Transcript TNF-a

Presented By:
Deepanjan Paul
M.Sc. (F), MHG.
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The term “PCD” was used in 1965 to describe developmental cell
deaths in insect systems by Lockshin & Williams.
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Apoptosis (Apo: Away; Ptosis: Falling off) was coined by John
F. Kerr, Andrew H. Wyllie & A. R. Currie in 1972.
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Cell shrinkage, karyorrhexis, blebbing, apoptotic bodies formation,
phagocytosis by macrophages.
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Autophagy- Portions of cytoplasm or organelles are sequestered
into a double-membrane autophagosome and delivered to the
lysosomes and autolysosomes for breakdown and recycling.
www.nature.com/reviews/molcellbio
APOPTOSIS TIMELINE
Cell Death and Differentiation (2002) 9, 349-354
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Pleiotropic cytokine.
Role in immune and inflammatory responses.
Induction of apoptosis.
TNF-a
Apoptosis
TNF R1 TNF R2 Mitochondrial
dysfunction
Extrinsic
pathway Intrinsic pathway
 Mediator
of TNF-a associated cellular response.
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Group of dimeric transcription factor.
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NF-kB/Rel family (p50, p52, p65, Rel-B & c-Rel).
NF-kB pathway
Canonical
Noncanonical
p50 & p65
p52 & Rel-B
IkB
NF-kB
IKK Kinases
IkB
Proteosomal degradation
Activated NF-kB
Nucleus
Binds to kB sequence
Target genes
NF-kB
NF-kB
c-FLIP,Bcl-2,Bcl-XL
cIAP2,A1/Bfl-2.
p53,Fas,Fas ligand
death receptor 4,death
receptor 5.
Protection from apoptosis.
Promotes apoptosis
p53- DEPENDENT
g-radiation
Drugs
p53
PUMA activation
p53- INDEPENDENT
Serum starvation,kinase inhibitors,
glucocorticoids, ER stress,
ischemia/reperfusion.
PUMA activation
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PUMA is essential for damage-induced & p53-dependent apoptosis.
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Phosphorylation of CREB-binding protein (CBP) by IKKa suppresses p53mediated gene expression by switching the binding preference of CBP from p53
to NF-kB.
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NF-kB can promote thymocyte and T-cell apoptosis, which is critical for shaping
the T-cell repertoire during thymocyte ontogeny.
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Glucocorticoid dexamethasone induces PUMA dependent apoptosis in mouse
thymocytes.
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PUMA can complement the function of Bim in controlling T-cell apoptosis in the
termination of immune response.
Mechanism & function of p53-independent PUMA induction
remain unclear.
PUMA is involved in ischemia/reperfusion-induced
intestinal injury, inducing inflammatory response ,
so, it may be regulated by inflammatory cytokines.
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Cell culture and drug treatment.
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Transfection and reporter assays.
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Western blotting and antibodies.
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RT-PCR and CHIP.
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Analysis of apoptosis.
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Animal experiments.
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TUNEL and Immunostaining.
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Bioinformatics and statistical analyses.
TNF-a (20ng/ml)
HCT116 colon cancer cells
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PUMA mRNA and protein were induced my TNF-a within several hours.
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Induction of PUMA mRNA and protein was unaffected in p53-KO or BS-KO.
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Induction of PUMA by TNF-a was also independent of FOXO3a.
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Bad, Bim and Noxa but not Bid were induced by TNF-a.
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Potential binding sites of several TF (NF-kB, ATF,IRF & CREB families)
known to mediate TNF-a response were identified .
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Only transfection of p65 subunit of NF-kB increased PUMA expression in
wild-type (WT), p53-KO, and BS-KO HCT116 cells.
p53-independent
induction of PUMA by p65
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BAY 11-7082
(inhibitor of IKK Kinases)
IkBa superrepressor mutant
(Non-degradable)
Suppression of PUMA induction & P65 nuclear
translocation.
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p65 si-RNA
PUMA induction
abrogated.
