Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage wwwe lsev ie rcom locate bbamcr

Mitogen-activated protein kinase kinases promote mitochondrial biogenesisin part through inducing peroxisome proliferator-activated receptor γcoactivator-1β expression

Minghui Gao a12 Junjian Wang a13 Na Lu a1 Fang Fang a Jinsong Liu a Chi-Wai Wong ba Guangzhou Institute of Biomedicine and Health Chinese Academy of Sciences 510530 Chinab NeuMed Pharmaceuticals Limited Hong Kong

Abbreviations ATP5b ATP synthase cyt-C cytochromrelated receptors α and γ ERK12 extracellular sigmitochondrial transcription factor A Mfn2 mitofusinprotein kinase kinases PGC-1α and PGC-1β peroxisome pcoactivator-1α and -1β Correspondence to Jinsong Liu Guangzhou Institu

Chinese Academy of Sciences 190 Kai Yuan Avenue SciChina Tel +86 20 3201 5317 Correspondence to Chi-Wai Wong NeuMed PharmTel +852 6748 7218

E-mail addresses liu_jinsonggibhaccn (J Liu) wo(C-W Wong)

1 These authors contributed equally to this study2 Present address Room 514 5F Choh-MingLi Basic

Chinese University of Hong Kong Shatin Hong Kong3 Present address Room 1400 Research III Building

Sciences 4645 2nd Avenue Sacramento CA 95817 USA

0167-4889$ ndash see front matter copy 2011 Elsevier BV Aldoi101016jbbamcr201103017

a b s t r a c t

a r t i c l e i n f o

Article historyReceived 26 January 2011Received in revised form 22 March 2011Accepted 23 March 2011Available online 31 March 2011

KeywordsMEK12PGC-1βMitochondrial biogenesis

Growth factor activates mitogen-activated protein kinase kinases to promote cell growth Mitochondrialbiogenesis is an integral part of cell growth How growth factor regulates mitochondrial biogenesis is not fullyunderstood In this study we found that mitochondrial mass was specifically reduced upon serum starvationand induced upon re-feeding with serum Using mitogen-activated protein kinase kinases inhibitor U0126we found that the mRNA expression levels of ATP synthase cytochrome-C mitochondrial transcription factorA and mitofusin 2 were reduced Since the transcriptional levels of these genes are under the control ofperoxisome proliferator-activated receptor γ coactivator-1α and -1β (PGC-1α and PGC-1β) we examinedand found that only themRNA and protein levels of PGC-1βwere suppressed Importantly over-expression ofPGC-1β partially reversed the reduction of mitochondrial mass upon U0126 treatment Thus we concludethat mitogen-activated protein kinase kinases direct mitochondrial biogenesis through selectively inducingPGC-1β expression

e-C ERRα and ERRγ estrogen-nal-regulated kinases Tfam2 MEK12 mitogen-activatedroliferator-activated receptor γ

te of Biomedicine and Healthence Park Guangzhou 510530

aceuticals Limited Hong Kong

ngcw123456yahoocom

Medical Sciences Building The

UC Davis Cancer CenterBasic

l rights reserved

copy 2011 Elsevier BV All rights reserved

1 Introduction

Growth factor activation of receptor tyrosine kinasewhicheventuallyactivates mitogen-activated protein kinase kinases (MEK12) and theirdown-stream targets extracellular signal-regulated kinases (ERK12)plays a central role in the regulation of cell growth and proliferation [1]Mutations or amplifications of these signaling molecules can contributeto the development of a broad spectrum of human cancers [2]

Suppression of ERK activation by specific inhibitors dominant-negativeforms of ERK as well as ERK antisense nucleotides have been shown toinhibit cell proliferation and induce apoptosis furthermore MEKinhibitors significantly altered the abilities of these cells to proliferate inresponse to growth factor stimulation [2]

The roles of mitochondrial biogenesis and function in cell growthand proliferation are actively being investigated In order to promotecell growth and division the MEKndashERK cascade needs to coordinatelyregulate protein nucleic acid and lipid biosynthesis for genome andorganelle duplications All of these processes consume high levels ofenergy thus mitochondrial biogenesis and energy metabolism mustalso be highly coordinated by the MEKndashERK cascade Recent studieshave shown that modulating ERK12 activities alters mitochondrialfunction [34] However how MEKndashERK cascade governs mitochon-drial biogenesis remains undefined

