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Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin.

Müllebner A, Moldzio R, Redl H, Kozlov AV, Duvigneau JC - Biomolecules (2015)

Bottom Line: The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress.The mechanisms by which HO mediates the described effects are not completely clear.However, the proteins that are targeted by BR still have to be identified.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Biochemistry, Veterinary University Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. andrea.muellebner@vetmeduni.ac.at.

ABSTRACT
Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.

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Effect of hemin and zinc protoprophyrin IX on mitochondrial function and morphology in hepatocytes. Liver cells (BRL3A) were incubated with JC-1 for 30 min, thereafter vehicle (DMSO, A) or hemin (20 µM); (B,C)) was added. Zinc protoporphyrin (ZnIXPP), a competitive inhibitor of HO (0.2 µM); was added 10 min before (C). Cells were analyzed after 2 h. A1, B1 and C1 show energized mitochondria (fluorescence at 590 nm). Competitive inhibition of HO by ZnIXPP resulted in delayed fragmentation of mitochondria (C1); (D) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I1, violet line) in the concentrations indicated above on quantification of mitochondrial potential (intensity of background normalized JC-1 fluorescence (emission at 590 nm) was used as a parameter for mitochondrial potential); (E) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I, violet line) in the concentrations indicated above on HO-1 mRNA expression in BRL3A cells determined by qPCR. Data are given as means (±SD) obtained from one representative experiment using n = 4 replicates.
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biomolecules-05-00679-f003: Effect of hemin and zinc protoprophyrin IX on mitochondrial function and morphology in hepatocytes. Liver cells (BRL3A) were incubated with JC-1 for 30 min, thereafter vehicle (DMSO, A) or hemin (20 µM); (B,C)) was added. Zinc protoporphyrin (ZnIXPP), a competitive inhibitor of HO (0.2 µM); was added 10 min before (C). Cells were analyzed after 2 h. A1, B1 and C1 show energized mitochondria (fluorescence at 590 nm). Competitive inhibition of HO by ZnIXPP resulted in delayed fragmentation of mitochondria (C1); (D) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I1, violet line) in the concentrations indicated above on quantification of mitochondrial potential (intensity of background normalized JC-1 fluorescence (emission at 590 nm) was used as a parameter for mitochondrial potential); (E) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I, violet line) in the concentrations indicated above on HO-1 mRNA expression in BRL3A cells determined by qPCR. Data are given as means (±SD) obtained from one representative experiment using n = 4 replicates.

Mentions: Heme toxicity is based on the oxidative modification and consequent damage exerted to membranes and associated proteins when exceeding critical levels. Heme induces HO-1 by directly interacting with the heme-sensitive transcription factors BACH1 and BACH2 [27], resulting in its subsequent degradation by the up-regulated HO activity. HO may provide protection in two ways, either by consumption of heme or by the release of BR, exerting putative anti-oxidative properties, or by both. In the following experiments we examined the effect of HO on hemin-mediated impairment of mitochondrial function. BRL3A cells were treated with 20 µM hemin and mitochondria were visualized using JC-1 (Figure 3), as a measure for membrane potential (Figure 3A1–C1). Membrane potential initially decreased in cells treated with hemin, and this effect was most pronounced when its degradation was inhibited by zinc protoprophyrin IX (ZnIXPP), a competitive inhibitor of HO (Figure 3D). Continuous inhibition of hemin degradation resulted in an increased mitochondrial fragmentation (Figure 3C1). Cells treated with hemin or vehicle alone did not show any effects on their mitochondria (Figure 3A,B).


Heme Degradation by Heme Oxygenase Protects Mitochondria but Induces ER Stress via Formed Bilirubin.

Müllebner A, Moldzio R, Redl H, Kozlov AV, Duvigneau JC - Biomolecules (2015)

Effect of hemin and zinc protoprophyrin IX on mitochondrial function and morphology in hepatocytes. Liver cells (BRL3A) were incubated with JC-1 for 30 min, thereafter vehicle (DMSO, A) or hemin (20 µM); (B,C)) was added. Zinc protoporphyrin (ZnIXPP), a competitive inhibitor of HO (0.2 µM); was added 10 min before (C). Cells were analyzed after 2 h. A1, B1 and C1 show energized mitochondria (fluorescence at 590 nm). Competitive inhibition of HO by ZnIXPP resulted in delayed fragmentation of mitochondria (C1); (D) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I1, violet line) in the concentrations indicated above on quantification of mitochondrial potential (intensity of background normalized JC-1 fluorescence (emission at 590 nm) was used as a parameter for mitochondrial potential); (E) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I, violet line) in the concentrations indicated above on HO-1 mRNA expression in BRL3A cells determined by qPCR. Data are given as means (±SD) obtained from one representative experiment using n = 4 replicates.
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Related In: Results  -  Collection

