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Updated: Mar 27, 2026
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Down-regulation of GRα expression and inhibition of its nuclear translocation by hypoxia
Pei Zhang1, Lei Fang1, HuiMei Wu1
1Department of Pulmonary, Anhui Geriatric Institute, The First Affiliated Hospital of Anhui Medical University.
Aims:
Glucocorticoids are the most effective anti-inflammatory agent in treating pulmonary diseases typically accompanied by hypoxia. Our previous study has demonstrated that glucocorticoid receptor α (GRα) expression is reduced in hypoxia but the underlying mechanism remains elusive. In this study we aim to identify the signaling pathway involved in hypoxia-induced down-regulation of GRα, and whether hypoxia affects nuclear translocation of GRα.
Main Methods:
Female C57BL/6 mice were sensitized with saline or ovalbumin (OVA) as the in vivo model. Mice were divided into control and OVA groups, and their lung histology and the expression of hypoxia inducible factor (HIF-1) and GRα were examined. A549 cells were exposed to chemical hypoxia as the in vitro model, where mitogen-activated protein kinases (MAPKs) were inhibited specifically by SB203580. Next, under normal or hypoxic conditions, the expression of GRα, MAPKs and HIF-1 signal protein were determined by Western blot analysis, and GRα translocation were observed through live-cell imaging.
Key Findings:
In OVA challenged mice the expression of GRα was down-regulated whereas HIF-1 was up-regulated. Hypoxia caused a time-dependent decrease of GRα expression, and activated multiple signaling pathways including MAPKs and HIF-1. Moreover, GRα expression increased with MAPK inhibition. Interestingly, only MAPK inhibitor SB203580, but not JNK inhibitor SP600125 or ERK inhibitor U0126 improved the expression of GRα under hypoxic condition. GRα nuclear translocation was also significantly inhibited by hypoxia.
Significance:
Hypoxia down-regulated the expression of GRα through p38 signaling pathway, as well as inhibited GRα nuclear translocation significantly.
Insights
Hypoxia reduces glucocorticoid receptor alpha (GRα) expression and nuclear translocation via the p38 signaling pathway. This finding is crucial for understanding pulmonary disease treatment during low oxygen conditions.
Area of Science:
- Pulmonary Medicine
- Cellular Signaling
- Molecular Biology
Background:
- Glucocorticoids are potent anti-inflammatory agents for pulmonary diseases, often occurring with hypoxia.
- Glucocorticoid receptor alpha (GRα) expression decreases in hypoxia, but the mechanism is unclear.
- Understanding GRα regulation in hypoxia is vital for optimizing anti-inflammatory therapies.
Purpose of the Study:
- To identify the signaling pathway responsible for hypoxia-induced GRα downregulation.
- To investigate whether hypoxia impairs GRα nuclear translocation.
- To explore therapeutic targets for maintaining GRα function under hypoxic conditions.
Main Methods:
- Established in vivo (ovalbumin-challenged mice) and in vitro (A549 cells) models of hypoxia.
- Examined lung histology, hypoxia-inducible factor (HIF-1), and GRα expression.
- Utilized Western blot analysis and live-cell imaging to assess protein expression and GRα translocation, with specific mitogen-activated protein kinase (MAPK) inhibition.
Main Results:
- Ovalbumin challenge induced GRα downregulation and HIF-1 upregulation in mice.
- Hypoxia decreased GRα expression and activated MAPKs and HIF-1 signaling.
- Inhibition of p38 MAPK (using SB203580) restored GRα expression under hypoxia; GRα nuclear translocation was significantly inhibited by hypoxia.
Conclusions:
- Hypoxia downregulates GRα expression primarily through the p38 MAPK signaling pathway.
- Hypoxia significantly inhibits the nuclear translocation of GRα.
- Targeting the p38 pathway may preserve GRα function in hypoxic pulmonary diseases.
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