Estrogen-related receptors α and γ (ERRα and ERRγ) togetherwith their coactivators peroxisome proliferator-activated receptor γcoactivator-1α and -1β (PGC-1α and PGC-1β) are key regulators ofmitochondria biogenesis and function [56] These transcriptionalregulators bind to response elements located on the promotersof their target genes such as ATP synthase (ATP5b) cytochrome-C(cyt-C) mitochondrial transcription factor A (Tfam) and mitofusin 2(Mfn2) to guide the expressions of these mitochondrial enzymes andregulators [7] In this study we investigated whether ERRα ERRγPGC-1α and PGC-1β participate in communicating the signal from

1240 M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

the MEKndashERK cascade to govern mitochondrial biogenesis We foundthat the mRNA expression level of PGC-1βwas specifically induced bythe MEKndashERK cascade

2 Material and methods

21 Cell culture

Human non-small cell lung cancer (NSCLC) A549 cells werepurchased from American Type Culture Collection (ATCC) and culturedin RIPM1640 medium (Gibco) supplemented with 10 fetal bovineserum (FBS) (Hyclone) at 37 degC in 5 CO2

22 Confocal laser scanning microscopy of mitochondrial mass

A549 cells were seeded onto coverslip in 6-well plates andcultured overnight after starvation or re-feeding treatment cellswere incubated in pre-warmed serum free medium with 400 nMMitotracker Green FM and 10 μgml 4prime6-diamidino-2-phenylindole(DAPI) for 20 min in the dark After staining cells were washed fourtimes with cold phosphate-buffered saline (PBS) and mounted onglass slides Imageswere subsequently takenwith a Leica SP2 confocalmicroscope (Leica Microsystems Mannheim Germany)

23 Mitochondrial mass measurement

Mitotracker green (Invitrogen) was added and quantified asdescribed [8] Briefly cells were incubated in serum free medium(pre-warmed to 37 degC) with 150 nMMitotracker Green FM for 20 minin the dark After staining cells were washed twice with coldphosphate-buffered saline (PBS) and suspended in 200 μl PBSSubsequently cells were analyzed on a flow cytometer (FAC-SCaliburBD Biosciences) with excitation at 490 nm and emission at 516 nmData were processed by using the CellQuest program (BD Bio-sciences) Assays done in triplicate were repeated at least three times

24 Cellular ATP level measurement

Total cellular ATP level was measured by ATPlite-glo (PerkinEl-mer) following the manufactures protocol using VERITASTM Micro-plate luminometer (Turner Biosystems) as described [9]

25 Quantitative real-time PCR

Total RNA extraction first-strand cDNA generation and quantita-tive real-time PCR analysis were performed as described [10] Relativegene expression was normalized to 18 S rRNA levels Primersequences and real-time PCR conditions are listed in Table S1 of thesupplementary data

26 Western blot analysis

Cell extracts weremade in RIPA buffer (50 mMTris pH75 150 mMNaCl 10 mM EDTA 1 NP-40 01 SDS 1 mM PMSF 10 μgmlAprotinin) and whole cell extracts (50ndash75 μg of protein) were treatedwith SDS sample buffer boiled for 5 min and subjected to SDSndashPAGEand Western analysis by ECL Western Blotting System (AmershamPharmacia UK) Membranes were incubated with rabbit anti-ERRα-ERRγ -PGC-1α and -PGC-1β antibodies [1112] or mouse anti-β-actin antibody (Boster) followed by horseradish peroxidase-conju-gated secondary antibody (Amersham) and developed with ECLreagent (Amersham)

27 Plasmids and transient transfection

A pGL3-PGC-1β-promoter reporter plasmid was cloned by PCRamplification of its 2 kb genomic region upstream of the transcrip-tional start site into a pGL3-luciferase vector (Promega) Transienttransfections were performed using Lipofectamine 2000 (Invitrogen)following the manufacturers instructions For luciferase reporterassays cells at 85ndash95 confluency in 96-well plates were cotrans-fected with reporter plasmids (25 ngwell) and Renilla luciferase(3 ngwell) as an internal control for transfection efficiency Six hoursafter transfection cells were starved in different amount of FBS ortreated with U0126 for 24 h Luciferase activity was measured asdescribed [1314]

