ROS, and quantitative real-time RT-PCR results wereexpressed as mean 6 SEM. For all analyses, two-tailed p values ,0.05were considered significant.

ResultsLPS treatment induces COX upregulation at the gene andprotein level

Because macrophages play a central role in recognition of TLRligands and initiating inflammatory responses, as well as ROSproduction, we have chosen murine macrophages to determine theeffects of LPS on the ETC. To show reproducibility in independentsystems, we conducted most experiments in two different celllines. Murine macrophage cells (B6-MCL) were generated aspreviously described (19). We also confirmed the results usingRAW 264.7 cells. In all experiments, cells were grown to reach80% confluency. B6-MCL cells were treated with 100 ng/mlultrapure LPS (to assure only TLR4-mediated signaling) fordifferent periods as indicated. Because the exact effects of LPSon the ETC and mitochondrial transcription factors in immunecells are unknown, we first investigated the kinetic responsesof important members of the ETC, including COX subunits Iand IV, as well as mitochondrial transcription factors NRF-1and T-fam, in response to LPS stimulation. Total cell lysateswere subjected to immunoblotting using specific Abs against COXsubunits I (mitochondrially encoded) and IV (nuclear encoded),NRF-1, and T-fam. As shown in Fig. 1A, LPS induced expressionof both subunits of COX in a time-dependent manner. We observedincreased protein expression for COX I/IV starting at 3 h after LPSchallenge, whereas increased expression of T-fam was observed asearly as 90 min after treatment. LPS stimulation had only a minimaleffect on NRF-1 protein expression at a later time point (after24 h; Fig. 1A). Fig. 1B summarizes the densitometric analysis offour different experiments. These data indicate that LPS increasedprotein expression of COX, and that there was a time-dependentprotein expression of mitochondrial transcription factors in re-sponse to LPS. To assess the dose-dependent upregulation of COXI, we treated cells with different log concentrations of LPS for 6 h.Supplemental Fig. 1 shows a dose-dependent but nonlinear re-sponse to LPS in terms of COX I upregulation. Fig. 1C showsmeans of relative gene expression for T-fam, COX IV, and NFR-1after 2-h LPS treatment. Although relative gene expression in-creased in response to LPS for all these genes, there was morevariation in terms of protein expression as determined by immu-

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noblotting, suggesting posttranscriptional regulation, a regulatorymechanism that seems to be important in mitochondria-relatedgenes (28).

LPS alters mitochondrial membrane potential, COX activity,and ATP production

The mitochondrial membrane potential (DCm) is generatedthrough pumping of protons across the mitochondrial inner mem-brane by ETC complexes I, III, and IV (COX) producing theproton motive force used by ATP synthase to generate ATP. Pre-vious studies have shown that DCm of monocytes is decreased insevere sepsis, and that this is associated with cell death (29). Weanalyzed the kinetic response of DCm in response to LPS treat-ment using a membrane potential-sensitive fluorescent probe(TMRM) and flow cytometry. As controls, the DCm was dissi-pated using 1 mM FCCP, resulting in a left shift of TMRM fluo-rescence, or increased DCm using 0.5 mM oligomycin with a rightshift of fluorescence (Fig. 2A). LPS treatment led to an early (at30 min) reduction in TMRM fluorescence indicating a decreasedDCm (Fig. 2B) followed by a recovery of DCm starting at 3 hafter LPS treatment (Fig. 2C). At 6 h after LPS treatment, DCmrecovered and was even slightly higher compared with untreatedcells (Fig. 2D), consistent with increased protein expression forCOX subunits at this time point (Fig. 1A, 1B).Next, we asked whether LPS-mediated increases in protein ex-

pression for COX I/IV are associated with a change in both theactivity of COX and changes of ATP content of cells. Murinemacrophages (B6-MCL) were cultured in the presence of LPS (100ng/ml) for 6 h as COX I and T-fam levels peaked at that time point(Fig. 1B). After solubilization, COX activity was measured byadding increasing amounts of its substrate cytochrome c. Asshown in Fig. 2E, LPS treatment led to a 56% increase of COXactivity at maximal turnover.To further assess the link between the change of protein ex-

pression in response to LPS treatment and changes in DCm, wemeasured the ATP content of B6-MCL cells. Fig. 2F shows thetime-dependent changes of ATP levels after LPS treatment of B6-MCL cells with a significant increase of ATP after 6 h and a fur-ther increase after 12 h. At 3 h, ATP levels were slightly de-creased, then increased .3-fold and 5-fold after 6 and 12 h,respectively. These data confirm the relationship between cellularATP and mitochondrial OxPhos capacity represented in the formof COX activity and expression.

