Cyclosporin A Prevents the Hypoxic Adaptation by Activating Hypoxia-inducible Factor-1α Pro-564 Hydroxylation

Abstract

Cells respond to low oxygen tensions by up-regulating the expression of genes involved in angiogenesis (e.g. vascular endothelial growth factor), erythropoiesis (e.g. erythropoietin), and glycolysis. The transcriptional activation of target genes is induced by a common transcription factor, hypoxia-inducible factor-1 (HIF-1). 1 HIF-1 was first identified as a heterodimeric transactivator that recognizes a specific DNA sequence, termed hypoxia-responsive element (HRE) in the 3Ј-untranslated region of the erythropoietin gene (1). HIF-1 consists of two subunits, HIF-1␣ and the aryl hydrocarbon receptor nuclear translocator, both of which belong to the large family of basic helixloop-helix-per-arnt-sim transcription factors (2, 3). Under normoxic conditions, HIF-1␣ subunits are unstable, being rapidly targeted to the ubiquitin-proteasome pathway. Degradation is mediated by a ubiquitin-protein isopeptide ligase (E3) complex, in which the von Hippel-Lindau protein (vHL) binds to a specific hydroxylated proline residue (Pro-564) within the oxygen-dependent degradation (ODD) domain of HIF-1␣ (4, 5). A major action of hypoxia is to suppress prolyl hydroxylation and degradation of HIF-1␣ by the proteasome. As a consequence, the protein accumulates, migrates to the nucleus, and associates with the aryl hydrocarbon receptor nuclear translocator, and the complex interacts with the HRE of target genes (6, 7). The importance of this mechanism is ascertained by the facts that vessel formation is hampered in HIF-1␣ Ϫ/Ϫ knock out mice (8) and that mutations in the vHL gene cause hereditary cancer syndrome associated with dysregulated angiogenesis and up-regulation of hypoxia-sensitive genes.Cyclosporin A (CsA) is a potent immunosuppressive agent used after organ transplantation and in the treatment of several autoimmune diseases. CsA is well known to be nephrotoxic probably as a consequence of the constriction of renal vessels and of the resulting hypoxia. Circumstantial evidence further suggests that CsA interferes with the hypoxic signaling cascade. (i) Maruyama et al. (9) reported that CsA inhibited vascular endothelial growth factor production by human lymphocytes. (ii) CsA inhibits erythropoietin production in anemic mice (10). (iii) Kang et al. (11) reported that vascular endothelial growth factor reverses some of the post-CsA-mediated hypertension and nephropathy in the rat. (iv) Erythropoietin deficiency has been proposed as a cause of the anemia observed in children following cardiac transplantation (12). These would suggest that CsA inhibits the cellular responses that are mediated by HIF-1␣ and HRE.In the present study, we examine the possible influence of CsA on the hypoxic signaling. Results show that CsA inhibits hypoxia-induced gene transcription by preventing hypoxia-induced and ODD-dependent stabilization of HIF-1␣. We also show that CsA stimulates a kidney prolyl hydroxylase activity that specifically modifies Pro-564 in the ODD domain of HIF-1␣. MATERIALS AND METHODSMaterials-Culture medium, restriction, and ...

Hypoxia-Inducible Factor-1-Dependent and -Independent Regulation of Insulin-Like Growth Factor-1-Stimulated Vascular Endothelial Growth Factor Secretion

“…To further address this possibility and determine whether IGF-1-induced VEGF expression was solely HIF-1-dependent, we used CsA as an inhibitor of Hif-1␣ stability (D'Angelo et al, 2003). We observed that CsA treatment inhibited IGF-1-stimulated Hif-1␣ protein expression and HIF-1 reporter activity, at the same time only partially reducing VEGF secretion.…”

“…CsA is a potent immunosuppressive agent that destabilizes Hif-1␣ by promoting hydroxylation of Pro-402 and Pro-564 within its oxygen-dependent degradation domain (D'Angelo et al, 2003). IGF-1 and CoCl 2 treatment each increased Hif-1␣ protein expression ( Fig.…”

The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway

“…However, it has been reported that infusion of CsA, a calcineurin/NFAT inhibitor, in rats during MCT-induced PAH decreases PA pressure and medial hypertrophy (4), which contradicts our observation that phenamil attenuates hypoxia-induced PAH and vascular remodeling by activating NFAT. It is known that CsA also inhibits the activity of hypoxia-inducible factor 1␣ (HIF-1␣), a transcription factor involved in hypoxia-mediated gene regulation (10). Thus, the therapeutic effect of CsA in a PAH animal model might be FIG.…”

Chrysin inhibits expression of hypoxia-inducible factor-1α through reducing hypoxia-inducible factor-1α stability and inhibiting its protein synthesis

“…The pCMV-ODD-GFP vector (encoding ODD-GFP) and pCMV-GFP are kind gifts from Dr. D'Angelo (Institut de Pharmacologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, France; ref. 30). The ODD fragment corresponds to amino acids 530 to 603 of HIF-1a.…”