The Mitochondrial Respiratory Chain Is Required for Organismal Adaptation to Hypoxia

Abstract

Hypoxia-inducible factors (HIFs) are crucial for cellular and organismal adaptation to hypoxia. The mitochondrial respiratory chain is the largest consumer of oxygen in most mammalian cells; however, it is unknown whether the respiratory chain is necessary for in vivo activation of HIFs and organismal adaptation to hypoxia. HIF-1 activation in the epidermis has been shown to be a key regulator of the organismal response to hypoxic conditions, including renal production of erythropoietin (Epo). Therefore, we conditionally deleted expression of TFAM in mouse epidermal keratinocytes. TFAM is required for maintenance of the mitochondrial genome, and TFAM-null cells are respiratory deficient. TFAM loss in epidermal keratinocytes reduced epidermal levels of HIF-1α protein and diminished the hypoxic induction of HIF-dependent transcription in epidermis. Furthermore, epidermal TFAM deficiency impaired hypoxic induction of renal Epo expression. Our results demonstrate that the mitochondrial respiratory chain is essential for in vivo HIF activation and organismal adaptation to hypoxia.

Figure 1

Mitochondrial generation of ROS is required for hypoxic induction of HIF-1α protein and target gene expression in mouse epidermal keratinocytes. (A) Representative Western blot analysis of HIF-1α and TFAM protein levels in primary mouse keratinocytes isolated from wild-type and TFAM EpiKO mice. Cells were exposed to normoxia (21% O₂) or hypoxia (1.5% O₂) for 4 hours. (B) Real Time PCR analysis of HIF-1α and TFAM mRNA expression in primary mouse keratinocytes isolated from wild-type and TFAM EpiKO mice. (C) Real Time PCR analysis of PGK1, CAIX, Pai1, VEGF, and BNIP3 mRNA expression in primary mouse keratinocytes isolated from wild-type and TFAM EpiKO mice. Cells were exposed to normoxia or hypoxia for 4 hours. (D) Representative Western blot analysis of HIF-1α protein levels in primary wild-type mouse keratinocytes after exposure to normoxia or hypoxia for 4 hours. Cells were treated with mitochondria-targeted vitamin E (MVE) or the control compound methyl-triphenylphosphonium (TPP; mitochondria-targeting moiety lacking antioxidant activity). (E) Representative Western blot analysis of HIF-1α protein levels in TFAM EpiKO primary mouse keratinocytes after exposure to normoxia, hypoxia, or 2,3-dimethoxy-4-naphthoquinone (DMNQ) for 4 hours. Charts represent means +/− SEM. N=4 independent keratinocyte preparations per genotype. mRNA expression was normalized to RPL19 levels. Significance was determined by (B) Students T-test or (C) one way ANOVA using Bonferonni’s post-test.

Figure 2

Hypoxic induction of HIF-1α protein and target gene expression is inhibited in the epidermis of TFAM EpiKO mice. (A) Representative immunohistochemical analysis of HIF-1α protein expression in the epidermis of wild-type and TFAM EpiKO mice. (B) Real Time PCR analysis of HIF-1α and TFAM mRNA expression in the epidermis of wild-type and TFAM EpiKO mice. (C) Representative Western blot analysis of HIF-1α and TFAM protein levels in epidermal lysates prepared from wild-type and TFAM EpiKO mice. Mice were exposed to normoxia (21% O₂) or hypoxia (9% O₂) for 16 hours. (D) Densitometric quantification of (C). HIF-1α protein levels were normalized to β-actin levels. (E) Real Time PCR analysis of PGK1, CAIX, Pai1, VEGF, and BNIP3 mRNA expression in epidermal lysates prepared from wild-type and TFAM EpiKO mice. Mice were exposed to normoxia or hypoxia for 16 hours. Charts represent means +/− SEM. N=5 mice per genotype per condition. mRNA expression was normalized to RPL19 levels. Significance was determined by one way ANOVA using Bonferonni’s post-test.

Figure 3

Hypoxic induction of HIF-1α target genes and erythropoietin is inhibited in the kidneys of TFAM EpiKO mice. (A) Real Time PCR analysis of Epo mRNA expression in renal lysates prepared from wild-type and TFAM EpiKO mice. Mice were exposed to normoxia or hypoxia for 16 hours. (B) Epo protein concentrations in the serum of wild-type and TFAM EpiKO mice as determined by ELISA. (C, D) Real Time PCR analysis of (C) HIF-1α, HIF-2α, or (D) TFAM mRNA expression in the kidney of wild-type and TFAM EpiKO mice. (E) Real Time PCR analysis of CTGF, Bnip3, Glut1, Pai1, and PGK1 mRNA expression in the kidneys of wild-type and TFAM EpiKO mice exposed to normoxia (21% O₂) or hypoxia (9% O₂) for 16 hours. (F) Real Time PCR analysis of Epo mRNA expression in the kidneys of wild-type and TFAM EpiKO mice exposed to epidermal application of either mustard seed oil (Allyl isothiocyanate) or EtOH as vehicle control. Charts represent means +/− SEM. mRNA expression was normalized to RPL19 levels. (A–E) N=5, (F) N=3 mice per genotype per condition. Significance was determined by one way ANOVA using Bonferonni’s post-test.

Figure 4

Deletion of VHL is sufficient to induce HIF-1α target gene expression in the epidermis of TFAM EpiKO mice. (A) Representative images of wild-type (KRT14-Cre⁻), TFAM EpiKO, VHL EpiKO, and TFAM+VHL EpiKO mice. (B–C) Real Time PCR analysis of (B) TFAM and VHL and (C) PGK1, CAIX, Pai1, VEGF, and BNIP3 mRNA expression in the epidermis of wild-type (KRT14-Cre⁻), TFAM EpiKO, VHL EpiKO, and TFAM+VHL EpiKO mice. (D) Real Time PCR analysis of Epo mRNA expression in the kidneys of wild-type (KRT14-Cre⁻), TFAM EpiKO, VHL EpiKO, and TFAM+VHL EpiKO mice. Charts represent means +/− SEM. mRNA expression was normalized to RPL19 levels. N=5 mice per genotype. Significance was determined by one way ANOVA using Dunnett’s post-test.