p53-dependent regulation of mitochondrial energy production by the rela nf-κb subunit. ini-isabée...

p53-dependent Regulation of Mitochondrial Energy Production by the RelA NF- B Subunit.κ

Ini-Isabée Witzel1,2, Renée Johnson1,3 and Neil Perkins1

1Institute for Cellular &Molecular Biosciences, Newcastle University, UK.2New York University Abu Dhabi, Saadiyat Campus, Abu Dhabi, UAE.3Victor Chang Cardiac Research Institute, Sydney, Australia.

NF- B Family MembersκRel Homology Domain

(RHD)(DNA binding and

dimerisation)

TransactivationNRelA (p65)

c-Rel

RelB

C

Proteolysis

NF-κB1 p105

p50

Ankyrin Repeats

Proteolysis

NF- κB2 p100

p52

NF- B and DiseaseκNF-kB under control(mainly cytoplasmic)

Normal cells

Out of control NF-kB (aberrantly nuclear)

Diseased cells

ArthritisAsthma

Atherosclerosis

Inflammatory bowel

diseases

IIINFLAMMATORY DISEASES

IIINFECTIONS

AIDSHeliobacter pylori-

associated gastritis

Renal diseasesGut diseasesSkin diseases

Heart Failure

Diabetes (type I & II)

Ectodermal dysplasia

Alzheimer’s diseases

Incontinentia pigmenti

CANCER

All subunits have been implicated in cancer.

NF-kB aberrantly active (nuclear):

LeukemiasLymphomas

Many solid tumours

Cancer Cells

Self-sufficiency in

growth signals

Resistance to anti-growth signals

Evasion of apoptosis

Sustained angiogensis

Limitless reproductive

potential

Tissue invasion and

metatasis

Abnormal metabolic pathways

Immune system evasion

InflammationUnstable DNA and

chromosome abnormalityCell. 2011 Mar

4;144(5):646-74.

Hallmarks of Cancer

IKK

NF-kB

Cancer therapies(chemotherapeutic

agentsγ-irradiation)

StressROI

inducers(hypoxia)

CarcinogensTumour

promoters(PMA, UV)

Oncogeneactivation

(Ras, Bcr-Abl)

Geneticalteration

(subunit expression,IkB deletion)

Cancer-associated inducers of aberrant NF-κB activity

CytokinesInfection

Inflammation(IL-1, TNF,

chemokines)

Angiogenesis(VEGF, TNF, IL-1, IL-

8) Metastasis(ICAM-1, VCAM-

1, ELAM-1)

Proliferation(Cyclin D1, c-

Myc)Survival(Bcl-xL, XIAP,

cIAP1 & 2)

Immortality(telomerase)

Tumour promotion(COX2, iNOS, MMP-9,

uPA)

Tumour promoting functions of NF-kB

Is NF-κB also involved in alteration of cellular metabolism in cancer

cells?

NF-κB and Cancer

RelA T505 Peptide (RelA):TTEPMLMEYPEAITRLVTGAQRPCScramble Peptide (S):LATRQETGPIRMPEVYTAETMPLC

428 521

Rel Homology Domain1

19551

301

RelA(p65)

TA1TA2

P

T505

Fr 3

-4

T505 Peptide Scramble

WB:Mortalin

kDa191

97

64

51

39

28

19

14

Scra

mbl

e

Mortalin

Fr 5

-6

Fr 3

-4Fr

5-6

HeLa cells

HeLa cells

T505

Pep

tide

Mortalin (mtHSP70) binds to the RelA T505

region

Mutation of RelA at T505 impairs binding of RelA to Mortalin

WB:RelA

Mortalin

mIg

G

IP:

Null T505A T505DIP Inputs:

Nul

l

Rel

AT

505A

T50

5D

Mor

talin

mIg

GM

orta

lin

mIg

G

Mor

talin

mIg

GM

orta

lin

Mortalin binds to endogenous RelA inU-2 OS and 293 cells

RelA reconstituted MEF cells

RelA interacts with mitochondrial HSP70, Mortalin

Cytoplasm Mitochondria

Nul

l WB:

RelA

Mortalin

VDAC

-tubulin

BiP

RelA

T505

AT5

05D

Nul

lRe

lAT5

05A

T505

D

RelA reconstituted MEF cells

Mitochondria Cytoplasm

U-2

OS

WB:

RelA

Mortalin

VDAC

-tubulin

BiP

Cont

rol

Mor

talin

U-2

OS

Cont

rol

Mor

talin

siRNA siRNA

U-2 OS cells

RelA import into mitochondria is facilitated by an interaction with mortalin that is impaired by mutation of RelA at T505.