28 Over-expression by transient transfection

A pcDNA31-PGC-1β-expression plasmid was cloned by PCRamplification MEK1 and MEK2 plasmids were gifts from Dr SylvainMeloche [15] Cells were over-expressedwith pcDNA31 or expressionplasmids using Lipofectamine 2000 (Invitrogen)

29 Statistical analysis

Data are presented as meanplusmnSE and analyzed by a variance test(ANOVA) Asterisks indicate significant differences Pb005 Pb001

3 Results

31 Growth factor signaling and mitochondrial biogenesis

In order to address the importance of mitochondrial biogenesis inmediating growth factor stimulated cell proliferation we first moni-tored the changes in mitochondrial mass in human non-small cell lungcancer A549 cells that were serum-starved and then re-fed with serumUsing Mitotracker Green a fluorescence dye that binds to mitochon-drion independent of its potential we found by confocal microscopythat the amount of stained mitochondria reduced upon serumstarvation but rebound partially upon serum re-fed (Fig 1A) In orderto quantify the changes we starved cells in different amounts of serumto induce cell cycle arrest (supplemental data Fig S1) followed byfluorescence-assisted cell sorting analysis (supplemental data Fig S2)We found that serum starvation quantitatively reduced mitochondrialmass and the decrease was negatively correlated to the amount ofserum supplemented (Fig 1B) We then asked how quickly mitochon-drial biogenesis would increase upon growth factor stimulation instarved cells re-fed with 10 FBS We found that mitochondrial massincreased gradually over time as cells were released from cell cyclearrest (Fig 1C) Similar observations were obtained using another lungcancer cell line 95D and a colon cancer cell line HCT-116 (supplementaldata Fig S3)

Besides quantifying mitochondrial mass by fluorescence dyestaining we also measured the mRNA expression levels of severalnuclear-encoded mitochondrial regulatory factors such as Mfn2 andTfam aswell as respiratory chain enzymatic components such as cyt-Cand ATP5b Correspondingly the mRNA expression levels of thesegenes which serve as markers to more globally reflect the changes inmitochondrial biogenesis and function were all reduced by serumstarvation and stimulated by re-feeding with serum (Fig 1D)Additionally we checked the mRNA expression levels of severalmitochondrial DNA-encoded respiratory chain enzymatic compo-nents such as cytochrome b (MT-CYB) cytochrome c oxidase subunit I(MT-COI) and ATP synthase 6 (MT-ATP6) and found similar modes ofregulation (Fig 1E)

Fig 1 Growth factor signaling and mitochondrial biogenesis (A) Separate and merged confocal images of A549 cells after staining with Mitotracker Green and 4prime6-diamidino-2-phenylindole (DAPI) under 10 FBS (Normal) 01 FBS (Starvation) and 01 FBS overnight followed by 18 h in 10 FBS (Re-fed) (BndashC)Mitochondrial mass wasmeasured as in Ref[9] (B) Cells were grown in different amounts of serum indicated for 24 h The relative amount in 10 FBS was set as 100 (C) Cells were first starved in 01 FBS overnight before re-feeding with 10 FBS for different amounts of time indicated The relative amount without re-feedingwas set as 100 (DndashE)mRNA expression levels of ATP5b cyt-C Mfn2 and Tfam(D) mitochondrial DNA-encoded respiratory chain enzymatic components MT-CYB MT-COI and MT-ATP6 (E) mRNA expression levels were measured under 10 FBS (Normal)01 FBS (Starvation) and 01 FBS overnight followed by 18 h in 10 FBS (Re-fed) Relative gene expression was normalized to 18 S rRNA and the relative level under normalcondition was set at 1 Data are presented as meanplusmnSE and analyzed by a variance test (ANOVA) Asterisks indicate significant differences Pb005 Pb001

1241M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

Fig 2 Suppressing MEK12 activities reduces mitochondrial mass and function (A) Mitochondrial mass levels were measured as in Fig 1 after cells were treated with differentamounts of U0126 for 24 h (B) Mfn2 and TfammRNA expression levels of cells in (A) were measured as in Fig 1 (C) Cellular ATP levels of cells in (A) were measured as in ldquoMaterialsand methodsrdquo (D) ATP5b and cyt-C mRNA expression levels of cells in (A) were measured as in Fig 1 Statistical analysis was performed as in Fig 1

