Vhlh Gene Deletion Induces Hif-1-Mediated Cell Death in Thymocytes

Abstract

The von Hippel-Lindau gene product (pVHL) targets the α subunit of basic helix-loop-helix transcription factor hypoxia-inducible factor (HIF) for proteasomal degradation. Inactivation of pVhl in the mouse germ line results in embryonic lethality, indicating that tight control of Hif-mediated adaptive responses to hypoxia is required for normal development and tissue function. In order to investigate the role of pVhl in T-cell development, we generated mice with thymocyte-specific inactivation of Vhlh resulting in constitutive transcriptional activity of Hif-1, as well as mice with thymocyte-specific repression of Hif-1 in a wild-type and Vhlh-deficient background. Thymi from Vhlh-deficient mice were small due to a severe reduction in the total number of CD4/CD8-double-positive thymocytes which was associated with increased apoptosis in vivo and in vitro. Increased apoptosis was a result of enhanced caspase 8 activity, while Bcl-2 and Bcl-X_L transgene expression had little effect on this phenotype. Inactivation of Hif-1 in Vhlh-deficient thymocytes restored thymic cellularity as well as thymocyte viability in vitro. Our data suggest that tight regulation of Hif-1 via pVhl is required for normal thymocyte development and viability and that an increase in Hif-1 transcriptional activity enhances caspase 8-mediated apoptosis in thymocytes.

SUPPLEMENTAL MATERIAL

This work was supported by a grant from the American Heart Association (0365342U) and in part by grants from the NIH (R03-DK-62060), the Charlotte Geyer Foundation, and seed money from the Department of Medicine, University of Pennsylvania (all to V.H.H.).

We are grateful to the members of the Morphology Core, in particular Gary Swain, Penn Center for Molecular Studies in Digestive and Liver Disease (P30-DK50306), for help with immunohistochemistry and image analysis. We thank Celeste Simon, Debra Higgins, and Erinn Bruno for critical reading of the manuscript.