RelA mitochondrial import is Mortalin dependent

O2

consumption(average

NRF)

**

***

0

0.5

1

1.5

siRNA: Control RelA Control RelA

Passage: Early Late

U-2 OS cells

0

0.5

1

1.5

0

0.4

0.8

1.2

1.6


O2

consumption(average

NRF)

Treatment: Control Oligomycin

********

Late Passage U-2 OS cells

0

0.6

1.2

1.8

ATP levels

(average RLU /cell)


Treatment: Control Oligomycin

** **Late Passage U-2 OS cells Late Passage

U-2 OS cells

0

1

2

3

siRNA: Control RelA

Glucoseconsumption

(μg/min/50 000 cells)

RelA-induced changes in ATP production have a physiological effect on the cells as indicated by phosphorylation of the energy sensing protein, AMPK.

RelA regulates cellular energy production

p12 p20 p12 p20


U-2 OS Cells

RelA

VDAC

Mot

α-tubulin

RelA regulates mitochondrial gene expression

Relative Enrichment

(ChIP Assay)

D-loop

0

1

2

3

Passage: Early Late

Antibody: Control RelA Control RelA

*

0

1

2

3

0

4

8

12

siRNA: Control RelA Control RelA Gene: D-loopAntibody: POLRMT

*

RelativeEnrichment

(ChIPAssay)

Passage: Early Late

Relative Cytochrome C Oxidase

I mRNA Level

Late Passage U-2 OS cells

siRNA: Control RelA0

0.04

0.08

0.12

*

0

0.02

0.04

0.06

0.08


AveragemRNA

Abundance

Gene: Cytochrome BPassage: Early Late

***

**

Loss of p53:• Results in reduced oxygen consumption and

increased glycolysis1

• Reduced SCO2 and TIGAR and increased PGM and GLUT31,2

• Enhances activation of NF-κB2

Increased glycolysis in the absence of p53 via up-regulation of GLUT3 is RelA dependent2

1. S. Matoba et al., Science 312, 1650 (Jun 16,2006).2. K. Kawauchi, K. Araki, K. Tobiume, N. Tanaka, Nat Cell Biol 10, 611 (May, 2008)

p53, RelA and Energy Production

Passage Number

U-2 OS cells

Phospho-RelA Thr505

Phospho-p53 Ser15

RelA

p53

Actin0

1

2

3

4

Relativeenrichment

(ChIPassay)

Treatment: Control IPTG

*

Cytochrome Bα-RelA

H1299wtp53 cells

Induction of p53 reduces RelA binding to the

mitochondrial genome

0

1

2

3

4


O2

consumption(average

NRF)

Treatment: Control IPTG

*

* *

H1299wtp53 cells MEF cells

0

0.4

0.8

1.2

Wild Type p53 -/-siRNA: Control RelA Control RelA

ATPlevels

(averageRLU/cell)

**

***

p53 mediates the switch in RelA regulation of cellular energy production

RelA regulation of mitochondrial energy production is p53 dependent

p53 prevents RelA import into mitochondria by disrupting the interaction of RelA with mortalin

IP:

IPT

G

Control IPTGInputs

WB:RelA

Mortalin

Con

trol

mIg

GM

orta

linm

IgG

Mor

talin

H1299wtp53 cells

WB:RelA

VDAC

Mortalin

α-tubulin

RSV

P53-R

SVRSV

P53-R

SV


H1299 cells

RelA regulation of mitochondrial energy production is p53 dependent

Conclusions

Early Passage (+p53)

Nucleus

Late Passage (-p53)

p53 GlycolysisRelA

AcknowledgementsNeil Perkins

Renée Johnson

Members of the Perkins Laboratory

Members of CTP at NYUAD

CRUK for funding our work!

p53-dependent regulation of mitochondrial energy production by the rela nf-κb subunit. ini-isabée...

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