Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity

Abstract

The inducible form of Heme Oxygenase-1 (HO-1), a major endoplasmic reticulum (ER) associated heme protein, is known to play important roles in protection against oxidative and chemical stress by degrading free heme released from degradation of heme proteins. In this study we show that induced expression of HO-1 by subjecting macrophage RAW-264.7 cells to chemical or physiological hypoxia resulted in significant translocation of HO-1 protein to mitochondria. Transient transfection of COS-7 cells with cloned cDNA also resulted in mitochondrial translocation of HO-1. Deletion of N-terminal ER targeting domain increased mitochondrial translocation under the transient transfection conditions. Mitochondrial localization of both intact HO-1 and N-terminal truncated HO-1 caused loss of heme aa-3 and cytochrome c oxidase (CcO) activity in COS-7 cells. The truncated protein, which localizes to mitochondria at higher levels, induced substantially steeper loss of CcO activity and reduced heme aa3 content. Furthermore, cells expressing mitochondria targeted HO-1 also induced higher ROS production. Consistent with dysfunctional state of mitochondria induced by HO-1, the mitochondrial recruitment of autophagy markers LC-3 and Drp-1 was also increased in these cells. Chronic ethanol feeding in rats also caused an increase in mitochondrial HO-1 and decrease in CcO activity. These results show that as opposed to the protective effect of the ER associated HO-1, mitochondria targeted HO-1 under normoxic conditions induces mitochondrial dysfunction.

Keywords: Autophagy; CcO, cytochrome c oxidase; Cytochrome c Oxidase; DCFH-DA, Dichlorofluorescein diacetate; ER, Endoplasmic reticulum; HO-1, Heme Oxygenase-1; Heme aa3 content; Heme oxygenase-1; Mitochondrial targeting; NPR, NADPH cytochrome P 450 reductase; ROS production; ROS, Reactive Oxygen Species.

Graphical abstract

Fig. 1

Hypoxia and CoCl₂ induced HO-1 localizes to mitochondria. (A) RAW 264.7 cells were treated with CoCl₂ for 0–96 h. Whole cell lysates (50 μg each) were prepared and subjected to immunoblot analysis using HO-1 antibody. Actin served as loading control. (B). Mitochondria and microsomes were prepared from cells treated with CoCl₂ for 0, 12, 24 and 36 h. The proteins (50 μg each) were resolved on SDS-PAGE and the immunoblot was developed with antibody to HO-1 (1:1500 dilution). The blot was also co-developed with antibody to NPR (1:2500 dilution) to detect cross-contamination. (C) Mitochondrial and microsome proteins from RAW 264.7 cells exposed to hypoxia (1% O₂) for 0, 12 and 24 h were resolved on SDS-PAGE and probed for HO-1 expression. 50 μg protein was used in each case. The purity of mitochondrial isolates was assessed by reprobing the blot with microsomal specific marker, NPR. (D) Histogram represents the % subcellular distribution of HO-1 protein in the mitochondria and microsomes after hypoxia treatment.

Fig. 2

Immunocytochemical localization of HO-1 in mitochondria: (A) and (B) RAW 264.7 cells without treatment (A) and with 150 μM CoCl₂ (B) for 48 h were stained with antibody to mitochondria specific marker, Cco I and antibody to HO-1. The cells were subsequently incubated with Alexa 488-conjugated anti-rabbit antibody and Alexa 594-conjugated anti-mouse goat IgG for colocalization of fluorescence signals. Slides were examined by confocal microscopy through Leica TCS SP5 microscope.

Fig. 3

Mitochondrial targeting of HO-1 protein: (A) Cartoon depicts the targeting domains of WT and truncated (ΔN16 and ΔN33) HO-1 cDNA's. The cDNA were cloned in PCMV4 using Hind 3 and Xba I restriction sites at 5′ and 3′ termini, respectively. The N-terminal 16 and 33 amino acids were deleted in ΔN16 and ΔN33, respectively. The ++ and +++ annotations on the extreme right represent the arbitrary units of mitochondrial targeting efficiencies. Mitochondrial and microsomal proteins from cells transfected with Mock, WT and N-terminal deletion mutant constructs cDNA were resolved on SDS-PAGE and probed for HO-1 expression. The purity of the mitochondrial isolates was assessed by reprobing the blot with microsomal specific marker, NPR.