1242 M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

32 Suppressing MEK12 activities reduces mitochondrial mass andfunction

To explore if inhibiting MEK12 alters mitochondrial mass wetreated normally growing A549 cells dose-dependently with a MEK12 inhibitor U0126 to induce cell cycle arrest in the G1 phase(supplemental data Fig S4) Concordantly U0126 dose-dependentlyreduced mitochondrial mass (Fig 2A) and suppressed the mRNAexpression levels of Tfam and Mfn2 (Fig 2B) In addition weinvestigated if ATP production was reduced due to the loss ofmitochondrial mass We indeed found that U0126 dose-dependentlylowered the total cellular ATP level (Fig 2C) and suppressed themRNA expression levels of ATP5b and cyt-C (Fig 2D) as well as MT-CYB MT-COI and MT-ATP6 expressions (supplemental data Fig S5)Although less effective compared to U0126 we also found thatanother inhibitor PD98059 which only inhibits MEK1 modestlyreduced mitochondrial mass and partially suppressed the expressionof these genes (supplemental data Fig S6)

Fig 3 Suppressing MEK12 activities selectively reduces PGC-1β expression (A) mRNA exprof U0126 for 24 h were measured The relative expression levels of these genes under DMSperformed for ERRα ERRγ PGC-1α and PGC-1β with β-actin as a control Statistical analy

33 Suppressing MEK12 activities selectively reduces PGC-1β expression

Since the transcriptional expression levels of ATP5b cyt-C Tfamand Mfn2 are in part controlled by ERRα ERRγ PGC-1α and PGC-1βwe next examined if the mRNA and protein levels of these factorswere altered by suppressing MEK12 activities We found that U0126did not significantly alter the mRNA or the protein levels of ERRαERRγ and PGC-1α (Fig 3A and B) On the other hand U0126selectively and dose-dependently suppressed the mRNA and proteinlevels of PGC-1β (Fig 3A and B)

Since suppressing MEK12 activities selectively reduces PGC-1βexpressionwe then checked if serum starvation and re-fed treatmentsaltered PGC-1β expression We found that PGC-1β mRNA level wasreduced by serum starvation and elevated upon re-feedingwith serum(Fig 4A) We then cloned the 2 kb promoter region of PGC-1βupstream of its transcriptional start site into a luciferase reporter [8]Using this PGC-1β-promoter-luciferase reporter we were able torecapture the responses to serum starvation (Fig 4B) and U0126

ession levels of ERRα ERRγ PGC-1α and PGC-1β in cells treated with different amountsO treatment as a control were set as 1 (B) Western blots for cell extracts in (A) weresis was performed as in Fig 1

Fig 1 Growth factor signaling and mitochondrial biogenesis (A) Separate and merged confocal images of A549 cells after staining with Mitotracker Green and 4prime6-diamidino-2-phenylindole (DAPI) under 10 FBS (Normal) 01 FBS (Starvation) and 01 FBS overnight followed by 18 h in 10 FBS (Re-fed) (BndashC)Mitochondrial mass wasmeasured as in Ref[9] (B) Cells were grown in different amounts of serum indicated for 24 h The relative amount in 10 FBS was set as 100 (C) Cells were first starved in 01 FBS overnight before re-feeding with 10 FBS for different amounts of time indicated The relative amount without re-feedingwas set as 100 (DndashE)mRNA expression levels of ATP5b cyt-C Mfn2 and Tfam(D) mitochondrial DNA-encoded respiratory chain enzymatic components MT-CYB MT-COI and MT-ATP6 (E) mRNA expression levels were measured under 10 FBS (Normal)01 FBS (Starvation) and 01 FBS overnight followed by 18 h in 10 FBS (Re-fed) Relative gene expression was normalized to 18 S rRNA and the relative level under normalcondition was set at 1 Data are presented as meanplusmnSE and analyzed by a variance test (ANOVA) Asterisks indicate significant differences Pb005 Pb001

