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The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia.

Poone GK, Hasseldam H, Munkholm N, Rasmussen RS, Grønberg NV, Johansen FF - Brain Sci (2015)

Bottom Line: As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia.On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001).As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Biotech Research & Innovation Centre (BRIC), University of Copenhagen, 2200, Denmark. gagandipkb@hotmail.com.

ABSTRACT
Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an in vitro and in vivo model of ischemic stroke, we investigated whether hypothermia regulates the unfolded protein response of CHOP and Endoplasmic reticulum oxidoreductin-α (Ero1-α), because Ero1-α is suggested to be a downstream CHOP target. The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR) in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C) and hypothermic (31 °C) conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course (p < 0.01, p < 0.0001), whereas Ero1-α expression peaked at three to six hours (p < 0.0001). Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001). On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001). Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

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Related in: MedlinePlus

Expression of CHOP and Ero1-α in hypoxic PC12 cells under normo- and hypothermic conditions. Quantitative PCR revealed increased expression levels of CHOP and Ero1-α over a 12 h time course under normothermic conditions. This is consistent with findings in the global ischemia in vivo model. Hypothermia attenuated the expression of CHOP significantly (p < 0.0001) at 3 (n = 6), 6 (n = 5) and 12 h (n = 5). The expression levels of Ero1-α were augmented by hypothermic conditions with marginally increased levels at 6 h (n = 6) and significantly increased levels at 12 h (n = 5). *** p < 0.001 and **** p < 0.0001.
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brainsci-05-00178-f003: Expression of CHOP and Ero1-α in hypoxic PC12 cells under normo- and hypothermic conditions. Quantitative PCR revealed increased expression levels of CHOP and Ero1-α over a 12 h time course under normothermic conditions. This is consistent with findings in the global ischemia in vivo model. Hypothermia attenuated the expression of CHOP significantly (p < 0.0001) at 3 (n = 6), 6 (n = 5) and 12 h (n = 5). The expression levels of Ero1-α were augmented by hypothermic conditions with marginally increased levels at 6 h (n = 6) and significantly increased levels at 12 h (n = 5). *** p < 0.001 and **** p < 0.0001.

Mentions: With the use of a SERCA (sarco/endoplasmatic reticulum Ca2+-ATPase) pump inhibitor, thapsigargin, a suitable concentration for induction of a UPR response in the PC12 cell line was found (data not shown), confirming the ability of the cell line to activate UPR upon stress. Since we saw a substantial increase in both CHOP and Ero1-α expression levels at 3 h in our in vivo model, we included an additional 1 h group subjected to hypothermic conditions. Messenger RNA expression of the two UPR markers was unaffected after 1 h of hypoxia, whereas 3, 6, and 12 h of hypoxia showed similar expression patterns as seen in vivo (Figure 3). However, results indicate that PC12 cells respond slightly different to hypoxia compared to hippocampal neurons, where 12 h of hypoxia further increases the expression levels of CHOP, in contrast to the decreased levels observed in our in vivo model (Figure 1). Ero1-α expression patterns in PC12 cells also resemble what we found in vivo; however, 6 h of hypoxia seemed to entail the greatest increase in expression. Upon incubation at hypothermic conditions, we found a significant decrease in CHOP expression at 3, 6 and 12 h compared to normothermic conditions (p < 0.0001). In contrast, expression levels of Ero1-α were increased as a result of hypothermia at 6 h (p < 0.001) and 12 h (p < 0.0001) (Figure 3).


The Hypothermic Influence on CHOP and Ero1-α in an Endoplasmic Reticulum Stress Model of Cerebral Ischemia.