LPS treatment activates both PI3K and AKT

The PI3K and AKT pathways are involved in many importantcellular processes including cell growth, cell survival, and apo-ptosis (30). Because of the central role of mitochondria as medi-ators of cell survival and apoptosis, we investigated the effect ofLPS-mediated AKT activation on the upregulation of T-fam, aswell as COX I. First, we examined the time-dependent phos-phorylation of PI3K and AKT. B6-MCL cells were challengedwith LPS (100 ng/ml) for different time points as indicated (Fig.3A). Total cell lysates were subjected to Western blot analysisusing a specific Ab against the phosphorylated form of the regu-latory domain of PI3K (phospho-p85) and an Ab detecting theSer473-phosphorylated form of AKT. Fig. 3A shows rapid phos-phorylation of p85 5 min after LPS challenge followed by AKTphosphorylation on Ser473. Next, we explored specific inhibitors ofthe PI3K/AKT pathway and their effect on LPS-mediated p85 andAKT phosphorylation. B6-MCL cells were treated with eitherLY294002 or wortmannin 30 min before LPS challenge or kept inmedia without treatment. Fig. 3B shows that both inhibitorsLY294002 and wortmannin prevented phosphorylation of p85 andAKT, indicating that LPS-induced AKT phosphorylation is PI3Kdependent.

Inhibition of the PI3K/AKT pathway abrogates LPS-mediatedupregulation of COX and T-fam

We next assessed the effect of inhibition of the PI3K/AKT pathwayon protein expression for COX I and T-fam. B6-MCL cells weretreated with vehicle (DMSO), LY294002, or wortmannin 30 minbefore LPS challenge. Immunoblotting on total cell lysates wasperformed using specific Abs against NRF-1, COX I, and T-fam.Fig. 4 shows that pretreatment with wortmannin inhibited LPS-mediated upregulation of both COX I and T-fam with a minimaleffect on NRF-1 expression. Similar results were observed in thepresence of LY294002 (Supplemental Fig. 2). We observed anincrease in expression for both proteins when cells were exposedto wortmannin or LY294002 alone. In the presence of AKT in-hibitors, however, LPS-mediated protein expression for T-fam andCOX I was strongly diminished. These data suggest that the ac-tivation of the PI3K/AKT pathway is required for LPS-mediatedupregulation of COX subunit I and T-fam. It is known that proteinphosphatase 2A plays a pivotal role in dephosphorylation of AKT,and thereby diminishes its activity (31). To confirm that phos-phorylation of AKT plays a critical role in LPS-mediated upreg-

FIGURE 1. Time-dependent effect of LPS on mitochondrial transcription factors (NRF-1, T-fam) and COX subunits I and IV. Murine macrophages (B6-

MCL) were cultured in a density of 2 3 106/well 24 h before LPS treatment. Cells were treated with LPS (100 ng/ml) for different periods. (A) Whole-cell

extracts were prepared, and 30 mg total protein was subjected to SDS-PAGE and Western blot analysis using Abs against COX subunits I and IV, NRF-1,

and T-fam. Equal loading was determined using b-actin. Cells responded to LPS stimulation with an early increase in expression of T-fam followed by

increased expression of both COX subunits (I and IV). NRF-1 increased at a later time point (24 h). (B) Densitometric analysis (mean) of NRF-1, COX I,

and T-fam of four independent experiments. (C) Relative gene expression for T-fam, COX IV, and NRF-1 in response to LPS. Cells were treated with LPS

(100 ng/ml) for 2 h, RNA was isolated, and quantitative real-time RT-PCR was carried out for T-fam, NRF-1, and COX IV. Values were normalized to

b-actin. Results represent mean values of four independent experiments each performed in triplicate. Using ANOVA Mann–Whitney U test, p , 0.05 was

considered significant. *p , 0.05, **p , 0.001.




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ulation of COX I and T-fam, we evaluated the effect of a proteinphosphatase 2A inhibitor (okadaic acid) to prevent the dephos-phorylation of AKT. Cells were incubated with okadaic acid (10nM) 30 min before LPS treatment for different time points. Asexpected, okadaic acid treatment led to AKT phosphorylationalone and to a prolonged activation of AKT in the presence of LPS(data not shown). Next, we evaluated the effect of okadaic acid inthe presence and absence of LPS on COX I. Okadaic acid pre-treatment led to an enhanced LPS effect in terms of COX I up-regulation (Fig. 4C), supporting the role of active AKT in thisprocess.To further confirm that the AKT pathway is a distinct intra-

cellular signal and specific to LPS-mediated upregulation of COX Iand T-fam, we assessed the effect of LPS challenge in the presenceand absence of LY294002 or wortmannin on MnSOD. MnSOD,a vital antioxidant enzyme localized to the mitochondrial matrix,catalyzes the dismutation of superoxide anions to H2O2. It has beenshown that LPS challenge leads to rapid induction of MnSOD atthe transcriptional and protein levels (32, 33). Because rapid up-regulation of the mitochondrial respiratory chain proteins in re-sponse to LPS is associated with increased ROS production (34),we investigated the effect of LPS on MnSOD protein expression.We confirmed in our experimental model that LPS challenge leadsto rapid induction of MnSOD (Fig. 4D, upper panel). Although ithas been shown that MnSOD expression is associated with mROSproduction during inflammation, the signaling pathways respon-sible for such a response are still unclear. We thus assessed the

effect of LPS on MnSOD expression in the presence or absence ofLY294002 or wortmannin. B6-MCL cells were cultured and pre-treated with either inhibitor for 30 min before LPS treatment. A6-h LPS treatment duration was chosen because it produced a ro-bust induction of MnSOD and COX I expression. MnSOD ex-pression was analyzed by immunoblotting of total cell lysatesusing a specific Ab. As shown in Fig. 4D (lower panel), neither ofthe two inhibitors prevented the LPS-mediated protein expressionfor MnSOD. Interestingly, the presence of wortmannin alone in-duced MnSOD, and a similar effect was found for LY294002.These data suggest that LPS-mediated upregulation of MnSODversus COX I and T-fam occurs through distinct intracellular signaltransduction pathways independent of the PI3K/AKT pathway.

