Understanding Theileria Parva's Impact on Bovine Lymphocytes: Signaling Pathways & Anti-Apoptotic Responses

1. By: Chris Carr

2. Background Information • Theileria parva is an intracellular protozoan parasite that is transmitted by ticks and causes East Coast fever, a disorder of cattle in East and Central Africa • They invade and immortalize bovine lymphocytes

3. The major players • C-Myc: a transcription factor that binds to specific sites in the promoters of at least 1382 different genes • JAK2/STAT3: a signalling pathway that contributes to c-Myc transcription • Mcl-1: target gene of c-Myc • CK2: a factor that increases the stability of c-Myc • BW720c: a drug that kills Theileria • AG 490: a drug that inhibits JAK2 activity • PARP: poly(ADP-ribose) polymerase • Z-VAD: Caspase inhibitor

4. What on earth is this about? • Theileria initiates a strong and sustained induction of c-Myc • This is caused by intervening with both c-Myc transcription and stability • Infection then results in a c-Myc mediated antiapoptotic response

5. How is this possible? • Activation of CK2 • Activation lf NF-κB signalling pathway • Induction of anti-apoptotic proteins • Decrease in the number of pro-apoptotic proteins • Infected lymphocytes use autocrine loops -TNF autocrine loop NF-κB activation -GM-CSF autocrine loop P13K activation AP-1 induction

6. What are autocrine loops? • A process of cell signaling whereby a cell both releases and responds to a soluble factor • The loops refer to the signal both originating and ending in the same location

7. Experiment 1: Theileria infection leads to phosphorylation of STAT3, and induction of c-Myc and Mcl-1 (part 1) • Theileria pursuade lymphocytes to proliferate uncontrollably via GM-CSF autocrine loop. Non-infected Infected Does this proliferation occur in tandem with a signalling pathway? Is the end result of this signalling pathway an increase in c-Myc levels? YES

8. Experiment 1: Part 2 • Are the phosphorylation of STAT3 and the induction of c-Myc due directly to infection of Theileria? Note: Stat3 is a protein that is present consistently with or without infection. However, the phosphorylation of this protein is dramatically increased upon infection. BW 720c was used to kill Theileria Of Course

9. Wait there’s moreExperiment 1: Part 3 • Does STAT3 signalling to c-Myc involve the JAK2 kinase? AG490 was used to inhibit JAK2 Over the course of the experiment, blocking JAK2 signalling resulted in a greater decrease in STAT3 phosphorylation than eliminating Theileria altogether Indubitably

10. Experiment 2: Theileria dependent post-translational stabilization of c-Myc involves CK2 • Does parasite infection affect the stability of the c-Myc protein? BW 720c was used to kill Theileria Cyclohexamide was used to inhibit the transcription of c-Myc In B-cells containing Theileria, c-Myc half-life was significantly prolonged Yes

11. Experiment 2: continued Apigenin was used as a CK2 inhibitor The presence of live Theileria parasites leads to a CK2-mediated increase in the stability of c-Myc

12. Experiment 3: Theileria-induced transcriptional activation of c-Myc is mediated in part by GMC-SF via activation of STAT3 • Does Theileria induce high c-Myc levels by transcriptional regulation? Theileria induces a four-fold increase in c-Myc driven luciferase activity Yup

13. Experiment 3: Continued C-Myc transactivation is dependent on live parasites C-Myc binds to the luciferase gene which results in luciferase activty

14. Experiment 3: Yes, there is more Addition of recombinant GM-CSF to infected cells increased endogenous c-Myc transactivation

15. Experiment 3: further continued A promotor containing 4 E-box elements were introduced upstream from the luciferae gene. Luciferase activity is decreased because there are an increased number of binding sites for c-Myc to attach to. Thus, target gene transcription is reduced.

16. Experiment 3: Wait, there is more control Kinase dead JAK2 The co-transfection of different mutants significantly lowered c-Myc driven luciferase activity Therefor, transcriptional induction of c-Myc clearly involves JAK2 and STAT3

17. Experiment 4: Theileria-transformed B cell survival is c-Myc dependent • How significant is the contribution of c-Myc to the survival of Theileria-infected B cells? Anti-sense C-Myc transcription was interfered with by the addition of antisense oligonucleotides Non-sense Very Significant

18. Experiment 4: continued Cell death correlated to a loss of c-Myc levels, Mcl-1 expression, and PARP cleavage Thus, Mcl-1 can be directly attributed to c-Myc activation

19. Experiment 5: Inhibition of JAK2 results in caspase-dependent apoptosis of Thjeileria- transformed B cells AG 490 induced apaptosis is caspase dependent

20. Experiment 5: continued LETD-AFC is a caspase 8 substrate DEVD-AFC is a general caspase substrate There is no LETD activity which means caspase 8 is not a part of this mechanism of cell death

21. Experiment 5: Can you handle it? In correlation with caspase 9 activation, there is PARP cleavage (fig 5c) There is also Annexin-V positivity and nuclear fragmentation (fig 5d) Therefore, the inhibition of the JAK2 signalling pathway leads to B-cell apoptosis

22. Experiment 6: Ectopic expression of c-Myc rescues Theileria infected B cells from AG 490 mediated apoptosis Apoptosis results when AG 490 blocks JAK2 activity, however, the addition of ectopic c-Myc reverts cell death.

23. Experiment 6: continued Ectopic expression of CMV-cMyc augments c-Myc levels, but does not effect STAT3 or its phosphorylation status

24. Results: Yes, the experiments are finally over Theileria infection leads to increased levels of c-Myc The life of the c-Myc transcription factor is prolonged due to CK2-mediated phosphorylation A JAK 2/STAT3 signalling pathway also contributes to increased c-Myc transcription This anti-apoptotic process can be inhibited at several junctures (Apigenin, BW 720c, AG 490)

25. Further studies • Other cytokines may be secreted that activate JAK2 and c-Myc by yet-to-be described autocrine loops • How do P13K activation and AP-1 induction lead to the phosphorylation of STAT3

26. The End Now you may start clapping