p53-dependent regulation of mitochondrial energy production by the rela nf-κb subunit. ini-isabée...
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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
siRNA: Control RelA Control RelA
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)
siRNA: Control RelA Control RelA
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
Cytoplasm Mitochondria
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
siRNA: Control RelA Control RelA
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
siRNA: Control RelA Control RelA
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
Cytoplasm Mitochondria
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!