| Hypoxia is a reduction in the normal level of oxygen tension and occurs during acute and chronic vascular disease, pulmonary disease, and cancer. The key transcription factor that regulates the cellular responses to hypoxia is hypoxia-inducible-factor-1 (HIF-1). Understanding how HIF-1 is regulated is fundamental for understanding the biology of tumor growth. My thesis demonstrates two novel signaling pathways that regulate HIF-1. First, I demonstrate that p38alpha-/- cells fail to activate HIF-1 under hypoxic conditions. Cells deficient in Mkk3 and Mkk6, the upstream regulators of p38alpha, also fail to activate HIF-1 under hypoxic conditions. Furthermore, the hypoxic activation of p38alpha and HIF-1 was abolished by myxothiazol, a mitochondrial complex III inhibitor, and glutathione peroxidase 1 (GPX1), a scavenger of hydrogen peroxide. Thus, the activation of p38alpha and HIF-1 is dependent on the generation of mitochondrial reactive oxygen species (ROS). These results provide genetic evidence that the p38 MAPK signaling pathway is essential for HIF-1 activation. To further elucidate the direct targets of the ROS or the upstream regulators of p38 MAPK pathway a candidate approach was taken. Interestingly, the hypoxic activation of HIF-1 is independent of SRC, ASK1, and AMPK.;The tumor suppressor PTEN is mutated or deleted in many tumors, causing the activation of the PI3K pathway. Here, I show that the loss of PTEN increases the transcriptional activity of HIF-1 independent of mTOR signaling, but dependent on the inactivation of Forkhead transcription factors (FOXO) in PTEN null cells. Reintroduction of PTEN in the nucleus, overexpression of a non-phosphorylatable FOXO3a, which accumulates in the nucleus, or inhibition of nuclear export of FOXO3a by leptomycin B repress HIF-1 transcriptional activity in PTEN null cells. PTEN and FOXO3a regulate the transactivation domain of HIF-1alpha. Co-immunoprecipitation shows that endogenous FOXO3a can associate with HIF-1alpha and p300 in the nucleus. Chromatin immunoprecipitation indicates that FOXO3a complexes with HIF-1alpha and p300 on the Glut-1 promoter, a HIF-1 target gene. Overexpression of p300 reverses FOXO3a mediated repression of HIF-1 transcriptional activity. Thus, FOXO3a negatively regulates HIF-1 transcriptional activity by interfering with p300's ability to function as a transcriptional co-factor. |
