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NF-κB signaling is essential for resistance to heat stress-induced early stage apoptosis in human umbilical vein endothelial cells.

Liu Y, Zhou G, Wang Z, Guo X, Xu Q, Huang Q, Su L - Sci Rep (2015)

Bottom Line: When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs).Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise.In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Southern Medical University, Guangzhou, China.

ABSTRACT
Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

No MeSH data available.


Related in: MedlinePlus

Relocalization of p65 from the cytosol into the nucleus of heat stressed HUVECs.Cells were incubated at 37 °C (CONT) or were subjected to a heat stress (HS) treatment at 43 °C for 90 min, followed by a recovery period at 37 °C for 0 h (R0), 2 h (R2), 6 h (R6), or 12 h (R12). (a) The cells were then fixed and processed for indirect immunofluorescence analysis using an antibody raised against the p65 subunit of NF-κB. Representative images are shown. (b) Expression of p65 was detected in cytoplasmic (CE) and nuclear (NC) fractions of HUVECs by Western blotting. The cropped images represent blotting experiments that were performed under the same experimental conditions. (c) HUVECs were pretreated with or without 0.5 μg/ml actinomycin D 5 min before being incubated at 37 °C (CONT) or being subjected a HS treatment. Whole cell extracts were prepared and NF-κB binding to DNA was quantified with the Trans-AMTMp65 transcription factor assay kit. Each value represents the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus control.
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f1: Relocalization of p65 from the cytosol into the nucleus of heat stressed HUVECs.Cells were incubated at 37 °C (CONT) or were subjected to a heat stress (HS) treatment at 43 °C for 90 min, followed by a recovery period at 37 °C for 0 h (R0), 2 h (R2), 6 h (R6), or 12 h (R12). (a) The cells were then fixed and processed for indirect immunofluorescence analysis using an antibody raised against the p65 subunit of NF-κB. Representative images are shown. (b) Expression of p65 was detected in cytoplasmic (CE) and nuclear (NC) fractions of HUVECs by Western blotting. The cropped images represent blotting experiments that were performed under the same experimental conditions. (c) HUVECs were pretreated with or without 0.5 μg/ml actinomycin D 5 min before being incubated at 37 °C (CONT) or being subjected a HS treatment. Whole cell extracts were prepared and NF-κB binding to DNA was quantified with the Trans-AMTMp65 transcription factor assay kit. Each value represents the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus control.

Mentions: The transcription factor, NF-κB, has been shown to be activated during the recovery period following heat stress in HeLa cells28. Therefore, in this study, it was investigated whether heat stress activates NF-κB in human umbilical vein endothelial cells (HUVECs). After HUVEC cells were grown in culture media for 48 h, the culture dishes were sealed with parafilm and immersed in a circulating water bath maintained at 43 °C to induce heat stress29. After 90 min, the culture media was replaced with fresh media and the cells were further incubated at 37 °C for various periods of time (e.g., 0, 2, 6, and 12 h) before being assayed. Indirect immunofluorescence studies demonstrated that the distribution of p65 in nucleus was obviously increased after 6–12 h of heat stress recovery at 37  °C (Fig. 1a). Then nuclear and cytoplasmic extracts of the same timepoints were collected and analyzed by western blot. The result revealed that, unexpectedly, the amount of p65 in nuclear and cytoplasm were both increased (Fig. 1b). It is possible that this increase of nucleus p65 is due to the increase of p65 synthesis, rather than the shuttling of p65 from cytoplasm to nucleus.


NF-κB signaling is essential for resistance to heat stress-induced early stage apoptosis in human umbilical vein endothelial cells.

Liu Y, Zhou G, Wang Z, Guo X, Xu Q, Huang Q, Su L - Sci Rep (2015)

Relocalization of p65 from the cytosol into the nucleus of heat stressed HUVECs.Cells were incubated at 37 °C (CONT) or were subjected to a heat stress (HS) treatment at 43 °C for 90 min, followed by a recovery period at 37 °C for 0 h (R0), 2 h (R2), 6 h (R6), or 12 h (R12). (a) The cells were then fixed and processed for indirect immunofluorescence analysis using an antibody raised against the p65 subunit of NF-κB. Representative images are shown. (b) Expression of p65 was detected in cytoplasmic (CE) and nuclear (NC) fractions of HUVECs by Western blotting. The cropped images represent blotting experiments that were performed under the same experimental conditions. (c) HUVECs were pretreated with or without 0.5 μg/ml actinomycin D 5 min before being incubated at 37 °C (CONT) or being subjected a HS treatment. Whole cell extracts were prepared and NF-κB binding to DNA was quantified with the Trans-AMTMp65 transcription factor assay kit. Each value represents the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Relocalization of p65 from the cytosol into the nucleus of heat stressed HUVECs.Cells were incubated at 37 °C (CONT) or were subjected to a heat stress (HS) treatment at 43 °C for 90 min, followed by a recovery period at 37 °C for 0 h (R0), 2 h (R2), 6 h (R6), or 12 h (R12). (a) The cells were then fixed and processed for indirect immunofluorescence analysis using an antibody raised against the p65 subunit of NF-κB. Representative images are shown. (b) Expression of p65 was detected in cytoplasmic (CE) and nuclear (NC) fractions of HUVECs by Western blotting. The cropped images represent blotting experiments that were performed under the same experimental conditions. (c) HUVECs were pretreated with or without 0.5 μg/ml actinomycin D 5 min before being incubated at 37 °C (CONT) or being subjected a HS treatment. Whole cell extracts were prepared and NF-κB binding to DNA was quantified with the Trans-AMTMp65 transcription factor assay kit. Each value represents the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus control.
Mentions: The transcription factor, NF-κB, has been shown to be activated during the recovery period following heat stress in HeLa cells28. Therefore, in this study, it was investigated whether heat stress activates NF-κB in human umbilical vein endothelial cells (HUVECs). After HUVEC cells were grown in culture media for 48 h, the culture dishes were sealed with parafilm and immersed in a circulating water bath maintained at 43 °C to induce heat stress29. After 90 min, the culture media was replaced with fresh media and the cells were further incubated at 37 °C for various periods of time (e.g., 0, 2, 6, and 12 h) before being assayed. Indirect immunofluorescence studies demonstrated that the distribution of p65 in nucleus was obviously increased after 6–12 h of heat stress recovery at 37  °C (Fig. 1a). Then nuclear and cytoplasmic extracts of the same timepoints were collected and analyzed by western blot. The result revealed that, unexpectedly, the amount of p65 in nuclear and cytoplasm were both increased (Fig. 1b). It is possible that this increase of nucleus p65 is due to the increase of p65 synthesis, rather than the shuttling of p65 from cytoplasm to nucleus.

Bottom Line: When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs).Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise.In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Southern Medical University, Guangzhou, China.

ABSTRACT
Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

No MeSH data available.


Related in: MedlinePlus