PUMA induction by TNF-a in mouse embryonic fibroblast (MEF) cells was also
found to be p65 dependent, but p53 independent.
p65
is necessary for PUMA induction by TNF-a
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A luciferase reporter construct containing the putative kB site in the PUMA
promoter was constructed.
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TNF-a treatment or co-transfection with p65 markedly activated the PUMA
reporter.
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BAY 11-7082, IkBa superrepressor mutant & introducing mutations into the kB
site abolished the responsiveness of the reporter.
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CHIP showed that recruitment of p65 to the PUMA promoter region containing
the kB site was enhanced following TNF-a treatment for 6 hrs.
p65
directly binds to the PUMA promoter to activate
PUMA transcription in response to TNF-a
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Knockdown of Bcl-XL (but not cIAP1 or Mcl-1) by siRNA, led to a significant
increase in TNF-a-induced apoptosis but apoptosis induction was much reduced
in PUMA-KO HCT116 cells.
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Caspase 3,8,9 activation, Bid cleavage & cytochrome-c release were also
suppressed in PUMA-KO cells.
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Dominant-negative (DN) FADD mutant slightly lowered TNF-a-induced
apoptosis, but did not affect PUMA-dependent apoptosis following Bcl-XL
knockdown.
So, NF-kB-mediated PUMA induction is a novel mechanism
mediating TNF-a-induced apoptosis.
PUMA-dependent apoptosis induced by TNF-a in
colon cancer cells
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TNF-a was injected intravenously at several doses (2, 4, and 10 mg) in WT and
PUMA-KO mice.
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PUMA, Bcl-XL & Bim were induced by TNF-a in the small intestine.
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TNF-a-induced apoptosis was blocked to a large extent (60–90%) in the crypts
and villus epithelium in PUMA-KO mice compared to the WT.
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Caspase-3 activation was blocked in PUMA-KO mice.
PUMA is necessary for TNF-a-induced apoptosis in
the intestinal epithelium
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PUMA (but not other BH3-only proteins) was increased in the liver of WT mice
following TNF-a treatment for 8 hr.
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TNF-a-induced apoptosis was blocked by 50–70% in the PUMA-KO mice.
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Caspase 3 activation induced by TNF-a at different doses was abrogated in the
hepatocytes of PUMA-KO mice.
So, PUMA mediates TNF-a-induced hepatocyte apoptosis.
PUMA mediates TNF-a-induced hepatocyte apoptosis
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TUNEL staining revealed more than two fold reduction in apoptosis in PUMAKO thymocytes compared to WT ones.
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Primary thymocytes from WT showed spontaneous apoptosis in culture but
TNF-a treatment had little effect on PUMA-KO primary thymocytes.
So, TNF-a-induced apoptosis in thymocytes is PUMA
dependent.
TNF-a-induced and PUMA-dependent apoptosis in
thymocytes
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1st case of direct regulation of a BH-3 only Bcl-2 family by NF-kB in the TNF-a
response.
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A completely p53 independent PUMA activation, inspite of the well-established
cross talk between p53 & NF-kB in regulating gene expression, is quite
unexpected.
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Phosphorylation of CBP & hence its preferential binding to NF-kB might be
directing p65, instead of p53, to the PUMA promoter in response to TNF-a.
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TNF-a-induced apoptosis often rely on using transcription or translation
inhibitors, which nonspecifically inhibit gene expression and often complicate
data interpretation.
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p53 & p65 can cooperatively induce apoptosis in some circumstances, resulting
from coordinated induction of PUMA by p53 & p65.
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PUMA functions as a novel link between the extrinsic & intrinsic apoptotic
pathways & hence necessary for TNF-a-induced apoptosis.
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In vivo analysis of PUMA induction by TNF-a needs to be further
examined using more physiological systems including genetic
models.
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PUMA was required for TNF-a-induced apoptosis in villus
epithelial cells but not in villus-inside cells.
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Inhibition of NF-kB has been explored as an attractive approach
for anticancer therapies.
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Again, NF-kB inhibition can compromise PUMA induction by
inflammatory cytokines, which may be involved in tumor
suppression and be beneficial for anticancer therapies.
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