AKT inhibition and failure of COX I and T-fam upregulation inresponse to LPS is associated with increased TNF-a, NO, andcell death, and decreased ROS and ATP levels

It has been suggested that the AKT pathway can play a positive anda negative role in TLR-mediated cytokine production (11, 13). Weinvestigated the effect of AKT inhibition on the response of LPSstimulation in our cell system. LPS stimulation (100 ng/ml for 24h) enhanced TNF-a secretion in supernatants, but secretion ofTNF-a was significantly higher (p , 0.05) in pretreated cells withwortmannin (Fig. 5A). Some studies suggested that NO may in-duce mitochondrial biogenesis (NRF-1, T-fam, and COX) andMnSOD expression (35, 36). Hence we investigated whether theinhibition of the AKT pathway in LPS-stimulated macrophages

FIGURE 2. LPS effect on mitochondrial membrane potential, COX activity, and ATP production. (A) As control, cells were either treated with FCCP (1

mM) to dissipate DCm, resulting in a left shift of TMRM fluorescence, or treated with 0.5 mM oligomycin to increase DCm showing a right shift of

fluorescence probe. All measurements were performed with a FACScan (Becton-Dickinson) flow cytometer equipped with a yellow fluorescence (exci-

tation, 532-nm laser; emission, 585 nm; band pass, 42 nm) analyzing 10,000 cells in each run. Data obtained were analyzed with the Cell Quest software

(n = 4). TMRM fluorescence was normalized to MitoTracker red fluorescence, a membrane potential-independent mitochondrial marker in these cells. (B–D)

Kinetic of DCm in response to LPS treatment. Cells were incubated with LPS (100 ng/ml), and relative membrane potentials were determined using the

fluorescent probe TMRM. Thirty-minute LPS treatment led to an early decrease of DCm (B) and recovery starting after 3-h treatment (C). After 6 h of LPS

treatment, cells showed an increase in fluorescent signal suggesting recovery of mitochondrial mass and function (D). (E) LPS increased COX activity. B6-

MCL cells were cultured with or without LPS (100 ng/ml). COX activity was measured 6 h after LPS treatment in solubilized cells by the addition of

increasing amounts of cytochrome c. Specific activity (TN, turnover number) is defined as consumed O2 ([mmol]/(min × total protein [mg]). Shown are

representative results of three independent experiments. (F) Time-dependent changes of ATP levels in response to LPS. B6-MCL cells were incubated with

LPS (100 ng/ml) for different time points as indicated. ATP concentrations were measured using the bioluminescence method. As indicated, LPS treatment

led to a time-dependent increase of ATP concentration. Using ANOVA Mann–Whitney U test, p , 0.05 was considered significant. **p , 0.001.




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changes the NO levels in these cells. Fig. 5B shows the effect ofLPS in the presence and absence of wortmannin on cumulativenitrite production 24 h after LPS treatment as an indirect measureof NO production. As expected, LPS treatment alone resulted ina 50% increase of nitrite levels. Interestingly, wortmannin alonehad no effect on nitrite production, whereas wortmannin incombination with LPS led to a 2-fold increase in nitrate levels(Fig. 5B). LPS stimulation of macrophages induces production ofROS such as peroxides and superoxide. ROS play an importantrole in cellular functions via the activation of signaling cascades,and it has been suggested that ROS affect mitochondrial biogen-esis, morphology, and function (37). Hence we investigated theeffect of LPS stimulation in the presence and absence of wort-mannin on the production of mROS in form of superoxide. Todetect mROS, we further investigated the effect of LPS in thepresence or absence of wortmannin using a fluorescence dye(MitoSox) that measures predominantly mitochondrial superoxide(3). As shown in Fig. 5C, LPS treatment led to a time-dependentaugmentation in mROS, which coincided with increased COX, aswell as mitochondrial membrane potential (Figs. 1, 2). The pres-ence of wortmannin reduced mROS. However, this mROS re-duction was associated with increased cell death (Fig. 5E). Thesedata suggest that mitochondrial biogenesis is required for mROSproduction. By inhibiting the AKT pathway, mitochondrial re-

generation is diminished, leading to decreased mROS productionand an increase in cell death. Next, we assessed whether the in-hibition of the AKT pathway translates into changes of ATPcontent of the cells. B6-MCL cells were cultured and pretreatedfor 30 min with either LY294002 or wortmannin before stimula-tion with LPS (100 ng/ml) for 6 h. As shown in Fig. 5D, ATPlevels were significantly decreased in the presence of both in-hibitors (LY294002 or wortmannin), whereas LPS treatment ledto an increase in cellular ATP content. To further explore the bi-ological effect of inhibition of AKT and subsequent failure ofupregulation of protein expression for COX I and T-fam, we as-sessed cell viability using the tetrazolium dye (MTT) assay (25).Fig. 5E shows that LPS treatment alone at the concentration ap-plied throughout this study did not change viability. Using AKTpathway inhibitors LY294002 or wortmannin led to a minimaldecrease in cell viability, whereas LPS challenge in the presence