1241M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

Fig 2 Suppressing MEK12 activities reduces mitochondrial mass and function (A) Mitochondrial mass levels were measured as in Fig 1 after cells were treated with differentamounts of U0126 for 24 h (B) Mfn2 and TfammRNA expression levels of cells in (A) were measured as in Fig 1 (C) Cellular ATP levels of cells in (A) were measured as in ldquoMaterialsand methodsrdquo (D) ATP5b and cyt-C mRNA expression levels of cells in (A) were measured as in Fig 1 Statistical analysis was performed as in Fig 1

1242 M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

32 Suppressing MEK12 activities reduces mitochondrial mass andfunction

To explore if inhibiting MEK12 alters mitochondrial mass wetreated normally growing A549 cells dose-dependently with a MEK12 inhibitor U0126 to induce cell cycle arrest in the G1 phase(supplemental data Fig S4) Concordantly U0126 dose-dependentlyreduced mitochondrial mass (Fig 2A) and suppressed the mRNAexpression levels of Tfam and Mfn2 (Fig 2B) In addition weinvestigated if ATP production was reduced due to the loss ofmitochondrial mass We indeed found that U0126 dose-dependentlylowered the total cellular ATP level (Fig 2C) and suppressed themRNA expression levels of ATP5b and cyt-C (Fig 2D) as well as MT-CYB MT-COI and MT-ATP6 expressions (supplemental data Fig S5)Although less effective compared to U0126 we also found thatanother inhibitor PD98059 which only inhibits MEK1 modestlyreduced mitochondrial mass and partially suppressed the expressionof these genes (supplemental data Fig S6)

Fig 3 Suppressing MEK12 activities selectively reduces PGC-1β expression (A) mRNA exprof U0126 for 24 h were measured The relative expression levels of these genes under DMSperformed for ERRα ERRγ PGC-1α and PGC-1β with β-actin as a control Statistical analy

33 Suppressing MEK12 activities selectively reduces PGC-1β expression

Since the transcriptional expression levels of ATP5b cyt-C Tfamand Mfn2 are in part controlled by ERRα ERRγ PGC-1α and PGC-1βwe next examined if the mRNA and protein levels of these factorswere altered by suppressing MEK12 activities We found that U0126did not significantly alter the mRNA or the protein levels of ERRαERRγ and PGC-1α (Fig 3A and B) On the other hand U0126selectively and dose-dependently suppressed the mRNA and proteinlevels of PGC-1β (Fig 3A and B)

Since suppressing MEK12 activities selectively reduces PGC-1βexpressionwe then checked if serum starvation and re-fed treatmentsaltered PGC-1β expression We found that PGC-1β mRNA level wasreduced by serum starvation and elevated upon re-feedingwith serum(Fig 4A) We then cloned the 2 kb promoter region of PGC-1βupstream of its transcriptional start site into a luciferase reporter [8]Using this PGC-1β-promoter-luciferase reporter we were able torecapture the responses to serum starvation (Fig 4B) and U0126

ession levels of ERRα ERRγ PGC-1α and PGC-1β in cells treated with different amountsO treatment as a control were set as 1 (B) Western blots for cell extracts in (A) weresis was performed as in Fig 1

Fig 2 Suppressing MEK12 activities reduces mitochondrial mass and function (A) Mitochondrial mass levels were measured as in Fig 1 after cells were treated with differentamounts of U0126 for 24 h (B) Mfn2 and TfammRNA expression levels of cells in (A) were measured as in Fig 1 (C) Cellular ATP levels of cells in (A) were measured as in ldquoMaterialsand methodsrdquo (D) ATP5b and cyt-C mRNA expression levels of cells in (A) were measured as in Fig 1 Statistical analysis was performed as in Fig 1

1242 M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

32 Suppressing MEK12 activities reduces mitochondrial mass andfunction

To explore if inhibiting MEK12 alters mitochondrial mass wetreated normally growing A549 cells dose-dependently with a MEK12 inhibitor U0126 to induce cell cycle arrest in the G1 phase(supplemental data Fig S4) Concordantly U0126 dose-dependentlyreduced mitochondrial mass (Fig 2A) and suppressed the mRNAexpression levels of Tfam and Mfn2 (Fig 2B) In addition weinvestigated if ATP production was reduced due to the loss ofmitochondrial mass We indeed found that U0126 dose-dependentlylowered the total cellular ATP level (Fig 2C) and suppressed themRNA expression levels of ATP5b and cyt-C (Fig 2D) as well as MT-CYB MT-COI and MT-ATP6 expressions (supplemental data Fig S5)Although less effective compared to U0126 we also found thatanother inhibitor PD98059 which only inhibits MEK1 modestlyreduced mitochondrial mass and partially suppressed the expressionof these genes (supplemental data Fig S6)

