An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.

The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1-PHD3) initiate oxygen-dependent degradation of HIF-alpha-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1alpha in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5' of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3'-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.

The transcription factor hypoxia-inducible factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published regarding the role of the mitochondrial electron transport chain (ETC) in the regulation of HIF-1␣. We assessed cellular hypoxia by pimonidazole staining and blotting of the O 2 -labile HIF-1 ␣-subunit in human osteosarcoma cell cultures (U2OS and 143B). In conventional, gas-impermeable cell culture dishes, ETC inhibitors had no effect on pimonidazole staining or HIF-1␣ abundance in a 20% O 2 atmosphere; both parameters were undetectable. Pimonidazole staining and HIF activity were substantial in 0.1% O 2 irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O 2 ) pimonidazole staining and HIF-␣ expression were detectable but strongly reduced after ETC inhibition in conventional cell cultures. All effects of ETC inhibition on HIF-1␣ regulation were eliminated in gas-permeable IntroductionAdaptation to low oxygen concentration leads to a series of responses through the transactivation of more than 70 target genes 1 mediated by the hypoxia-inducible factor-1 (HIF-1). The hematopoietic growth factor erythropoietin (EPO) and the angiogenic peptide vascular endothelial growth factor (VEGF) are the most prominent examples of HIF-1 target genes. Actually, the HIF binding site embedded in the 3Ј enhancer of the EPO gene allowed purification and characterization of HIF-1. 2 HIF is also known to be involved in angiogenesis, tumor growth, and apoptosis, 3,4 which makes it a challenging target for therapeutic manipulation. HIF-1 is a heterodimeric transcription factor composed of 2 subunits. 2 Each HIF-1 subunit contains an N-terminal basic helix-loop-helix domain responsible for dimerization and DNA binding followed by a PAS domain (Per, aryl hydrocarbon nuclear translocator [ARNT], and Sim were the first members of this protein family) that is involved in protein interactions. 5,6 Both subunits are constitutively expressed. While the ␤-subunit is present in the nucleus at detectable levels under normoxia and hypoxia, HIF-1␣ levels are affected by changes in the cellular oxygen partial pressure (pO 2 ). 7,8 In hypoxia, HIF-1␣ accumulates and is translocated to the nucleus where it dimerizes with HIF-1␤, forming an active DNA-binding complex (reviewed by Semenza 1 ). In contrast, atmospheric pO 2 leads to a rapid destruction by the ubiquitin-proteasome system. 9,10 Initially, HIF-1␣ is hydroxylated at Pro564 and Pro402 by 2-oxoglutarate-dependent dioxygenases, 11,12 which have been termed "prolyl hydroxylase domain," containing protein 1, 2, and 3 (PHD-1, 2, 3) 13 or HIF-1␣ prolyl hydroxylase 3, 2, and 1, 14 which prevent the stabilization. Furthermore, HIF-1␣ is hydroxylated at Asn803 by an enzyme first identified as "factor inhibiting HIF-1" (FIH-1), 15 which abrogates the transactivation by preventing the binding of transcriptional coactivators such as cyclic adenosine monophosphate response...

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