ABSTRACT
Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.
Subject(s)
Autophagy , Hypoxia-Inducible Factor 1/metabolism , Hypoxia , Mitochondria/metabolism , Animals , Apoptosis Regulatory Proteins , Autophagy-Related Protein 5 , Beclin-1 , Cell Death , Cytoplasm/metabolism , Membrane Proteins/biosynthesis , Mice , Mice, Knockout , Microtubule-Associated Proteins/biosynthesis , Mitochondrial Proteins/biosynthesis , Models, Biological , Molecular Conformation , Proteins/metabolism , Reactive Oxygen SpeciesABSTRACT
Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1alpha gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.
Subject(s)
Aging/metabolism , Cell Movement/physiology , Hypoxia-Inducible Factor 1/metabolism , Ischemia/genetics , Ischemia/therapy , Lower Extremity/blood supply , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/therapy , Aging/genetics , Aging/pathology , Animals , Cell Movement/genetics , Cells, Cultured , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/therapeutic use , Ischemia/metabolism , Ischemia/pathology , Lower Extremity/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Neovascularization, Pathologic/metabolism , Reperfusion/methodsABSTRACT
Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1alpha subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O(2)-dependent prolyl hydroxylation of HIF-1alpha is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1alpha and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1alpha even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1alpha, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1alpha in an O(2)-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1alpha and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha.
Subject(s)
Acetyltransferases/physiology , GTP-Binding Proteins/chemistry , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neoplasm Proteins/chemistry , Receptors, Cell Surface/chemistry , Ubiquitin/chemistry , Acetyltransferases/metabolism , Elongin , HSP90 Heat-Shock Proteins/metabolism , Humans , Hypoxia , Models, Biological , Oxygen/metabolism , Protein Binding , Protein Interaction Mapping , RNA, Small Interfering/metabolism , Receptors for Activated C Kinase , Transcription Factors/chemistry , Two-Hybrid System TechniquesABSTRACT
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that functions as a master regulator of oxygen homeostasis. The HIF-1alpha subunit is subjected to O(2)-dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N(1)-acetyltransferase (SSAT) 2 plays an essential role in this process. SSAT2 binds to HIF-1alpha, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1alpha by stabilizing the interaction of VHL and Elongin C. Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1alpha that is observed in oxygenated cells.