Poone GK, Hasseldam H, Munkholm N, Rasmussen RS, Grønberg NV, Johansen FF - Brain Sci (2015)

Expression of CHOP and Ero1-α in hypoxic PC12 cells under normo- and hypothermic conditions. Quantitative PCR revealed increased expression levels of CHOP and Ero1-α over a 12 h time course under normothermic conditions. This is consistent with findings in the global ischemia in vivo model. Hypothermia attenuated the expression of CHOP significantly (p < 0.0001) at 3 (n = 6), 6 (n = 5) and 12 h (n = 5). The expression levels of Ero1-α were augmented by hypothermic conditions with marginally increased levels at 6 h (n = 6) and significantly increased levels at 12 h (n = 5). *** p < 0.001 and **** p < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493463&req=5

brainsci-05-00178-f003: Expression of CHOP and Ero1-α in hypoxic PC12 cells under normo- and hypothermic conditions. Quantitative PCR revealed increased expression levels of CHOP and Ero1-α over a 12 h time course under normothermic conditions. This is consistent with findings in the global ischemia in vivo model. Hypothermia attenuated the expression of CHOP significantly (p < 0.0001) at 3 (n = 6), 6 (n = 5) and 12 h (n = 5). The expression levels of Ero1-α were augmented by hypothermic conditions with marginally increased levels at 6 h (n = 6) and significantly increased levels at 12 h (n = 5). *** p < 0.001 and **** p < 0.0001.
Mentions: With the use of a SERCA (sarco/endoplasmatic reticulum Ca2+-ATPase) pump inhibitor, thapsigargin, a suitable concentration for induction of a UPR response in the PC12 cell line was found (data not shown), confirming the ability of the cell line to activate UPR upon stress. Since we saw a substantial increase in both CHOP and Ero1-α expression levels at 3 h in our in vivo model, we included an additional 1 h group subjected to hypothermic conditions. Messenger RNA expression of the two UPR markers was unaffected after 1 h of hypoxia, whereas 3, 6, and 12 h of hypoxia showed similar expression patterns as seen in vivo (Figure 3). However, results indicate that PC12 cells respond slightly different to hypoxia compared to hippocampal neurons, where 12 h of hypoxia further increases the expression levels of CHOP, in contrast to the decreased levels observed in our in vivo model (Figure 1). Ero1-α expression patterns in PC12 cells also resemble what we found in vivo; however, 6 h of hypoxia seemed to entail the greatest increase in expression. Upon incubation at hypothermic conditions, we found a significant decrease in CHOP expression at 3, 6 and 12 h compared to normothermic conditions (p < 0.0001). In contrast, expression levels of Ero1-α were increased as a result of hypothermia at 6 h (p < 0.001) and 12 h (p < 0.0001) (Figure 3).

Bottom Line: As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia.On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001).As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Biotech Research & Innovation Centre (BRIC), University of Copenhagen, 2200, Denmark. gagandipkb@hotmail.com.

ABSTRACT
Hypoxia induced endoplasmic reticulum stress causes accumulation of unfolded proteins in the endoplasmic reticulum and activates the unfolded protein response, resulting in apoptosis through CCAAT-enhancer-binding protein homologous protein (CHOP) activation. In an in vitro and in vivo model of ischemic stroke, we investigated whether hypothermia regulates the unfolded protein response of CHOP and Endoplasmic reticulum oxidoreductin-α (Ero1-α), because Ero1-α is suggested to be a downstream CHOP target. The gene expression of CHOP and Ero1-α was measured using Quantitative-PCR (Q-PCR) in rat hippocampi following global cerebral ischemia, and in hypoxic pheochromocytoma cells during normothermic (37 °C) and hypothermic (31 °C) conditions. As a result of ischemia, a significant increase in expression of CHOP and Ero1-α was observed after three, six and twelve hours of reperfusion following global ischemia. A stable increase in CHOP expression was observed throughout the time course (p < 0.01, p < 0.0001), whereas Ero1-α expression peaked at three to six hours (p < 0.0001). Induced hypothermia in hypoxia stressed PC12 cells resulted in a decreased expression of CHOP after three, six and twelve hours (p < 0.0001). On the contrary, the gene expression of Ero1-α increased as a result of hypothermia and peaked at twelve hours (p < 0.0001). Hypothermia attenuated the expression of CHOP, supporting that hypothermia suppress endoplasmic reticulum stress induced apoptosis in stroke. As hypothermia further induced up-regulation of Ero1-α, and since CHOP and Ero1-α showed differential regulation as a consequence of both disease (hypoxia) and treatment (hypothermia), we conclude that they are regulated independently.

No MeSH data available.


Related in: MedlinePlus