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biomolecules-05-00679-f003: Effect of hemin and zinc protoprophyrin IX on mitochondrial function and morphology in hepatocytes. Liver cells (BRL3A) were incubated with JC-1 for 30 min, thereafter vehicle (DMSO, A) or hemin (20 µM); (B,C)) was added. Zinc protoporphyrin (ZnIXPP), a competitive inhibitor of HO (0.2 µM); was added 10 min before (C). Cells were analyzed after 2 h. A1, B1 and C1 show energized mitochondria (fluorescence at 590 nm). Competitive inhibition of HO by ZnIXPP resulted in delayed fragmentation of mitochondria (C1); (D) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I1, violet line) in the concentrations indicated above on quantification of mitochondrial potential (intensity of background normalized JC-1 fluorescence (emission at 590 nm) was used as a parameter for mitochondrial potential); (E) Effect of hemin (H, grey line), HO-inhibitor ZnIXPP (I, orange line), and hemin plus inhibitor (H+I, violet line) in the concentrations indicated above on HO-1 mRNA expression in BRL3A cells determined by qPCR. Data are given as means (±SD) obtained from one representative experiment using n = 4 replicates.
Mentions: Heme toxicity is based on the oxidative modification and consequent damage exerted to membranes and associated proteins when exceeding critical levels. Heme induces HO-1 by directly interacting with the heme-sensitive transcription factors BACH1 and BACH2 [27], resulting in its subsequent degradation by the up-regulated HO activity. HO may provide protection in two ways, either by consumption of heme or by the release of BR, exerting putative anti-oxidative properties, or by both. In the following experiments we examined the effect of HO on hemin-mediated impairment of mitochondrial function. BRL3A cells were treated with 20 µM hemin and mitochondria were visualized using JC-1 (Figure 3), as a measure for membrane potential (Figure 3A1–C1). Membrane potential initially decreased in cells treated with hemin, and this effect was most pronounced when its degradation was inhibited by zinc protoprophyrin IX (ZnIXPP), a competitive inhibitor of HO (Figure 3D). Continuous inhibition of hemin degradation resulted in an increased mitochondrial fragmentation (Figure 3C1). Cells treated with hemin or vehicle alone did not show any effects on their mitochondria (Figure 3A,B).

Bottom Line: The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress.The mechanisms by which HO mediates the described effects are not completely clear.However, the proteins that are targeted by BR still have to be identified.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Biochemistry, Veterinary University Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. andrea.muellebner@vetmeduni.ac.at.

ABSTRACT
Heme oxygenase (HO), in conjunction with biliverdin reductase, degrades heme to carbon monoxide, ferrous iron and bilirubin (BR); the latter is a potent antioxidant. The induced isoform HO-1 has evoked intense research interest, especially because it manifests anti-inflammatory and anti-apoptotic effects relieving acute cell stress. The mechanisms by which HO mediates the described effects are not completely clear. However, the degradation of heme, a strong pro-oxidant, and the generation of BR are considered to play key roles. The aim of this study was to determine the effects of BR on vital functions of hepatocytes focusing on mitochondria and the endoplasmic reticulum (ER). The affinity of BR to proteins is a known challenge for its exact quantification. We consider two major consequences of this affinity, namely possible analytical errors in the determination of HO activity, and biological effects of BR due to direct interaction with protein function. In order to overcome analytical bias we applied a polynomial correction accounting for the loss of BR due to its adsorption to proteins. To identify potential intracellular targets of BR we used an in vitro approach involving hepatocytes and isolated mitochondria. After verification that the hepatocytes possess HO activity at a similar level as liver tissue by using our improved post-extraction spectroscopic assay, we elucidated the effects of increased HO activity and the formed BR on mitochondrial function and the ER stress response. Our data show that BR may compromise cellular metabolism and proliferation via induction of ER stress. ER and mitochondria respond differently to elevated levels of BR and HO-activity. Mitochondria are susceptible to hemin, but active HO protects them against hemin-induced toxicity. BR at slightly elevated levels induces a stress response at the ER, resulting in a decreased proliferative and metabolic activity of hepatocytes. However, the proteins that are targeted by BR still have to be identified.

Show MeSH
Related in: MedlinePlus