RESEARCH REPORT 2001-2003

Mechanisms of apoptosis in metazoan cells

The integration of pro-apoptotic (death) and anti-apoptotic (survival) signals is essential for the development and normal existence of multi-cellular organisms.  Developmental regulators provide survival cues by integrating with anti-apoptotic pathways or suppressing molecules that trigger death. Emerging evidence has indicated that these interactions persist in differentiated cells in the adult organism as well. In my laboratory we study these interactions using model cell lines, which permit us to explore molecular and biochemical correlates of cellular responses to death or survival cues. The mammalian immune system comprises many cell types, which depend on complex homeostatic mechanisms to ensure the production and survival of each cell types. The maintenance of T-cell number in the mammalian immune system is one example of the physiological role of apoptosis in adult organisms. This lineage presents a unique model system to investigate survival mechanisms as well as maturation-stage specific apoptotic signaling in mammalian cells.

Cellular strategies signaling survival:

Cells depend on extracellular growth factors and nutrients for their survival, proliferation and differentiation. In their absence, cells undergo neglect-induced death through the intrinsic pathway of apoptosis that impinges on and is regulated by mitochondria. Several aspects of this process can be recapitulated in vitro permitting analysis of underlying cellular and molecular mechanisms (panel on left).

Our experiments have revealed a novel cascade activated by the trans-membrane receptor Notch, in the coordination of the survival response to nutritional cues in mammalian cell lines and T-cells.

The Notch family of receptors regulates a number of processes, including fate determination, differentiation, proliferation and cell survival in metazoans. Ectopic expression of recombinant Notch protected cells from neglect-induced apoptosis (Fig-1), which prompted an analysis of the mechanism underlying Notch activity. Signaling via the Notch family of transmembrane receptors is typically initiated following interactions with ligands that trigger the proteolytic cleavage and nuclear localization of the Notch intracellular domain (NIC), culminating in transcriptional outputs1.  Previous work from our laboratory and others has demonstrated that Notch activity converged on the serine-threonine kinase Akt for the inhibition of apoptosis2-4. However, the targets of Notch activity and the mechanism by which NIC triggered the activation of Akt were not characterized.

We used gain-of-function and site-directed mutants, recombinants with addressing tags, which

restricted spatial distribution and RNA interference approaches in our experiments, to identify novel interactions of NIC with the lipid kinase PI3K, the nutrient sensing kinase mammalian Target Of Rapamycin (mTOR) and the associated protein Rictor, to trigger the activation of Akt. Two residues in Akt – S473 and T308 – are implicated in its activity. Site-directed mutants of Akt indicated a specific requirement for Akt-S473 dependent activity in this transduction cascade, which is summarized in the schematic (Fig-2)5. 

T-cell survival depends on their ability to respond to appropriate cytokines in their microenvironment such that even in nutrient-enriched environments, the removal of cytokines has deleterious consequences.T-cells activated in vitro if cultured in the absence of appropriate growth factors undergo apoptosis, which was inhibited in cells that express recombinant nuclear-excluded NIC recombinants, or by the ectopic expression of an Akt-S473 active construct. Furthermore, these activities were sensitive to regulation by Rapamycin a specific inhibitor of mTOR5. These experiments suggest that the model we propose based on in vitro data most likely operates in the in vivo situation to control cell survival.

Molecular mechanisms underlying T-cell apoptosis: Reactive oxygen species (ROS) regulate activated T-cell death

T cell apoptosis not only shapes the immune repertoire but is essential for immune responses to new and repeated antigenic challenges.
T-cells in circulation activated following encounter with cognate antigen, experience several rounds of proliferation contributing to the inflammatory response. Following antigen clearance the immune system resets numbers by the programmed deletion of the bulk of antigen-specific activated T-cells (panel on the left). This event is thought to be triggered by the depletion of cytokines from the extracellular milieu (activated T-cell neglect-induced death/ANID). Mice with targeted disruptions in specific genes have established a role for the Bcl-2 family proteins Bax, Bak and BIM in activated T-cell apoptosis6. Broad spectrum antioxidants, which prevent the accumulation of ROS and apoptotic damage indicated ROS participation in this process7. However, the identity of redox complex implicated in this process or the molecular targets of ROS activity and the interactions with Bcl-2 family proteins are poorly characterized.

Our experiments have identified a role for the phagocytic-type NADPH oxidase (phox) complex in

activated T-cell apoptosis. NADPH oxidase is a multicomponent enzyme system comprising two membrane associated proteins (gp91phox and p22 phox); three soluble proteins – p67phox, p47phox, p40phox – present in a heterotrimeric complex and Rac, a small GTPase. We explored the biochemical and molecular links to apoptosis using mice with a genetic deficiency of the catalytic subunit gp91phox. This approach, coupled with shRNA-mediated ablation of other subunits, positioned the phox complex as a key intermediate in the generation of ROS, regulation of pro-apoptotic Bcl-2 family intermediates and cellular damage triggered by neglect (Fig-3). Furthermore, activated Tgp91phox-/- cells demonstrated improved survival in vivo and consistent with defects in post- activation apoptosis, memory T-cell response to antigens were elevated in null mice compared to their wild-type or heterozygous litter mates8.   Thus, by impinging on mitochondrial integrity the phox complex may serve as a crucial intermediate in the targeted deletion of activated T-cells, a step that is critical for the maintenance of T-cell homeostasis. Ongoing experiments are exploring the spatio-temporal dynamics and cytokine-dependence of this signaling cascade in T-cells.

References:

1.    Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Science 284:770-776.
2.    Sade H, Krishna S, Sarin A (2004) J Biol Chem 279:2937-2944.
3.    Ciofani M, Zuniga-Pflucker JC (2005) Nat Immunol 6:881-888.
4.    Androutsellis-Theotokis A et al. (2006) Nature 442:823-826.
5.    Perumalsamy LR et al. (2009) Cell Death Differ 16:879-889.
6.    Lindsten, T et al. Molecular Cell 6, 1389-1399.
7.    Hildeman, DA et al. (1999). Immunity 10, 735-744.
8.    P. Divya & Sarin A (2009) J. Exp. Med. 206, 1515-1523

Faculty