Under low oxygen tension, cells increase the transcription of specific genes that are involved in angiogenesis, erythropoiesis, and glycolysis. Hypoxia-induced gene expression primarily depends on the stabilization of the alpha-subunit of hypoxia-inducible factor-1 (HIF-1alpha), which acts as a heterodimeric trans-activator. Our results indicate that stabilization of HIF-1alpha protein by treatment of proteasome inhibitors, is not sufficient for hypoxia-induced gene activation, and an additional hypoxia-dependent modification is necessary for gene expression by HIF-1alpha. Here, we demonstrate that mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 does not change either the stabilization or DNA binding ability of HIF-1alpha but it inhibits the trans-activation ability of HIF-1alpha, thereby it reduces the hypoxia-induced transcription of both an endogenous target gene and a hypoxia-responsive reporter gene. We found that hypoxia induced p42/p44 mitogen-activated protein kinases (MAPKs) that are target protein kinases of MEK-1, and that expression of dominant-negative p42 and p44 MAPK mutants reduced HIF-1-dependent transcription of the hypoxia-responsive reporter gene. Our results are the first to identify that hypoxia-induced trans-activation ability of HIF-1alpha is regulated by different mechanisms than its stabilization and DNA binding, and that these processes can be experimentally dissociated. MEK-1/p42/p44 MAPK regulates the trans-activation, but not the stabilization or DNA binding ability, of HIF-1alpha.

Under low oxygen tension, cells increase the transcription of specific genes involved in angiogenesis, erythropoiesis, and glycolysis. Hypoxia-induced gene expression depends primarily on stabilization of the ␣ subunit of hypoxia-inducible factor-1 (HIF-1␣), which acts as a heterodimeric trans-activator with the nuclear protein known as the aryl hydrocarbon receptor nuclear translocator (Arnt). The resulting heterodimer (HIF-1␣/Arnt) interacts specifically with the hypoxia-responsive element (HRE), thereby increasing transcription of the genes under HRE control. Our results indicate that the 90-kDa heat-shock protein (Hsp90) inhibitor radicicol reduces the hypoxia-induced expression of both endogenous vascular endothelial growth factor (VEGF) and HRE-driven reporter plasmids. Radicicol treatment (0.5 g/ml) does not significantly change the stability of the HIF-1␣ protein and does not inhibit the nuclear localization of HIF-1␣. However, this dose of radicicol significantly reduces HRE binding by the HIF-1␣/Arnt heterodimer. Our results, the first to show that radicicol specifically inhibits the interaction between the HIF-1␣/Arnt heterodimer and HRE, suggest that Hsp90 modulates the conformation of the HIF-1␣/Arnt heterodimer, making it suitable for interaction with HRE. Furthermore, we demonstrate that radicicol reduces hypoxia-induced VEGF expression to decrease hypoxia-induced angiogenesis.Cells adapt to hypoxia by up-regulating the transcription of specific genes involved in angiogenesis, erythropoiesis, and glycolysis. Pathologically, tumor hypoxia contributes directly to enhanced glucose metabolism and angiogenesis, which are major features of malignant progression. The genes up-regulated during hypoxia include vascular endothelial growth factor (VEGF), erythropoietin, and several glycolytic enzymes. These diverse, targeted genes are induced by a common trans-activator, hypoxia-inducible factor 1 (HIF-1) (Iyer et al., 1998;Bruick and McKnight, 2001b;Semenza, 2002).HIF-1 was first identified as a heterodimeric trans-activator composed of two subunits, HIF-1␣ and -␤, both of which belong to the growing family of basic-helix-loop-helix-PAS (bHLH-PAS) proteins, including period (Per), Arnt, and single-minded (Sim). The bHLH-PAS proteins share common characteristics: first, a bHLH-PAS protein dimerizes with a specific partner protein through the HLH-PAS domain. Second, a partner such as the aryl hydrocarbon receptor (AhR) or HIF-1␣ is activated by specific stimuli (i.e., xenobiotics or low oxygen tension, respectively) before translocating to the nucleus, where it heterodimerizes with a partner protein. Alternatively, Arnt, another bHLH-PAS protein, is constitutively located in the nucleus and interacts with several

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