PUMA is directly activated by NF-kB and contributes to TNF-a-induced apoptosis

P Wang, W Qiu, C Dudgeon, H Liu, C Huang, GP Zambetti, J Yu and L Zhang.

Cell Death and Differentiation (2009) 16, 1192-1202.Published online 15 May 2009.

Presented By:Deepanjan PaulM.Sc. (F), MHG.

Apoptosis

The term “PCD” was used in 1965 to describe developmental cell deaths in insect systems by Lockshin & Williams.

Apoptosis (Apo: Away; Ptosis: Falling off) was coined by John F. Kerr, Andrew H. Wyllie & A. R. Currie in 1972.

Cell shrinkage, karyorrhexis, blebbing, apoptotic bodies formation, phagocytosis by macrophages.

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

Cell Death and Differentiation (2002) 9, 349-354

APOPTOSIS TIMELINE

TNF-a Pleiotropic cytokine. Role in immune and inflammatory responses. Induction of apoptosis.

TNF-a

Apoptosis

TNF R1 TNF R2 Mitochondrial dysfunction Extrinsic pathway Intrinsic pathway

NF-kB Mediator of TNF-a associated cellular response.

Group of dimeric transcription factor.

NF-kB/Rel family (p50, p52, p65, Rel-B & c-Rel).

NF-kB pathway Canonical Noncanonical

p50 & p65 p52 & Rel-B

NF-kB activation

IkB NF-kB IKK Kinases

IkB

Proteosomal degradation

Activated NF-kB Nucleus Binds to kB sequence Target genes

NF-kB & Apoptosis

NF-kB

c-FLIP,Bcl-2,Bcl-XL cIAP2,A1/Bfl-2.

Protection from apoptosis.

NF-kB

p53,Fas,Fas liganddeath receptor 4,death

receptor 5.

Promotes apoptosis

PUMA

BH 3-only Bcl-2 family member.Downstream target of p53.

p53- DEPENDENT

g-radiation

Drugs p53

PUMA activation

p53- INDEPENDENT

Serum starvation,kinase inhibitors, glucocorticoids, ER stress, ischemia/reperfusion.

PUMA activation

Work Done

PUMA is essential for damage-induced & p53-dependent apoptosis.

Phosphorylation of CREB-binding protein (CBP) by IKKa suppresses p53-mediated gene expression by switching the binding preference of CBP from p53 to NF-kB.

NF-kB can promote thymocyte and T-cell apoptosis, which is critical for shaping the T-cell repertoire during thymocyte ontogeny.

Glucocorticoid dexamethasone induces PUMA dependent apoptosis in mouse thymocytes.

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.

Hypothesis

PUMA is involved in ischemia/reperfusion-induced intestinal injury, inducing inflammatory response , so, it may be regulated by inflammatory cytokines.

Materials & Methods

Cell culture and drug treatment. Transfection and reporter assays. Western blotting and antibodies. RT-PCR and CHIP. Analysis of apoptosis. Animal experiments. TUNEL and Immunostaining. Bioinformatics and statistical analyses.

REsults

p53-independent induction of PUMA by TNF-a

TNF-a (20ng/ml) HCT116 colon cancer cells

PUMA mRNA and protein were induced my TNF-a within several hours.

Induction of PUMA mRNA and protein was unaffected in p53-KO or BS-KO.

Induction of PUMA by TNF-a was also independent of FOXO3a.

Bad, Bim and Noxa but not Bid were induced by TNF-a.

p53-independent induction of PUMA by p65

Potential binding sites of several TF (NF-kB, ATF,IRF & CREB families) known to mediate TNF-a response were identified .

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

p65 is necessary for PUMA induction by TNF-a

BAY 11-7082 IkBa superrepressor mutant p65 si-RNA(inhibitor of IKK Kinases) (Non-degradable)

Suppression of PUMA induction & P65 nuclear PUMA induction translocation. 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

p65 directly binds to the PUMA promoter to activate PUMA transcription in response to TNF-a

A luciferase reporter construct containing the putative kB site in the PUMA promoter was constructed.

TNF-a treatment or co-transfection with p65 markedly activated the PUMA reporter.

BAY 11-7082, IkBa superrepressor mutant & introducing mutations into the kB site abolished the responsiveness of the reporter.

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 activatePUMA transcription in response to TNF-a

PUMA-dependent apoptosis induced by TNF-a in colon cancer cells

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.

Caspase 3,8,9 activation, Bid cleavage & cytochrome-c release were also suppressed in PUMA-KO cells.

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

PUMA is necessary for TNF-a-induced apoptosis in the intestinal epithelium

TNF-a was injected intravenously at several doses (2, 4, and 10 mg) in WT and PUMA-KO mice.

PUMA, Bcl-XL & Bim were induced by TNF-a in the small intestine.

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.

Caspase-3 activation was blocked in PUMA-KO mice.

PUMA is necessary for TNF-a-induced apoptosis inthe intestinal epithelium

PUMA mediates TNF-a-induced hepatocyte apoptosis

PUMA (but not other BH3-only proteins) was increased in the liver of WT mice following TNF-a treatment for 8 hr.

TNF-a-induced apoptosis was blocked by 50–70% in the PUMA-KO mice.

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

TNF-a-induced and PUMA-dependent apoptosis in thymocytes

TUNEL staining revealed more than two fold reduction in apoptosis in PUMA-KO thymocytes compared to WT ones.

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

Discussion

1st case of direct regulation of a BH-3 only Bcl-2 family by NF-kB in the TNF-a response.

A completely p53 independent PUMA activation, inspite of the well-established cross talk between p53 & NF-kB in regulating gene expression, is quite unexpected.

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.

TNF-a-induced apoptosis often rely on using transcription or translation inhibitors, which nonspecifically inhibit gene expression and often complicate data interpretation.

p53 & p65 can cooperatively induce apoptosis in some circumstances, resulting from coordinated induction of PUMA by p53 & p65.

PUMA functions as a novel link between the extrinsic & intrinsic apoptotic pathways & hence necessary for TNF-a-induced apoptosis.

FUTURE PROSPECTS

In vivo analysis of PUMA induction by TNF-a needs to be further examined using more physiological systems including genetic models.

PUMA was required for TNF-a-induced apoptosis in villus epithelial cells but not in villus-inside cells.

Inhibition of NF-kB has been explored as an attractive approach for anticancer therapies.

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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