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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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Physiologic range of BR induces ER stress response in BRL 3A cells. (A) Cells were treated with vehicle (DMSO) or BR in the concentrations indicated, and incubated for 8 h. RNA was extracted and expression of cell stress markers X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78), interleukin 6 (IL6), HO-1, CRBP homologous protein (CHOP) and the internal reference genes (cyclophilin A, hypoxanthine ribosyltransferase, glycerinaldehyde dehydrogenase) used for basket normalisation was determined by means of real-time PCR. Target mRNA was normalized to the internal references and calculated relative to the vehicle control (DMSO); (B) BR induced unconventional XBP1 splicing. PCR products were separated using electrophoresis and visualized by ethidium bromide staining. PCR products consisting of spliced (sXBP1) and unspliced variants (usXBP1) were quantified by means of densitometry using the public domain Scion Image program (http://www.scioncorp.com/), and intensities were expressed as a ratio (spliced to unspliced isoforms). Data are given as means (±SD) obtained from one experiment with n = 2 replicates, indicating significant differences (*).
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biomolecules-05-00679-f008: Physiologic range of BR induces ER stress response in BRL 3A cells. (A) Cells were treated with vehicle (DMSO) or BR in the concentrations indicated, and incubated for 8 h. RNA was extracted and expression of cell stress markers X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78), interleukin 6 (IL6), HO-1, CRBP homologous protein (CHOP) and the internal reference genes (cyclophilin A, hypoxanthine ribosyltransferase, glycerinaldehyde dehydrogenase) used for basket normalisation was determined by means of real-time PCR. Target mRNA was normalized to the internal references and calculated relative to the vehicle control (DMSO); (B) BR induced unconventional XBP1 splicing. PCR products were separated using electrophoresis and visualized by ethidium bromide staining. PCR products consisting of spliced (sXBP1) and unspliced variants (usXBP1) were quantified by means of densitometry using the public domain Scion Image program (http://www.scioncorp.com/), and intensities were expressed as a ratio (spliced to unspliced isoforms). Data are given as means (±SD) obtained from one experiment with n = 2 replicates, indicating significant differences (*).

Mentions: We have found that BR concentrations reduced the metabolic activity of BRL3A indicative for enhanced cell stress. It is known that induction of ER stress decelerates growth rate, involving sXBP1 [46] and promotes apoptosis via CHOP [47,48]. We therefore analyzed the expression of markers for ER stress, X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78) HO-1, CRBP homologous protein (CHOP), and interleukin 6 (IL6) as a marker for an inflammatory response, in BR-treated BRL3A cells. Already after 8 h at concentrations between 4 µM and 20 µM BR elicited an ER stress response (Figure 8), which was accompanied by elevated levels of the XBP1 splice variant, a typical ER-stress marker [49]. Additionally, we determined increased levels of IL6, suggesting onset of an inflammatory response, a pathologic reaction mediated by classical ER stressors [50]. Our data indicate that BR may affect proper function of ER. BR may induce protein mis-folding and aggregation due to its particular chemical properties. We showed that BR, which is newly formed in the HO reaction, tightly binds to proteins. At higher concentrations BR is known to lead to aggregates which are favored at lower pH [51]. Additionally, BR is able to associate with calcium [52], which is high in the ER lumen, and to precipitate with other amphiphilic compounds. This phenomenon is known to occur in the bile leading to the formation of pigmented gallstones containing calcium bilirubinate [53]. In neuronal cells, both mitochondrial and ER function are sensitive to elevated concentrations of BR [54]. In parenchymal hepatocytes, in contrast, BR affects primarily the ER. If the concept remains valid that BR works as a potent anti-oxidant within the cell, an elevated level of BR is supposed to disturb the finely tuned redox equilibrium. However, it is well possible that under conditions of excessive oxidative stress BR would help to reinstall a disturbed equilibrium.


