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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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Related in: MedlinePlus

The suitability of the newly designed primers was verified in separate experiments by performing dilution series using the PCR products (Table A2) as well as dilution series of a cDNA pool (A). In melt curve (B) and amplification plots (B) samples are shown in green while controls (no reverse transcription control and no template control) are shown in yellow and orange respectively.
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biomolecules-05-00679-f009: The suitability of the newly designed primers was verified in separate experiments by performing dilution series using the PCR products (Table A2) as well as dilution series of a cDNA pool (A). In melt curve (B) and amplification plots (B) samples are shown in green while controls (no reverse transcription control and no template control) are shown in yellow and orange respectively.

Mentions: RNA was isolated from BRL3A treated with BR (0.8 µM, 4 µM, 20 µM) for 8 h and processed as described elsewhere [56]. Primer sequences used for amplification are given in Table 1. Primer sequences for XBP-1 were newly designed (and amplification efficiency was verified by dilution series (accessory information is given in the AppendixFigure A1, Table A1 and Table A2). Expression of target genes was measured using a CFX96™ (Bio-Rad, Hercules, CA, USA). Each reaction contained SYBR® green I as reporter (0.5×), iTaq™ polymerase™ (0.625 U/reaction; BioRad), the primers (250 nmol/L each, Invitrogen) with a final concentration of 200 μmol/L dNTP (each) and 3 mmol/L MgCl2 in the provided reaction buffer with a final volume of 12 μL. Data were collected in the regression mode and calculated against an internal standard (IS) consisting of pooled cDNA samples of all experiments. We used a modified comparative ΔΔCq method. First the gene specific Cqs were subtracted from the mean Cq of the IS obtained for the same gene giving rise to ΔCq. The values were then subtracted from the normalization factor, which was calculated by averaging the ΔCqs of the internal reference genes (cyclophilin A, hypoxanthinribosyl transferase, glycerinaldehyde dehydrogenase) of the same sample (ΔΔCq). The obtained ΔΔCq values of the replicates were averaged and expressed as 2−ΔΔCq in fold changes relative to the IS.


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)

The suitability of the newly designed primers was verified in separate experiments by performing dilution series using the PCR products (Table A2) as well as dilution series of a cDNA pool (A). In melt curve (B) and amplification plots (B) samples are shown in green while controls (no reverse transcription control and no template control) are shown in yellow and orange respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496691&req=5

biomolecules-05-00679-f009: The suitability of the newly designed primers was verified in separate experiments by performing dilution series using the PCR products (Table A2) as well as dilution series of a cDNA pool (A). In melt curve (B) and amplification plots (B) samples are shown in green while controls (no reverse transcription control and no template control) are shown in yellow and orange respectively.
Mentions: RNA was isolated from BRL3A treated with BR (0.8 µM, 4 µM, 20 µM) for 8 h and processed as described elsewhere [56]. Primer sequences used for amplification are given in Table 1. Primer sequences for XBP-1 were newly designed (and amplification efficiency was verified by dilution series (accessory information is given in the AppendixFigure A1, Table A1 and Table A2). Expression of target genes was measured using a CFX96™ (Bio-Rad, Hercules, CA, USA). Each reaction contained SYBR® green I as reporter (0.5×), iTaq™ polymerase™ (0.625 U/reaction; BioRad), the primers (250 nmol/L each, Invitrogen) with a final concentration of 200 μmol/L dNTP (each) and 3 mmol/L MgCl2 in the provided reaction buffer with a final volume of 12 μL. Data were collected in the regression mode and calculated against an internal standard (IS) consisting of pooled cDNA samples of all experiments. We used a modified comparative ΔΔCq method. First the gene specific Cqs were subtracted from the mean Cq of the IS obtained for the same gene giving rise to ΔCq. The values were then subtracted from the normalization factor, which was calculated by averaging the ΔCqs of the internal reference genes (cyclophilin A, hypoxanthinribosyl transferase, glycerinaldehyde dehydrogenase) of the same sample (ΔΔCq). The obtained ΔΔCq values of the replicates were averaged and expressed as 2−ΔΔCq in fold changes relative to the IS.

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