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Published on: August 21, 2013
CHMP4B and VSP4A reverse GSDMD-mediated pyroptosis by cell membrane remodeling in endometrial carcinoma
Ye Yang1, Hai-Lian Chen2, Su Fang Wu1
1Obstetrics and Gynecology Department, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85 Wujin Road, Hongkou, Shanghai 200080, PR China.
Background:
In advanced and recurrent endometrial carcinoma (EC), the current state of immuno- or targeted therapy remains in the clinical research phase. Our study aimed to explore the role of the ESCRT machinery in maintaining cell membrane integrity and reversing pyroptotic cell death.
Methods:
Immunohistochemistry, western blotting, and co-immunoprecipitation were performed to determine the expression and relationship between GSDMD, CHMP4B, and VPS4A. We employed techniques such as FITC Annexin V/propidium iodide staining, Ca2+ fluorescence intensity, IL-1β enzyme-linked immunosorbent assay, and lactate dehydrogenase release assay to detect pyroptosis in endometrial cancer cells. Plasma membrane perforations and CHMP4B/VPS4A puncta were observed through electron and fluorescence confocal microscopy.
Results:
We showed that GSDMD, CHMP4B, and VPS4A were differentially expressed in the pyroptotic EC xenograft mouse model group, as well as high, moderate, and mild expression in EC cells treated with LPS and nigericin compared to endometrial epithelial cells. Co-IP confirmed the interaction between GSDMD, CHMP4B, and VPS4A. We found that GSDMD knockdown reduced PI-positive cells, Ca2+ efflux, IL-1β, and LDH release, while CHMP4B and VPS4A depletion enhanced these indicators in HEC1A and AN3CA cells. Electron microscopy showed membrane perforations correspondingly decreased with inactivated GSDMD and increased or decreased after CHMP4B and VPS4A depletion or overexpression in EC cells. Fluorescence confocal microscopy detected CHMP4B protein puncta associated with VPS4A at the injured plasma membrane in GSDMDNT cells.
Conclusions:
We preliminary evidenced that CHMP4B and VPS4A reverses GSDMD-mediated pyroptosis by facilitating cell membrane remodeling in endometrial carcinoma. Targeting CHMP4B related proteins may promote pyroptosis in endometrial tumors.
Insights
The ESCRT machinery, specifically CHMP4B and VPS4A, can reverse pyroptosis in endometrial cancer cells by remodeling the cell membrane. Targeting these proteins may offer a new therapeutic strategy for endometrial tumors.
Area of Science:
- Oncology
- Cell Biology
- Molecular Medicine
Background:
- Advanced and recurrent endometrial carcinoma (EC) lacks effective immuno- or targeted therapies.
- The role of the ESCRT machinery in EC cell membrane integrity and pyroptosis is unexplored.
Purpose of the Study:
- To investigate the function of the ESCRT machinery in pyroptosis in endometrial cancer.
- To explore the potential of targeting ESCRT components for EC treatment.
Main Methods:
- Immunohistochemistry, western blotting, co-immunoprecipitation to assess protein expression and interactions.
- Assays for pyroptosis detection: Annexin V/PI staining, Ca2+ imaging, IL-1β ELISA, LDH release.
- Electron and fluorescence confocal microscopy to visualize membrane integrity and protein localization.
Main Results:
- GSDMD, CHMP4B, and VPS4A expression was altered in a pyroptotic EC model.
- GSDMD knockdown reduced pyroptosis indicators, while CHMP4B/VPS4A depletion enhanced them.
- CHMP4B and VPS4A localized to the plasma membrane and were involved in membrane repair during pyroptosis.
Conclusions:
- CHMP4B and VPS4A appear to reverse GSDMD-mediated pyroptosis in EC via cell membrane remodeling.
- Targeting CHMP4B-related proteins could be a strategy to induce pyroptosis in endometrial tumors.