Fig 3 Suppressing MEK12 activities selectively reduces PGC-1β expression (A) mRNA exprof U0126 for 24 h were measured The relative expression levels of these genes under DMSperformed for ERRα ERRγ PGC-1α and PGC-1β with β-actin as a control Statistical analy

33 Suppressing MEK12 activities selectively reduces PGC-1β expression

Since the transcriptional expression levels of ATP5b cyt-C Tfamand Mfn2 are in part controlled by ERRα ERRγ PGC-1α and PGC-1βwe next examined if the mRNA and protein levels of these factorswere altered by suppressing MEK12 activities We found that U0126did not significantly alter the mRNA or the protein levels of ERRαERRγ and PGC-1α (Fig 3A and B) On the other hand U0126selectively and dose-dependently suppressed the mRNA and proteinlevels of PGC-1β (Fig 3A and B)

Since suppressing MEK12 activities selectively reduces PGC-1βexpressionwe then checked if serum starvation and re-fed treatmentsaltered PGC-1β expression We found that PGC-1β mRNA level wasreduced by serum starvation and elevated upon re-feedingwith serum(Fig 4A) We then cloned the 2 kb promoter region of PGC-1βupstream of its transcriptional start site into a luciferase reporter [8]Using this PGC-1β-promoter-luciferase reporter we were able torecapture the responses to serum starvation (Fig 4B) and U0126

ession levels of ERRα ERRγ PGC-1α and PGC-1β in cells treated with different amountsO treatment as a control were set as 1 (B) Western blots for cell extracts in (A) weresis was performed as in Fig 1

Fig 4 Growth regulation on PGC-1β expression (A) mRNA expression levels of PGC-1βin cells from Fig 1D were measured The relative amount in 10 FBS was set as 1 (BndashC)Cells were transiently transfected with the PGC-1β-promoter luciferase reporter as inRef [8] Luciferase activity was measured as in ldquoMaterials and methodsrdquo (B) Six hoursafter transfection cells were starved in different amounts of FBS for 24 h (C) Six hoursafter transfection cells were treated with different amounts of U0126 for 24 hStatistical analysis was performed as in Fig 1

Fig 5 PGC-1β over-expression partially reverses the effects of U0126 onmitochondrial massfor 24 h and then starved overnight in 01 FBS Non-starved cells (10 FBS) were set at 10024 h and then starved overnight before re-fed with 10 FBS for 12 h Different doses of U012Statistical analysis was performed as in Fig 1

1243M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

treatments (Fig 4C) suggesting that regulatory elements within thispromoter region may mediate the signal derived from the MEKndashERKcascade to govern mitochondrial biogenesis

34 Over-expressing PGC-1β partially blunts the suppression ofmitochondrial mass mediated by U0126

Finally we examined the importance of this MEKndashERK cascade-stimulated PGC-1β expression to mitochondrial biogenesis We firstchecked if over-expression of MEK1 or MEK2 would enhancemitochondrial biogenesis A549 cells were transiently transfectedwith a control MEK1 or MEK2 expression vector for 24 h beforeserum starvation overnight Over-expression of MEK1 or MEK2 wassufficient to restore the level of mitochondrial mass similar to fullserum level (Fig 5A) Concordantly over-expression of PGC-1βincreased the basal level of mitochondrial mass and partially reversedthe reduction in mitochondrial mass mediated by U0126 (Fig 5B)confirming a role for PGC-1β in promoting mitochondrial biogenesisunder the influence of MEKndashERK cascade

4 Discussion

Mitochondrial biogenesis and function are intimately linked to cellcycle progression Tumor suppressor Rb promotes mitochondrialbiogenesis [16] whereas p53 influences mitochondrial function [17]In our current study we provide evidences that the MEKndashERK cascadegoverns mitochondrial biogenesis through selectively stimulatingPGC-1β expression thus priming cells for entry into rapidly growingphase Specifically the expression level of PGC-1β correlated withserum starvation and re-fed treatments Additionally MEK12inhibitor U0126 dose-dependently reduced PGC-1β expression level