FIGURE 3. LPS treatment activates the PI3K/AKT pathway in murine

macrophages. (A) Murine macrophages (B6-MCL) were cultured and

treated with LPS (100 ng/ml) for different time points. Total cell lysates

were prepared, and 20 mg total cellular proteins was subjected to SDS-

PAGE and Western blot analysis using phospho-epitope–specific PI3K p85

(Tyr458/p55 Tyr199) and AKT (Ser473) Abs. Equal loading of protein was

confirmed using total p85 and AKT Abs. The results shown are repre-

sentative of three independent experiments. LPS treatment led to an early

phosphorylation of the PI3K p85/AKT pathway. (B) Murine macrophages

(B6-MCL) were treated with LY294002 (50 ng/ml) or wortmannin (100

ng/ml) 30 min before LPS (100 ng/ml) stimulation. Thirty min after LPS

stimulation, total cell lysates were prepared and 20 mg total cellular protein

was subjected to Western blot analysis using phospho-epitope–specific

PI3K detecting dual-phosphorylation sites on p85 (Tyr458/p55 Tyr199) and

AKT (Ser473). Equal loading of protein was confirmed using total AKT and

p85 Abs. Both inhibitors blocked the effect of LPS on phosphorylation

of p85 and AKT. (C and D) Densitometric analyses (mean 6 SEM)

of phosphorylated form of p85 and AKT in response to LPS treatment

in presence and absence of wortmannin and LY294002. Using ANOVA

Mann–Whitney U test, p , 0.05 was considered significant. *p , 0.05,

**p , 0.001.

FIGURE 4. Inhibition of the PI3K/AKT pathway abrogates LPS-medi-

ated upregulation of COX I and T-fam, but not MnSOD. (A) Murine

macrophages (B6-MCL) were pretreated with wortmannin (100 ng/ml) for

30 min before adding LPS (100 ng/ml) to the media for 6 h. After incu-

bation, whole-cell extracts were prepared and 20 mg total protein was

subjected to SDS-PAGE and Western blot analysis for NRF-1, COX I,

T-fam, and b-actin as loading control. (B) Densitometric analysis (mean 6SEM) of COX I in four independent experiments in response to LPS

stimulation in the presence of wortmannin. Using ANOVA Mann–Whitney

U test, p , 0.05 was considered significant. *p , 0.05, **p , 0.001. (C)

Murine macrophages (B6-MCL) were pretreated with okadaic acid (10

nM) 30 min before adding LPS (100 ng/ml) to the media for 6 h. After

incubation, whole-cell extracts were prepared, and 20 mg total protein was

subjected to SDS-PAGE and Western blot analysis using an Ab against

COX I; b-actin was used to confirm equal loading. Figure is representative

of four independent experiments. (D, upper panel) Time-dependent ex-

pression of MnSOD in B6-MCL cells. Cells were treated with LPS (100

ng/ml) for different periods as indicated. Total cell lysates were prepared

and 20 mg protein was subjected to SDS-PAGE and Western blot analysis

using an Ab against MnSOD. (D, lower panel) Murine macrophages (B6-

MCL) were pretreated with wortmannin (100 ng/ml) or LY294002 (50 ng/

ml) for 30 min before adding LPS (100 ng/ml) to the media for 6 h. After

incubation, whole-cell extracts were prepared, and 20 mg total protein was

subjected to SDS-PAGE and Western blot analysis using an Ab against

MnSOD. Equal loading was assessed using a b-actin Ab. The presence of

wortmannin or LY294002 did not abrogate LPS-mediated upregulation of

MnSOD.




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of AKT pathway inhibitor led to a significant loss of cell viability(p , 0.001). Collectively, these data suggest that LPS-mediatedupregulation of COX requires PI3K/AKT activation, and that thisactivation is important in the prevention of cell death and exces-sive proinflammatory mediators (NO and TNF-a).