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)

Physiologic range of BR induces ER stress response in BRL 3A cells. (A) Cells were treated with vehicle (DMSO) or BR in the concentrations indicated, and incubated for 8 h. RNA was extracted and expression of cell stress markers X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78), interleukin 6 (IL6), HO-1, CRBP homologous protein (CHOP) and the internal reference genes (cyclophilin A, hypoxanthine ribosyltransferase, glycerinaldehyde dehydrogenase) used for basket normalisation was determined by means of real-time PCR. Target mRNA was normalized to the internal references and calculated relative to the vehicle control (DMSO); (B) BR induced unconventional XBP1 splicing. PCR products were separated using electrophoresis and visualized by ethidium bromide staining. PCR products consisting of spliced (sXBP1) and unspliced variants (usXBP1) were quantified by means of densitometry using the public domain Scion Image program (http://www.scioncorp.com/), and intensities were expressed as a ratio (spliced to unspliced isoforms). Data are given as means (±SD) obtained from one experiment with n = 2 replicates, indicating significant differences (*).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496691&req=5

biomolecules-05-00679-f008: Physiologic range of BR induces ER stress response in BRL 3A cells. (A) Cells were treated with vehicle (DMSO) or BR in the concentrations indicated, and incubated for 8 h. RNA was extracted and expression of cell stress markers X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78), interleukin 6 (IL6), HO-1, CRBP homologous protein (CHOP) and the internal reference genes (cyclophilin A, hypoxanthine ribosyltransferase, glycerinaldehyde dehydrogenase) used for basket normalisation was determined by means of real-time PCR. Target mRNA was normalized to the internal references and calculated relative to the vehicle control (DMSO); (B) BR induced unconventional XBP1 splicing. PCR products were separated using electrophoresis and visualized by ethidium bromide staining. PCR products consisting of spliced (sXBP1) and unspliced variants (usXBP1) were quantified by means of densitometry using the public domain Scion Image program (http://www.scioncorp.com/), and intensities were expressed as a ratio (spliced to unspliced isoforms). Data are given as means (±SD) obtained from one experiment with n = 2 replicates, indicating significant differences (*).
Mentions: We have found that BR concentrations reduced the metabolic activity of BRL3A indicative for enhanced cell stress. It is known that induction of ER stress decelerates growth rate, involving sXBP1 [46] and promotes apoptosis via CHOP [47,48]. We therefore analyzed the expression of markers for ER stress, X-Box binding protein 1 (XBP1), glucose regulated protein 78 (GRP78) HO-1, CRBP homologous protein (CHOP), and interleukin 6 (IL6) as a marker for an inflammatory response, in BR-treated BRL3A cells. Already after 8 h at concentrations between 4 µM and 20 µM BR elicited an ER stress response (Figure 8), which was accompanied by elevated levels of the XBP1 splice variant, a typical ER-stress marker [49]. Additionally, we determined increased levels of IL6, suggesting onset of an inflammatory response, a pathologic reaction mediated by classical ER stressors [50]. Our data indicate that BR may affect proper function of ER. BR may induce protein mis-folding and aggregation due to its particular chemical properties. We showed that BR, which is newly formed in the HO reaction, tightly binds to proteins. At higher concentrations BR is known to lead to aggregates which are favored at lower pH [51]. Additionally, BR is able to associate with calcium [52], which is high in the ER lumen, and to precipitate with other amphiphilic compounds. This phenomenon is known to occur in the bile leading to the formation of pigmented gallstones containing calcium bilirubinate [53]. In neuronal cells, both mitochondrial and ER function are sensitive to elevated concentrations of BR [54]. In parenchymal hepatocytes, in contrast, BR affects primarily the ER. If the concept remains valid that BR works as a potent anti-oxidant within the cell, an elevated level of BR is supposed to disturb the finely tuned redox equilibrium. However, it is well possible that under conditions of excessive oxidative stress BR would help to reinstall a disturbed equilibrium.

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