The expression level of PGC-1β is likely under the guidance ofdifferent transcription factors that recognizes specific responseelements on its promoter Bioinformatics analysis of the 2 kb PGC-1β promoter region reveals several estrogen-related receptor re-sponse elements (ERREs) (data not shown) We indeed found thatboth ERRα and ERRγ enhanced the expression of the PGC-1β-promoter-luciferase reporter and U0126 dose-dependently sup-pressed these enhancements (supplemental data Fig S7) Althoughthe mRNA and protein levels of both ERRα and ERRγ were notsignificantly altered by U0126 treatment (Fig 3) their transcriptionalactivitiesmay nonetheless be suppressed Of note the activity of ERRαis enhanced by epidermal growth factor in breast cancer cells [18]Incidentally knockingdownERRαbyRNAi reduces the inductionof cyt-C in primary Schwann cells stimulated with neuregulin and insulin-like

(A) Cells were transiently transfected with a control MEK1 or MEK2 expression vector (B) Cells were transiently transfected with a control or PGC-1β expression vector for6 were added during serum re-fed (A and B) Relative mitochondrial mass were shown

1244 M Gao et al Biochimica et Biophysica Acta 1813 (2011) 1239ndash1244

growth factor 1 [19] It is therefore possible that the MEKndashERK cascadecommunicates to these receptors through post-translational modifica-tion such as phosphorylation on the receptors themselves or theircofactors to influence DNA binding and transcriptional activities

The differential roles of ERRα ERRγ PGC-1α and PGC-1β in cancerare just beginning to be investigated ERRα has been suggested to be aprognostic marker for breast ovarian prostate and colon cancers[20] Using different ERRα inverse agonists we and others haveshown that suppressing its activity leads to cell cycle arrest orapoptosis in vitro and reduces tumor growth in vivo [921ndash23] Incontrast over-expressing ERRγ has been demonstrated to reduceprostate cancer growth [24] On the other hand the roles of PGC-1αand PGC-1β in cancer have not been fully investigated Intriguinglyour data suggest that PGC-1α and PGC-1β respond differentially togrowth factor signaling Namely only the expression of PGC-1β isunder the influence of the MEKndashERK cascade It would be informativeto examine if the expression levels of PGC-1β are elevated in cancersthat are characterized by over-activation of receptor tyrosine kinase

Conflicts of interest

The authors declare no conflict of interest

Acknowledgments

We are grateful to Dr Sylvain Meloche for contributing the MEK1and MEK2 expression plasmids This work was supported by grantsfrom the One Hundred Person Project of the Chinese Academy ofSciences and the Knowledge Innovation Program of the ChineseAcademy of Sciences (KSCX2-YW-R-084)

Appendix A Supplementary data

Supplementary data to this article can be found online atdoi101016jbbamcr201103017

References

[1] MM McKay DK Morrison Integrating signals from RTKs to ERKMAPKOncogene 26 (2007) 3113ndash3121

[2] PJ Roberts CJ Der Targeting the Raf-MEKndashERK mitogen-activated proteinkinase cascade for the treatment of cancer Oncogene 26 (2007) 3291ndash3310

[3] MM Monick LS Powers CW Barrett S Hinde A Ashare DJ Groskreutz TNyunoya M Coleman DR Spitz GW Hunninghake Constitutive ERK MAPKactivity regulates macrophage ATP production and mitochondrial integrityJ Immunol 180 (2008) 7485ndash7496

[4] JM Flynn DA Lannigan DE Clark MH Garner PR Cammarata RNAsuppression of ERK2 leads to collapse of mitochondrial membrane potential

with acute oxidative stress in human lens epithelial cells Am J PhysiolEndocrinol Metab 294 (2008) E589ndashE599

[5] V Giguere Transcriptional control of energy homeostasis by the estrogen-relatedreceptors Endocr Rev 29 (2008) 677ndash696

[6] C Handschin BM Spiegelman Peroxisome proliferator-activated receptorgamma coactivator 1 coactivators energy homeostasis and metabolism EndocrRev 27 (2006) 728ndash735