Fig. 4

Measurement of Cytochrome c oxidase activity and heme aa3 contents: (A) CcO activity was measured by incubating 10 μg of freeze-thawed mitochondrial extract from cells transfected with Mock, WT, ΔN16 and ΔN33 cDNA in 1 ml of assay medium (25 mM potassium phosphate, pH 7.4, containing 0.45 mM dodecyl maltoside and 15 μM reduced cytochrome c. The CcO activity was measured as described in the “Materials and methods”. (B) Mitochondrial proteins from mock, WT and ΔN16 transfected cells were solubilized in lauryl maltoside containing buffer and used for spectral analysis as described in the Materials and methods section. Difference spectra of reduced minus air oxidized samples were recorded in the range of 400–700 nm and heme aa3 contents were calculated also as described in the Materials and methods section. ^⁎⁎ represents statistical significance of p<0.05.

Fig. 5

ROS production by mitochondria targeted HO-1 (A) ROS levels in mock, WT, ΔN16 and ΔN33 transfected cells were measured using DCFH-DA substrate. 48 h post transfection, the media was aspirated and the cells were rinsed with 1X PBS. The cells were loaded with 15 μM DCFH DA for 15 min in dark to allow intracellular conversion of DCFH. At the end of incubation, cells were scraped off gently in 1 ml ice cold PBS. 2×10⁶ cells in 1 ml of PBS were incubated and fluorescence was recorded using LPS-220B spectroflourometer (Photon Technology International, Bermingham, NJ) with an excitation wavelength of 485 nm and emission wavelength of 535 nm (for 20 min). As controls, cells were also treated with membrane permeable SOD (300 U/ml), catalase (200 U/ml) and N-acetyl cysteine, NAC (25 mM). ^⁎⁎ indicates p<0.05.

Fig. 6

Intramitochondrial localization of HO-1: (A) Immunofluorescence microscopy was carried out with permeabilized Cos-7 cells transfected with WT, ΔN16 and ΔN33 cDNA's as described in the Materials and methods section. The cells were washed, blocked with 5% goat serum and incubated with primary HO-1 (anti-rabbit) antibody and mitochondria specific marker, CcO I (anti-mouse). The cells were subsequently incubated with Alexa 488-conjugated anti-rabbit antibody and Alexa 594-conjugated anti-mouse goat IgG for colocalization of fluorescence signals. (B) The transfected cells were also co-stained with mitotracker green for 30 min at 37 °C prior to imaging.

Fig. 7

Induction of mitochondrial fission and autophagy: (A) and (B) The immunofluorescence microscopy was carried out with permeabilized Cos-7 cells transfected with WT, ΔN16 and ΔN33 cDNA's. Cells were incubated with primary HO-1 (anti-rabbit) antibody, and were co-stained with mitochondrial fission marker DRP-1 (A) and autophagy marker LC-3 (B) antibodies. The slides were subsequently stained with Alexa conjugated antibodies and examined through Olympus microscope.

Fig. 8

Mitochondrial HO-1 level in livers of rats fed with ethanol for 10 weeks: (A) Mitochondria were prepared from control rats and pair fed ethanol for 10 weeks using Lieber decarli diet. 50 μg mitochondrial protein each was subjected to immunoblot analysis using antibody to HO-1. The blot was also co-developed with mitochondrial specific marker, Porin as a loading control. (B) The HO-1 band intensities from controls and ethanol treated rats (n=4)were averaged using Image J and plotted. (C) CcO activity of rat liver mitochondria from control and pair-fed rats shown in (A) was measured as described in “Materials and methods”. Data are presented as±S.E. from three experiments, and groups were compared using an unpaired, two-tailed Student's t test. ^⁎⁎ indicates p<0.05.