LPS-mediated AKT activation and subsequent COX I andT-fam upregulation are MyD88/TRIF dependent

Although several studies have shown that LPS stimulation leads toPI3K/AKTactivation, the mechanism by which LPS activates AKTis not well understood and remains controversial. In addition, PI3K/AKT pathway activation can occur in response to diverse growthfactors and ligand stimulation through diverse receptor activation(30). Macrophages from MyD88/TRIF2/2 mice are defective inmany TLR4-mediated responses, such as LPS-induced secretionof cytokines (e.g., IL-6, TNF-a, and IL-1b) (38). A previous studyhas shown that LPS-mediated AKT activation is TLR4/MyD88dependent and requires PI3K activation (13). Therefore, we as-sessed whether the LPS-mediated increased expression for COXI and T-fam requires MyD88/TRIF adaptor proteins. Phenotypesof WT and MyD88/TRIF2/2 cells were verified by a range offunctional parameters, including responsiveness to TLR4 and cy-tokine production (Supplemental Fig. 3). We stimulated side-by-

side WT and MyD88/TRIF2/2 cells with LPS for different timepoints to assess AKT activation. Fig. 6A shows phosphorylation ofp85 and AKT in response to LPS stimulation in WT and MyD88/TRIF2/2. As shown, WT cells responded to LPS stimulation withphosphorylation of PI3K and AKT on Ser473, whereas no phos-phorylation of either kinase was observed in MyD88/TRIF2/2

macrophages. These data suggest that LPS-induced activation ofthe PI3K/AKT pathway is dependent on the presence of MyD88/TRIF adaptor protein. To determine whether the lack of AKTactivation in MyD88/TRIF2/2 would parallel the effect on LPS-mediated T-fam and COX I expression, we stimulated WT andMyD88/TRIF2/2 cells side by side with LPS for different timepoints (90 min, 3 h, and 6 h). As shown in Fig. 6B, WT cellsresponded to LPS treatment with increased expression of bothT-fam and COX I, whereas MyD88/TRIF2/2 cells failed to up-regulate either one of them. These data suggest that LPS-inducedphosphorylation of AKT on Ser473 requires the presence ofMyD88 and TRIF adaptor proteins, and that the LPS-inducedexpressions of T-fam and COX I require activation of the PI3K/AKT pathway. Next, we asked whether absence of either MyD88or TRIF would be sufficient to prevent LPS-mediated PI3K/AKTpathway activation and subsequent T-fam and COX expression.BMDMs from WT and macrophages deficient for either MyD88

FIGURE 5. Effect of LPS and PI3K inhibitor on cellular ATP and nitrite levels, ROS production, and cell viability. (A) B6-MCL cells were pretreated

with wortmannin (100 ng/ml) for 30 min before adding LPS (100 ng/ml) or kept in media for 24 h. Supernatants were analyzed for TNF-a via ELISA. Data

are presented as means of four independent experiments and error bars indicate the SEM. p , 0.01. (B) Cumulative nitrite levels in the supernatant were

measured as described in Materials and Methods. Data presented as mean6 SEM of percentage changes of untreated cells. Using ANOVA Mann–Whitney

U test, p , 0.05 was considered significant. *p , 0.05, **p , 0.001. (C) mROS were measured using the probe MitoSox. As positive control, cells were

treated with antimycin A (AA) for 30 min before treatment with the MitoSox fluorescence probe. Cells were treated with LPS (100 ng/ml) in presence and

absence of wortmannin (100 ng/ml). Data are presented as fold change in mean intensity of MitoSOX fluorescence compared with PBS plus MitoSOX

alone (negative control). LPS treatment led to a time-dependent increase of mROS after 6 and 24 h. Pretreatment of cells 30 min before LPS stimulation led

to a significant decrease in mROS production. Using ANOVA Mann–Whitney U test, p , 0.05 was considered significant. *p , 0.05, **p , 0.001. (D)

Murine macrophages (B6-MCL) were treated with LPS (100 ng/ml) for 6 h in the presence or absence of wortmannin (100 ng/ml) or LY294002 (50 ng/ml)

30 min before LPS treatment. ATP concentrations were measured using the bioluminescence method. ATP levels increased in response to LPS. Pretreatment

with wortmannin or LY294002 abrogated the LPS-mediated ATP upregulation. Data presented are the result of three independent experiments measured in

triplicates. Using ANOVA Mann–Whitney U test, p , 0.05 was considered significant. *p , 0.05, **p , 0.001. (E) Cell viability. A total of 2 3 105

B6-MCL cells was plated in 96-well plate and treated with LPS (100 ng/ml) for 24 h in the presence or absence of wortmannin (100 ng/ml) or LY294002

(50 ng/ml). Cell viability was assessed using MTT assay. Data presented as mean 6 SEM of percentage changes of untreated cells. Using ANOVA Mann–

Whitney U test, p , 0.05 was considered significant. *p , 0.05, **p , 0.001.




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or TRIF were stimulated with LPS for the indicated times. Asshown, LPS-mediated PI3K/AKT pathway activation requiredboth MyD88 and TRIF. In macrophages lacking TRIF, we ob-served an attenuated and delayed response in AKT phosphor-ylation, whereas cells lacking MyD88 did not respond to LPSwith AKT phosphorylation (Fig. 6C). Similarly, WT macrophagesresponded to LPS with an increased expression for T-fam (Fig.6D) and COX IV (Fig. 6E), whereas macrophages deficient inMyD88 or TRIF did not increase expression for T-fam (Fig. 6D)and COX IV (Fig. 6E).

