Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia

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.

FIGURE 1.

Regulation of mitochondrial mass and respiration by HIF-1 ex vivo and in vivo. A, the ratio of mitochondrial:nuclear DNA was determined by quantitative real-time PCR in wild type (WT) and Hif1a^-/- (KO) MEFs exposed to 20 or 1% O₂ for 48 h and normalized to the results obtained for WT cells at 20% O₂. Mean values are shown (±S.E.). ^*, p < 0.05 by Student's t test compared with WT MEFs at 20% O₂;#, p < 0.05 compared with WT MEFs at 1% O₂. B, WT and KO MEFs were exposed to 20 or 1% O₂ for 48 h. Equal numbers of cells were stained with nonyl acridine orange (NAO) and analyzed by flow cytometry to measure mitochondrial mass. C and D, O₂ consumption (C) and ATP levels (D) were measured in WT and KO MEFs exposed to 20 or 1% O₂ for 48 h and normalized to the results obtained for WT MEFs at 20% O₂. Mean values are shown (±S.E.). ^*, p < 0.05 by Student's t test compared with WT MEFs at 20% O₂;#, p < 0.05 compared with WT MEFs at 1% O₂. E, WT and KO MEFs were exposed to 20 or 1% O₂ for 48 h. Equal numbers of cells were stained with ER-Tracker and analyzed by flow cytometry to measure endoplasmic reticulum mass. F, WT and KO MEFs were transduced with empty retroviral vector (EV) or vector encoding constitutively active HIF-1α (CA5). After 3 days the ratio of mitochondrial:nuclear DNA was determined. Mean values are shown (±S.E.). ^*, p < 0.05 for indicated comparison. G and H, DNA was isolated from lungs of WT and Hif1a^+/- HIF-1α-HET littermate mice (G) or Arnt^flox/flox HIF-1β-conditional-knock-out mice that were either transgenic (Cre⁺) or non-transgenic (Cre^-) for Tie2-Cre (H). The ratio of mitochondrial: nuclear DNA was determined by real-time PCR and normalized to the results obtained for WT (G) or Cre^- (H) mice. ^*, mean (± S.E., n = 3) that is significantly different from WT or Cre^-.

FIGURE 2.

HIF-1-dependent induction of BNIP3 expression in hypoxic MEFs. A, BNIP3 mRNA was measured by quantitative real-time RT-PCR in WT and KO MEFs exposed to 20 or 1% O₂ for 24 h. Mean values (±S.E.) are shown. ^*, p < 0.05 by Student's t test compared with WT MEFs at 20% O₂;#, p < 0.05 compared with WT MEFs at 1% O₂. B, BNIP3 and β-actin protein expression was measured by immunoblot assay using lysates from WT and KO MEFs exposed to 20 or 1% O₂ for 48 h. C and D, BNIP3 mRNA (C) and protein (D) expression were analyzed in WT and HET mouse lung tissues. ^*, mean (±S.E., n = 3) that is significantly different from WT. Anti-tubulin immunoblot assay was performed to confirm equal protein loading. E and F, BNIP3 mRNA (E) and protein (F) levels were analyzed in Cre^- and Cre⁺ mouse lung tissues. ^*, mean (±S.E., n = 3) that is significantly different from Cre^-. Tubulin served as loading control. NS indicates a nonspecific band.

FIGURE 3.

Effect of BNIP3 loss-of-function on mitochondrial mass and respiration in WT and KO MEFs. A and B, quantitative real-time RT-PCR (A) and immunoblot analysis (B) showed down-regulation of BNIP3 mRNA and protein, respectively, by short hairpin RNAs sh80 and sh82 in cells incubated at 20 or 1% O₂ for 24 h (A) or 48 h (B). β-Actin blot showed equal protein loading. NS, nonspecific band. C, D, E, and F, mitochondrial DNA content (C), mitochondrial mass (D), O₂ consumption (E), and ATP levels (F) were measured in MEF subclones that were stably transfected with EV or vector encoding sh80 or sh82 and cultured at 20 or 1% O₂ for 48 h. Data are presented as the mean (±S.E.). ^*, p < 0.05 by Student's t test compared with WT-EV MEFs at 20% O₂;#, p < 0.05 compared with WT-EV MEFs at 1% O₂.

FIGURE 4.

Effect of BNIP3 gain-of-function on mitochondrial mass and respiration in WT and KO MEFs. A, immunoblot analysis showed expression of BNIP3 protein in KO-BNIP3 MEFs cultured at 20 or 1% O₂ for 48 h. β-Actin served as a loading control. NS, nonspecific band. Mitochondrial DNA content (B), mitochondrial mass (C), O₂ consumption (D), and ATP levels (E) were measured in WT-EV, KO-EV, and KO-BNIP3 MEFs cultured at 20 or 1% O₂ for 48 h. Data are presented as the mean (±S.E.). ^*, p < 0.05 by Student's t test compared with WT-EV MEFs at 20% O₂;#, p < 0.05 for the indicated comparison (bent lines).

FIGURE 5.

Beclin-1 is required for HIF-1-dependent regulation of mitochondrial mass and respiration in MEFs. Subclones of WT and KO MEFs expressing short hairpin RNA directed against Beclin-1 (shBeclin) or a scrambled negative control (SNC) were cultured at 20 or 1% O₂ for 48 h. Mitochondrial DNA content (A), mitochondrial mass (B), O₂ consumption (C), and ATP levels (D) were measured. Mean values are shown (±S.E.). ^*, p < 0.05 by Student's t test compared with WT-SNC at 20% O₂;#, p < 0.05 compared with WT-SNC at 1% O₂.

