Presented By : Deepanjan Paul M.Sc. (F), MHG.

1. Presented By: Deepanjan Paul M.Sc. (F), MHG. PUMA is directly activated by NF-kB and contributes to TNF-a-induced apoptosisP 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.

2. Apoptosis • The term “PCD” was usedin 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.

3. www.nature.com/reviews/molcellbio

4. APOPTOSIS TIMELINE Cell Death and Differentiation (2002) 9, 349-354

5. Pleiotropic cytokine. Role in immune and inflammatory responses. Induction of apoptosis. TNF-a Apoptosis TNF R1 TNF R2 Mitochondrial dysfunction Extrinsic pathway Intrinsic pathway TNF-a

6. 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-kBpathway Canonical Noncanonical p50 & p65 p52 & Rel-B

7. NF-kB activation IkB NF-kB IKK Kinases IkB Proteosomal degradation Activated NF-kB Nucleus Binds to kB sequence Target genes

8. NF-kB & Apoptosis NF-kB c-FLIP,Bcl-2,Bcl-XL cIAP2,A1/Bfl-2. Protection from apoptosis. NF-kB p53,Fas,Fas ligand death receptor 4,death receptor 5. Promotes apoptosis

9. PUMABH 3-only Bcl-2 family member.Downstream target of p53. p53- dependent p53- independent Serum starvation,kinaseinhibitors, glucocorticoids, ER stress, ischemia/reperfusion. PUMA activation g-radiation Drugs p53 PUMA activation

10. 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. • Glucocorticoiddexamethasone 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.

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

12. 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.

13. REsults

14. 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.

16. 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.

17. p53-independent induction of PUMA by p65

18. p65 is necessary for PUMA induction by TNF-a • BAY 11-7082 IkBasuperrepressor 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.

19. p65 is necessary for PUMA induction by TNF-a

20. 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, IkBasuperrepressor 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.

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

22. 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.

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

25. 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.

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

28. 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.

29. PUMA mediates TNF-a-induced hepatocyte apoptosis

31. 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.

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

33. 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.

34. 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-kBinhibition can compromise PUMA induction by inflammatory cytokines, which may be involved in tumor suppression and be beneficial for anticancer therapies.

35. THANK YOU