Expression of AKT1 is pivotal in LPS-mediated COXupregulation in BMDMs

Three AKT isoforms have been described with distinct biologicalfunctions. To further investigate mechanistically which AKT iso-form plays a major role in the LPS response and mitochondrialbiogenesis in macrophages, we used cells derived from knockoutmice lacking AKT1 or AKT2. Whereas AKT1 and AKT2 areubiquitously expressed, including immune cells, endothelial cells,and in hematopoietic cells, AKT3 expression is largely confined tothe testes and brain (39–41). Thus, we focused on AKT1 andAKT2 isoforms. BMDMs from mice deficient in AKT1, AKT2, or

WT mice were cultured side by side under equal conditions. Cellswere treated with LPS (100 ng/ml) for different time points, andwhole-cell extracts were subjected to immunoblotting. As seen isFig. 7A, LPS stimulation of BMDMs isolated from WTAKT12/2

and AKT22/2 led to rapid phosphorylation of p85 and AKTphosphorylation on phospho-Ser473. This Ab detects phosphory-lated AKT on Ser473 regardless of the isoforms expressed (eitherAKT1, AKT2, or AKT3). Next, we used isoform-specific AKT1and AKT2 Abs to detect the level of AKT1 and AKT2 expressionin WT, AKT12/2, and AKT22/2 BMDMs. Fig. 7B confirmed thelack of expression of AKT1 protein in AKT12/2 and of AKT2 inAKT22/2 macrophages, respectively. As described earlier, in ourcell model, we detected the highest expression for COX 6 h afterLPS treatment. Thus, immunoblotting was done using the 6 h timepoint after LPS treatment. Fig. 7C shows that the cells lackingAKT1 had severe impairment in COX I and COX IV expressionat baseline, as well as in response to LPS. In contrast, the induc-tion of COX I was unimpaired in response to LPS treatment inAKT22/2 macrophages (Fig. 7C). Furthermore, the basal level ofCOX IV was lower in these macrophages as compared with thoseof WT, but AKT22/2 macrophages responded to LPS with anenhanced expression for COX IV. These findings indicate that the

FIGURE 6. LPS-induced COX I and T-fam expression is MyD88/TRIF dependent. (A) WT murine macrophages and MyD882/2 TRIF2/2 cells were

cultured side by side in the presence and absence of LPS (100 ng/ml) for 30 or 60 min. Total cell lysates were prepared, and 20 mg total protein was

subjected to SDS-PAGE and Western blot analysis using phospho-epitope–specific Abs for p85 and AKT (Ser473). Equal loading of protein was confirmed

using total p85 and AKTAbs. (B) WT murine macrophages and MyD88/TRIF2/2 cells were cultured side by side in the presence and absence of LPS for 90

min, 3 h, and 6 h. Total cell lysates were prepared and 20 mg total protein was subjected to SDS-PAGE and Western blot analysis using Ab against T-fam

and COX I. Equal loading of protein was confirmed using b-actin Ab. WT cells responded to LPS stimulation with a significant increase in T-fam starting at

90 min throughout 6 h after treatment with LPS. Upregulation of COX I in WTwas seen starting at 3 h after LPS stimulation. In contrast, MyD88/TRIF2/2

cells did not respond to LPS with increased expression for T-fam and COX I. (C) BMDMs isolated from WT, TRIF-, or MyD88-deficient mice were

cultured in parallel with LPS for 5, 30, and 60 min. Total cell lysates were prepared, and 20 mg total protein was subjected to SDS-PAGE and Western blot

analysis using phospho-epitope–specific Abs for p85 and AKT (Ser473). Equal loading was confirmed using total AKT and total p85 Abs. LPS treatment

evoked a weak and delayed AKT phosphorylation in TRIF2/2 cells, but MyD88-deficient cells did not show any response to LPS treatment. (D) BMDMs

isolated fromWT, TRIF-, or MyD88-deficient mice were either kept in media or treated with LPS for 3 h. Twenty micrograms of total protein was subjected

to SDS-PAGE and Western blot analysis using T-fam Ab. Equal loading was confirmed using b-actin Ab. Although LPS treatment led to an increase in

T-fam expression in WT, neither TRIF2/2 nor MyD882/2 cells responded with an increased expression for T-Fam. (E) BMDMs isolated from WT, TRIF-,

or MyD88-deficient mice were either kept in media or treated with LPS for 3 and 6 h. Twenty micrograms of total protein was subjected to SDS-PAGE

and Western blot analysis using a COX IVAb. Equal loading was confirmed using total AKT Ab. Although LPS treatment led to an increase in COX IV

expression in WT after 6 h, TRIF2/2 and MyD882/2 cells did not show any expression of COX IV.




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AKT1 isoform plays a central role in LPS-mediated COX I and IVexpression, whereas AKT2 contributes only to COX IV upregu-lation by LPS in BMDMs.

