IL-7, a cytokine produced by thymic stromal cells, has been shown to be essential for normal T cell development in humans and mice. Our goal is to understand the basis of this IL-7 requirement. We have shown that IL-7 supports VDJ recombination in several different ways: IL-7 induces expression of the Rag proteins (which cleave DNA), it acts as a trophic factor for pro-T cells, protecting them from cell death during the VDJ recombination events, and for some loci, IL-7 signals the opening of chromatin, rendering the locus accessible to cleavage. The trophic survival effect of IL-7 is a major area of current interest. Understanding the trophic activity of IL-7 may shed light on a general mechanism for the action of trophic factors and in fact we have extended several of our findings to the trophic effects of IL-3. We initially observed that IL-7 induced the synthesis of Bcl-2 family members that protect mitochondria. We now find that mitochondria are actively attacked by the death protein Bax after IL-7 withdrawal. In the presence of IL-7, Bax is located in cytosol. Six hours after IL-7 withdrawal, Bax is activated via a conformational change that exposes a hydrophobic region, triggering Bax to associate with mitochondrial membrane. The Bax conformational change in turn is a result of a sharp rise in intracellular pH occurring after IL-7 withdrawal. This transient rise in pH (to over 8.0) apparently results from disregulation of membrane exchangers, primarily NHE1. Cytosolic alkalinization additionally causes hyperpolarization of mitochondria membranes through an effect of high pH on the FoF1 ATP synthase. Current efforts are to define the mechanisms upstream of the disregulation of NHE1. We have studied early signaling events following IL-7 withdrawal and identified the protein kinase Pyk2, which is associated with the receptor complex, as an important signal in maintaining survival. Another phosphoprotein, p200, is associated with the IL-7 receptor complex and efforts are underway to sequence this protein. We also found that following IL-7 withdrawal, the MAP kinase stress pathway is activated, and this will be evaluated as a potential trigger of NHE1. AIDS TITLE: Early Events in the Development of T Lymphocytes.
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