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Effect of downregulated citrate synthase on oxidative phosphorylation signaling pathway in HEI-OC1 cells
Xiaowen Xu1,2, Yue Liu1,3, Jun Luan1,4
1Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, People's Republic of China.
Background:
Citrate Synthase (Cs) gene mutation (locus ahL4) has been found to play an important role in progressive hearing loss of A/J mice. HEI-OC1 cells have been widely used as an in vitro system to study cellular and molecular mechanisms related to hearing lose. We previously reported the increased apoptosis and the accumulation of reactive oxygen species in shRNACs-1429 cells, a Cs low-expressed cell model from HEI-OCI. The details of the mechanism of ROS production and apoptosis mediated by the abnormal expression of Cs needed to research furtherly.
Methods:
iTRAQ proteomics was utilized to detect the differentially expressed proteins (DEPs) caused by low expression of Cs. The GO and KEGG pathways analysis were performed for annotation of the differentially expressed proteins. Protein-protein interaction network was constructed by STRING online database. Immunoblotting was utilized to confirm the protein levels of the the differentially expressed proteins.
Results:
The differentially expressed proteins were significantly enriched in various signaling pathways mainly related to mitochondrial dysfunction diseases including Parkinson's disease, Alzheimer's disease, Huntington's disease, et al. Most noteworthy, the oxidative phosphorylation pathway was most significantly suppressed in the shRNACs-1429 cells,, in which a total of 10 differentially expressed proteins were enriched and were all downregulated by the abnormal expression of Cs. The downregulations of Ndufb5, Ndufv1 and Uqcrb were confirmed by immunoblotting. Meanwhile, the ATP levels of shRNACs-1429 cells were also reduced.
Conclusions:
These results suggest that low level expression of Cs induces the inhibition of oxidative phosphorylation pathway, which is responsible for the high level production of reactive oxygen species and low level of ATP, leading to the apoptosis of cochlear cells. This study may provide new theories for understanding and therapy of progressive hearing loss.
Insights
Low Citrate Synthase (Cs) expression in cochlear cells inhibits oxidative phosphorylation, increasing reactive oxygen species and decreasing ATP. This leads to apoptosis and progressive hearing loss, offering new therapeutic insights.
Area of Science:
- Oto-genetics and molecular biology of hearing loss.
- Mitochondrial dysfunction and cellular apoptosis.
Background:
- Citrate Synthase (Cs) gene mutations are linked to progressive hearing loss in A/J mice.
- HEI-OC1 cells are a model for studying hearing loss mechanisms.
- Previous studies showed increased apoptosis and reactive oxygen species (ROS) in Cs low-expressed cells.
Purpose of the Study:
- To elucidate the mechanism of ROS production and apoptosis mediated by abnormal Citrate Synthase (Cs) expression.
- To identify differentially expressed proteins (DEPs) in Cs low-expressed cochlear cells.
Main Methods:
- iTRAQ proteomics to detect DEPs in shRNACs-1429 cells (Cs low-expressed).
- Gene Ontology (GO) and KEGG pathway analysis for DEP annotation.
- STRING database for protein-protein interaction network construction.
- Immunoblotting to confirm protein level changes.
Main Results:
- DEPs were enriched in pathways related to mitochondrial dysfunction, including neurodegenerative diseases.
- The oxidative phosphorylation pathway was significantly suppressed, with 10 downregulated DEPs.
- Downregulation of Ndufb5, Ndufv1, and Uqcrb was confirmed; ATP levels were reduced.
Conclusions:
- Low Cs expression inhibits oxidative phosphorylation, causing excessive ROS production and ATP depletion.
- This mechanism leads to cochlear cell apoptosis and progressive hearing loss.
- Findings offer novel theoretical frameworks for understanding and treating hearing loss.
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