DiscussionIn this study, we analyzed the molecular mechanisms underlyingthe LPS-mediated upregulation of ETC components focusing onCOX and mitochondrial transcription factors (T-fam and NRF-1) asmarkers of mitochondrial biogenesis. We show that LPS-mediatedupregulation of COX and T-fam is dependent on AKT phos-phorylation (Figs. 1, 3), and that inhibition of the PI3K/AKTpathway leads to abrogation of this effect (Fig. 3). Increased ex-pression of COX subunits I and IV in response to LPS challengewas associated with restoration of COX activity and increasedcellular ATP levels (Fig. 2). Inhibition of the PI3K/AKT pathwayabrogated the effect of LPS on both COX and T-fam, and led todecreased ATP production and increased cell death. We also showthat the effect of LPS on both COX and T-fam is dependent onMyD88 and TRIF adaptor proteins. LPS challenge of MyD88/TRIF knockout cells failed to phosphorylate AKT and upregu-late T-fam and COX (Fig. 6A, 6B). Cells lacking MyD88 did notrespond to LPS with AKT activation, whereas macrophages defi-cient in TRIF showed a delayed AKT activation (Fig. 6C). NeitherMyD88 nor TRIF deficient cells showed an increase in T-fam orCOX expression (Fig. 6D, 6E). These data suggest that MyD88/TRIF is required both for AKT phosphorylation and for LPS-mediated increased expression of COX and T-fam. Importantly,macrophages from AKT12/2 mice showed a lack of response toLPS in terms of upregulation of COX I and IV (Fig. 7C). Thesedata collectively indicate that AKT1 regulates and maintains mi-tochondrial function in response to TLR4 agonists.Mitochondria are highly dynamic organelles and essential in

providing most cellular ATP. They have emerged as importantintegrators of several vital signaling cascades (42). Mitochondrialmembrane potential (DCm) builds the proton motive force forgeneration of ATP through OxPhos and is critical for the integrityof mitochondrial function. Bacteria and several bacterial productssuch as LPS or bacterial toxins have been shown to affect DCmand alter the OxPhos, as well as mROS production (3, 43, 44). Weshow in this study the dynamic changes of DCm of murinemacrophages in response to LPS, an initial decrease of DCmfollowed by a delayed restoration of DCm that mirrored the up-regulation of COX (Figs. 1, 2). This process may be specifically

important in the response of macrophages to bacterial productsand may facilitate the required energy for cytokine production, aswell as phagocytic activity of these cells. One recent study foundthat mROS production in response to specific TLRs is required forphagocytic activity of macrophages (3). Another study found thatAKT1 and AKT2 isoforms regulate the intracellular ROS contentin hematopoietic stem cells necessary for their function and pro-liferation (40).COX biogenesis is a critical part of mitochondrial biogenesis.

Among the 13 subunits of COX, the largest subunits, COX I–III, areencoded by the mitochondrial DNA and regulated at the tran-scriptional level by T-fam (45). COX is the terminal enzyme of theETC and plays a pivotal role in electron transfer to molecularoxygen, and generation and maintenance of DCm and ATP. COXactivity, as well as expression, is tightly regulated by metaboliccellular demand (5, 42). Our laboratory has shown that TNF-achallenge leads to rapid inhibition of COX, and this inhibition isdue to tyrosine phosphorylation of the catalytic subunit I of COX(5). This finding may explain, at least in part, the observed met-abolic switch from aerobic to glycolytic energy metabolism duringsepsis. However, not all septic patients experience organ failurewith a poor outcome. Thus, additional mechanisms may be es-sential for the recovery from sepsis. Our current data indicate thatLPS stimulation of murine macrophages leads to 20-fold increasein expression of COX I (Fig. 1A), and that such an increase wasassociated with a 56% increase in COX activity.Evidence suggest that nonsurvivors of sepsis exhibit more ex-

tensive mitochondrial damage and failure of cellular energeticscompared with survivors as determined from muscle biopsies ofcritically ill patients (46). Interestingly, Staphylococcus aureus-induced sepsis in mice with a lower bacterial load led to a recov-ery from bioenergetic failure with orderly (sequential) inductionof NRF-1, NRF-2, PGC-1, and T-fam m-RNAs in the liver ofseptic animals (47), whereas animals with sepsis induced witha higher bacterial count failed to recover and exhibited lack ofmitochondrial biogenesis. We have observed that treatment ofmacrophages with a higher dose of LPS (.1 mg/ml) failed toincrease expression for COX I (Supplemental Fig. 1) and T-fam.In contrast with Haden et al.’s observation (47), we show ex-pression at both the gene and protein levels that murine macro-phages exhibit higher baseline NRF-1 mRNA expression andminimal change in response to LPS treatment as compared withPGC-1a (data not shown), T-fam, and COX I and IV. It should be

FIGURE 7. Critical role of AKT1 in LPS-mediated COX expression. BMDMs were isolated from WT, AKT1-, or AKT2-deficient mice, cultured under

equal conditions, and treated with LPS for different time points as indicated. (A) Detection of phospho-p85 and -AKT. Twenty micrograms total protein was

subjected to SDS-PAGE and Western blot analysis using phospho-epitope–specific Abs for p85 and AKT (Ser473). Equal loadings were confirmed using

either total p85 or total AKT Abs. (B) Detection of AKT isoforms. Immunoblot analysis of whole-cell lysate performed using Abs detecting AKT1 and

