title: Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation.
abstract: The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug.
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, 660 route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France. dangelo@ipmc.cnrs.fr
authors.forename: Gisela
authors.initials: G
authors.lastname: D'Angelo
authors.forename: Eric
authors.initials: E
authors.lastname: Duplan
authors.forename: Paul
authors.initials: P
authors.lastname: Vigne
authors.forename: Christian
authors.initials: C
authors.lastname: Frelin
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects* / Q000235:genetics; D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D000860:Hypoxia / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007668:Kidney / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014157:Transcription Factors; D016572:Cyclosporine; D011392:Proline; D011393:Procollagen-Proline Dioxygenase