[7] CR Dufour BJ Wilson JM Huss DP Kelly WA Alaynick M Downes RMEvans M Blanchette V Giguere Genome-wide orchestration of cardiac functionsby the orphan nuclear receptors ERR[alpha] and [gamma] Cell Metab 5 (2007)345ndash356

[8] Y Wang F Fang CW Wong Troglitazone is an estrogen-related receptor alphaand gamma inverse agonist Biochem Pharmacol 80 (2010) 80ndash85

[9] F Wu J Wang Y Wang T-T Kwok S-K Kong C Wong Estrogen-related receptor[alpha] (ERR[alpha]) inverse agonist XCT-790 induces cell death in chemothera-peutic resistant cancer cells Chem Biol Interact 181 (2009) 236ndash242

[10] WWang CWWong Statins enhance peroxisome proliferator-activated receptorgamma coactivator-1alpha activity to regulate energy metabolism J Mol Med 88(2010) 309ndash317

[11] X Liao Y Wang CW Wong Troglitazone induces cytotoxicity in part bypromoting the degradation of peroxisome proliferator-activated receptor gammaco-activator-1alpha protein Br J Pharmacol 161 (2010) 771ndash781

[12] Z Huang F Fang J Wang CW Wong Structural activity relationship offlavonoids with estrogen-related receptor gamma FEBS Lett 584 (2010) 22ndash26

[13] J Xu X Liao C Wong Downregulations of B-cell lymphoma 2 and myeloid cellleukemia sequence 1 bymicroRNA 153 induce apoptosis in a glioblastoma cell lineDBTRG-05MG Int J Cancer 126 (2010) 1029ndash1035

[14] J Wang F Fang Z Huang Y Wang C Wong Kaempferol is an estrogen-relatedreceptor [alpha] and [gamma] inverse agonist FEBS Lett 583 (2009) 643ndash647

[15] K Gopalbhai G Jansen G Beauregard MWhiteway F Dumas C Wu S MelocheNegative regulation of MAPKK by phosphorylation of a conserved serine residueequivalent to Ser212 of MEK1 J Biol Chem 278 (2003) 8118ndash8125

[16] VG Sankaran SH Orkin CR Walkley Rb intrinsically promotes erythropoiesisby coupling cell cycle exit with mitochondrial biogenesis Genes Dev 22 (2008)463ndash475

[17] S Matoba J-G Kang WD Patino A Wragg M Boehm O Gavrilova PJ Hurley FBunz PM Hwang p53 regulates mitochondrial respiration Science 312 (2006)1650ndash1653

[18] JB Barry V Giguere Epidermal growth factor-induced signaling in breast cancercells results in selective target gene activation by orphan nuclear receptorestrogen-related receptor alpha Cancer Res 65 (2005) 6120ndash6129

[19] P Echave G Machado-da-Silva RS Arkell MR Duchen J Jacobson R Mitter ACLloyd Extracellular growth factors and mitogens cooperate to drive mitochon-drial biogenesis J Cell Sci 122 (2009) 4516ndash4525

[20] RA Stein DP McDonnell Estrogen-related receptor alpha as a therapeutic targetin cancer Endocr Relat Cancer 13 (Suppl 1) (2006) S25ndashS32

[21] MJ Chisamore HA Wilkinson O Flores JD Chen Estrogen-related receptor-alpha antagonist inhibits both estrogen receptor-positive and estrogenreceptor-negative breast tumor growth in mouse xenografts Mol Cancer Ther8 (2009) 672ndash681

[22] S Bianco O Lanvin V Tribollet C Macari S North JM Vanacker Modulatingestrogen receptor-related receptor-alpha activity inhibits cell proliferation J BiolChem 284 (2009) 23286ndash23292

[23] J Wang Y Wang C Wong Oestrogen-related receptor alpha inverse agonist XCT-790 arrests A549 lung cancer cell population growth by inducing mitochondrialreactive oxygen species production Cell Prolif 43 (2010) 103ndash113

[24] S Yu X Wang C-F Ng S Chen FL Chan ERRgamma suppresses cellproliferation and tumor growth of androgen-sensitive and androgen-insensitiveprostate cancer cells and its implication as a therapeutic target for prostate cancerCancer Res 67 (2007) 4904ndash4914