AKT2. Equal loading was confirmed using b-actin Ab. As shown, AKT12/2 macrophages lack expression of AKT1, whereas AKT22/2 macrophages lack

AKT2. (C) BMDMs isolated from WT, AKT12/2, and AKT22/2 mice kept in media or treated with LPS for 6 h. Immunoblot analysis of whole-cell lysate

performed using Abs detecting COX I (upper panel) and COX IV (lower panel). Equal loading was confirmed using b-actin Ab. Cells lacking AKT1 had

severe impairment in COX I and COX IV expression at baseline, as well as in response to LPS. Induction of COX I was unimpaired in response to LPS

treatment in AKT22/2 macrophages. Furthermore, the basal level of COX IV was lower in these macrophages as compared with those of WT, but AKT22/2

macrophages responded to LPS with an enhanced expression for COX IV.




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noted, however, that tissue and cell-specific differences may exist,and that various mitochondria-related transcription factors likelyplay different roles in specific tissues and cells. In addition, diverseposttranscriptional modifications of mitochondrial transcription fac-tors such as phosphorylation or redox regulation (e.g., in the case ofNRF-1) have been reported (15). Such events may be important inregulation of mitochondrial transcripts in response to LPS.Recognition of LPS by TLR4 plays a pivotal role in the initiation

of Gram-negative sepsis and septic shock through diverse in-flammatory processes in mammals. LPS challenge leads to acti-vation of diverse cell signaling pathways such as inflammatory,apoptotic, but also prosurvival pathways. It has been shown thatLPS challenge results in activation of the PI3K/AKT pathway,which is required for activation of survival pathways (48). BothPI3K and AKT pathway activation have been reported to be as-sociated with TLR2, TLR3, TLR4, and TLR5 in different cells(13, 49). Although it has been suggested that the activation of thePI3K/AKT pathway has a negative regulatory function on TLRligation, the level of interaction between the TLRs and the PI3K/AKT pathway is not well understood (13, 49, 50). Several studiessuggested that the inhibition of this pathway leads to decreasedphosphatase activity, resulting in an increased production of in-flammatory mediators such as IL-1b and IL-6 with an adverseeffect on cell survival (11). Our data indicate that inhibition ofAKT pathway in presence of LPS increases proinflammatorycytokines (IL-6 and TNF-a), ROS, NO, and decreased cell sur-vival in murine macrophages, and these findings paralleled thelack of mitochondrial biogenesis. Although we have observed in-creased ROS (both cellular and mROS) in response to LPS, thepresence of wortmannin led to an increased fluorescence in CM-H2DCFDA (data not shown) but a decrease in the MitoSox signal.We propose that the enhanced CM-H2DCFDA probe in the pres-ence of wortmannin is due to increased peroxynitrite in the cy-toplasm because of cell death processes. As shown in Fig. 5C,wortmannin led to a decreased mROS, but also increased celldeath (Fig. 5E).Moreover, in macrophages, TLR ligation initiates autophagy,

a process that provides metabolic intermediates to maintain bio-energetics necessary to carry out the killing of pathogens and topromote survival (51, 52). It has been shown that mammalian cellswith hyperactive AKT accumulate ATP, whereas cells with re-duced AKT activity exhibit markedly reduced ATP content (53).Such effects have been explained by the role of activated AKT onmitochondria-associated hexokinase and glycolysis (54). Our dataindicate that presence of AKT1 and its activation in response toLPS is critical for the upregulation of mitochondrial complexes, inparticular, COX. Previously, AKT1 has been implicated as a crit-ical regulator in homeostasis of hematopoietic stem cell functionand ROS (40). Interestingly, another study associated the loss ofAKT1 with severe atherosclerosis in a mice model of coronaryartery disease (55). Macrophages of animals lacking AKT1 weremore susceptible to apoptosis and exhibited defective clearanceof dead foam cells (55). Our study sheds some light on theseobservations because our data mechanistically suggest that sup-pressed mitochondrial energetics cause an impaired stress re-sponse in AKT12/2 macrophages. PI3K is involved in manyimportant cellular pathways including cell growth, migration, andapoptosis (30). It has been shown that the PI3K/AKT pathway isactivated in response to growth factors but also in response to TLRligands (13). However, PI3K activation in absence of AKT1 wasnot sufficient to upregulate COX in response to LPS. Our resultsindicate that the PI3K/AKT pathway is required for upregulationof mitochondrial OxPhos and T-fam, and both processes requirethe adaptor protein MyD88/TRIF (Fig. 6A, 6B). Previously, it has

been shown that MyD88-deficient animals exhibit poorer outcomewhen infected with various microorganisms that were associatedwith an enhanced bacterial burden (11, 56–58). This paradigm, atleast in part, may be explained by their inability to enhance mi-tochondrial biogenesis in response to microbial stimuli.

AcknowledgmentsWe thank Dr. Eicke Latz (University of Massachusetts Medical School,

Worcester, MA) for providing the macrophage cell lines.

DisclosuresThe authors have no financial conflicts of interest.

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