Hypoxia inducible factor-1alpha regulates apoptosis and T cell receptor signaling in thymocytes
ABSTRACT Hypoxia occurs in normal, healthy tissues as well as during a wide variety of pathological events, and lymphocytes must encounter and adapt to varying levels of oxygen tension throughout their life cycle. T lineage lymphocytes experience hypoxia in vivo during their development in the thymus, where selection of the mature T cell repertoire occurs and signals received through the T cell receptor (TCR) determine the survival or apoptotic deletion of individual thymocytes. Furthermore, mature T cells also experience hypoxia in peripheral tissues where their activation via the TCR is central to adaptive immunity. Von Hippel-Lindau protein (pVHL) causes the rapid polyubiquitination and degradation of the transcription factor Hypoxia Inducible Factor-1α (HIF-1α) under normoxic conditions but permits its stabilization under hypoxic conditions. In this highly conserved pathway, HIF-1α induces adaptation to hypoxia through transactivation of genes that influence angiogenesis, erythropoiesis, cellular metabolism, proliferation, and apoptosis. We have utilized a murine genetic model with conditional thymic deletion of Vhl (Vhl-/- ) to observe the effects of constitutive Hif-1α stabilization on apoptosis and TCR signaling. Furthermore, conditional deletion of both Vhl and Hif-1 α (Vhl-/- , Hif-1 α-/- ) allowed a clear delineation of the role of Hif-1α in this model. We observed an ∼80% reduction in thymic cellularity of Vhl -/- mice accompanied by increased apoptosis in vivo and in vitro , and these phenotypes were clearly reversed in Vhl ,-/-HASH(0x28c07d24)Hif-1 α-/- mice. Hif-1α dependent apoptosis was not rescued by overexpression of Bcl-2 or Bcl-xL , but was dependent upon caspase 8. Since TCR signaling and apoptosis are closely linked in T lymphocytes, we examined the effect of Hif-1α stabilization on a key element of TCR-mediated activation, Ca2+ signaling. Constitutive stabilization of Hif-1α caused substantially diminished TCR-mediated Ca2+ mobilization. TCR-proximal tyrosine phosphorylation events and intracellular Ca2+ store operated Ca2+ entry across the plasma membrane were unaffected in Vhl -/- cells. We observed that accelerated clearance of cytoplasmic Ca2+ into ER and mitochondrial compartments contributes to Hif-mediated blunted Ca2+ mobilization. Consistent with this finding, Hif-1α stabilization dramatically increased protein expression of the ER Ca2+ pump, Serca2. Our findings suggest that Hif-1α may modulate T lymphocyte survival and activation under conditions of tissue hypoxia.