Role of TNFa and IGF-1 in neuronal apoptosis

Project Associates / Assistants
Ajay Yadav
K Prashanth (till Oct 2002)
Debjani Kundu

Collaborators
Ejaz Hussain, Jamia Millia Islamia

Tumor necrosis factor-a (TNFa) is known to mediate various forms of neurodegeneration. On the other hand insulin like growth factor-1 (IGF-1) plays a pivotal role in regulating inflammatory events in the brain. Originally it was thought that the mechanism of action of IGF-1 and TNFa were unrelated and disparate. But recently it has been shown that signaling pathways of these cytokines may interact during neurodegeneration. The proposed project is aimed at understanding the signaling pathways, mechanisms of actions and cross talk between TNFa and IGF-1. The specific objectives of the project are (i) to study the effect of TNFa in the system under study and identify the signal transduction pathways associated with action of TNFa, (ii) to establish the effect of IGF-1 in the system under study and identify the signal transduction pathways associated with action of IGF-1 and (iii) to identify potential cross talk points between the various signaling cascades initiated by TNFa and IGF-1 leading to inhibition of neurodegeneration by IGF-1

In the reporting year we first looked at the mechanisms of TNFa mediated cell death. We were able to demonstrate that this cell death caused by TNFa was mainly because of generation of reactive oxygen species and a free radical scavenger butylated hydroxyanisole (BHA) was able to completely reverse this process.

We then addressed the question of rescue mediated by IGF-1 of TNFa inhibited cells. Previously we had observed that JNK kinase got activated preferentially by IGF-1 and thus could be one of the pathways of IGF-1 mediated rescue. In order to prove this we inhibited this enzyme with a specific JNK kinase inhibitor- dicumarol. This could completely reverse the protective effects of IGF-1 proving that this kinase indeed plays an important role in IGF-1 mediated rescue. We then searched for the upstream and downstream molecules involved in JNK kinase pathway. We observed an activation of SEK1/MKK4 (known to directly phosphorylate and activate JNK kinase in many cells) in response to IGF-1 both in the presence and absence of TNFa but not by TNFa alone. JNK kinase is known to activate various downstream transcription factors like c-jun and ATF-1. We observed an increase in phospho levels of both c-jun as well as ATF-1.

We then explored another pathway which is known to play an important role in cell survival, both during IGF-1 as well as TNFa signaling i.e. the akt kinase (PKC) pathway. akt is activated by phospholipid binding and activation loop phosphorylation at thr308 by PDK1 and also by phosphorylation within the C terminus at ser473. We observed that both phospho-akt (ser473) as well as phospho-akt (thr308) was activated by IGF-1 and remained so upon co-stimulation with TNFa. To confirm what activates akt we looked at role of PDK-1. We observed an activation of PDK-1 by IGF-1 alone as well as in the presence of TNFa. The common targets that akt phosphorylates and inactivates are GSK-3b(ser9) and forkhead transcription factor-FKHR(ser256) which are known to promote apoptosis. We observed an increase in levels of phospho-GSK-3b (ser9) post IGF-1 stimulation as well as after co-stimulation with TNFa. Our initial studies show no apparent role of FKHR. The involvement of PI3 kinase in the rescue of TNFa inhibited cells by IGF-1 was confirmed by addition of PI3 kinase specific inhibitor- LY294002 as IGF-1 was not able to rescue cell growth.

In summary both akt pathway as well as PI3 kinase pathways play a crucial role in IGF-1 mediated rescue of TNFa triggered apoptosis.

Publications

Original peer-reviewed articles

1.     Shahjee H Md, Banerjee K and Ahmad F (2002) Comparative analysis of naturally occurring L-amino acid osmolytes and their D-isomers on protection of Escherichia coli against environmental stresses. J Biosci 27:515-520.

Reviews/Proceedings

1.     Ghosh S, Parvez MK, Banerjee K, Sarin SK and Hasnain SE (2002) Baculovirus as mammalian cell expression vector for gene therapy:an emerging strategy. Mol Ther 6:5-11.