FIGURE 6.

Atg5 is required for HIF-1-dependent regulation of mitochondrial mass and respiration in MEFs. WT and KO MEFs were transfected with siRNA directed against Atg5 (siAtg5) or a control siRNA (siCTR) and cultured at 20 or 1% O₂ for 48 h. Mitochondrial DNA content (A), mitochondrial mass (B), O₂ consumption (C), and ATP levels (D) were measured. Mean values are shown (±S.E.). ^*, p < 0.05 by Student's t test compared with WT-siCTR at 20% O₂;#, p < 0.05 compared with WT-siCTR at 1% O₂.

FIGURE 7.

HIF-1 activates BNIP3-, Beclin-1-, and Atg5-dependent autophagy in hypoxic MEFs. A, WT and KO MEFs were incubated at 20 or 1% O₂ for 48 h, and whole cell lysates were subjected to immunoblot assay using an anti-LC3 antibody. B, WT and KO MEFs were transiently transfected with vector encoding GFP or GFP-LC3, incubated at 20% or 1% O₂, and analyzed by fluorescence microscopy. C, KO-EV MEFs and WT MEF subclones, which were stably transfected with EV or vector expressing short hairpin RNA directed against BNIP3 (sh82), were transiently transfected with vector GFP-LC3, cultured at 20 or 1% O₂, and analyzed by fluorescence microscopy. The percentage of cells exhibiting punctate fluorescence was calculated relative to all GFP-positive cells. Mean data (±S.E.) are shown. ^*, p < 0.05 compared with GFP-LC3-transfected WT-EV MEFs at 20% O₂;#, p < 0.05 compared with GFP-LC3-transfected WT-EV MEFs at 1% O₂. D, the percentage of cells with punctuate GFP-LC3 fluorescence was calculated relative to all fluorescent cells in WT-EV, KO-EV, and KO-BNIP3 MEF subclones. ^*, p < 0.05 compared with WT-EV at 20% O₂. E, the percentage of cells with punctuate GFP-LC3 fluorescence was calculated in WT MEF subclones expressing short hairpin RNA directed against Beclin1 (shBeclin) or a SNC. Mean data (±S.E.) are shown. ^*, p < 0.05 compared with WT-SNC at 20% O₂;#, p < 0.05 compared with WT-SNC at 1% O₂. F, the percentage of cells with punctuate GFP-LC3 fluorescence was calculated in WT MEF subclones expressing small interfering RNA against Atg5 (siAtg5) or a negative control siRNA (siCTR). Mean data (±S.E.) are shown. ^*, p < 0.05 compared with WT-siCTR at 20% O₂;#, p < 0.05 compared with WT-siCTR at 1% O₂.

FIGURE 8.

BNIP3 competes with Beclin-1 for binding to Bcl2. A, MEFs were exposed to 20 or 1% O₂ for 48 h, whole cell lysates (WCL) were prepared, and aliquots were subjected to direct immunoblot assays (IB, left panel) or after immunoprecipitation (IP) with anti-Bcl2 antibody (right panel). B and C, MEFs stably transfected with EV or vector encoding Bcl2 were exposed to 20 or 1% O₂ for 48 h and analyzed for mitochondrial:nuclear DNA ratio (B) or punctate fluorescence of GFP-LC3 (C). Mean data (±S.E.) are shown. ^*, p < 0.05 compared with WT-EV at 20% O₂;#, p < 0.05 compared with WT-EV at 1% O₂.

FIGURE 9.

Protective effect of HIF-1/BNIP3/Beclin/Atg6-induced autophagy in hypoxic cells. A, B, C, D, and E, the indicated MEF subclones were cultured at 20 or 1% O₂ for 48 h, and the number of dead cells as a percentage of total cell number was determined by trypan blue staining. Mean data (±S.E.) are shown. ^*, p < 0.05 by Student's t test compared with the control WT MEF subclone in the first column of each bar graph. #, p < 0.05 for indicated comparison (A and B) or compared with WT-SNC (C), siCTR (D), or WT-EV (E) at 1% O₂. F, MEFs were cultured at 20 or 1% O₂ for 48 h and then incubated with 7-AAD and phosphatidylethanolamine-labeled anti-annexin V antibody for flow cytometric analysis of apoptosis. The percentage (mean ± S.E.) of annexin⁺/7-AAD^- cells are shown.

FIGURE 10.

Analysis of ROS levels. Equal numbers of the indicated MEF subclones were cultured at 20 or 1% O₂ for 48 h and stained with 1 μm dichlorodihydrofluorescein diacetate, and oxidative metabolism to dichlorofluorescein (DCF) was determined by flow cytometry.

FIGURE 11.

ROS scavenger rescues HIF-1α-deficient MEFs from hypoxia-induced cell death. MEFs were exposed to 20 or 1% O₂ for 48 h in the presence of 25 μm MnTMPyP, a superoxide dismutase mimetic, or vehicle control (CTR). A, ROS levels were quantified by dichlorofluorescein (DCF) fluorescence. B, percent cell death (mean ± S.E.) was quantified by trypan blue staining. ^*, p < 0.05 by Student's t test compared with WT-CTR at 20% O₂;#, p < 0.05 compared with WT-CTR at 1% O₂; ^**, p < 0.05 for indicated comparison. C, molecular pathway regulating mitochondrial autophagy, cell respiration, ROS levels, and cell survival in MEFs subjected to prolonged hypoxia.