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USP5 Deletion Inhibits KD Serum Induced-Human Coronary Artery Endothelial Cell Dysfunction by Regulating the
Lidan Yao1, Yupeng Lai2, Heng Li1
1Department of Pediatric Cardiology, Maternal and Child Health Hospital of Hubei Province, Tongji Medical College, Huazhong University of Science and Technology, Wuhan City, Hubei Province, P.R. China.
Insights
Nuclear factor of activated T cells, cytoplasmic 1 (NFATC1) is crucial in Kawasaki disease (KD) vasculitis. USP5 deubiquitinates NFATC1, promoting its interaction with TLR4, activating NF-κB signaling and causing KD-induced endothelial injury.
Area of Science:
- Immunology
- Molecular Biology
- Cardiovascular Research
Background:
- Kawasaki disease (KD) is an acute febrile illness causing systemic vasculitis, particularly affecting coronary arteries.
- Nuclear factor of activated T cells, cytoplasmic 1 (NFATC1) is implicated in KD pathogenesis, but its precise molecular mechanisms remain unclear.
Purpose of the Study:
- To elucidate the molecular mechanism of NFATC1 in Kawasaki disease (KD) pathogenesis.
- To investigate the role of Ubiquitin-specific peptidase 5 (USP5) and Toll-like receptor 4 (TLR4) in NFATC1-mediated KD endothelial injury.
Main Methods:
- Human coronary artery endothelial cells (HCAECs) were treated with KD patient serum to model inflammatory injury.
- NFATC1, USP5, TLR4, and NF-κB pathway components were analyzed using RT-qPCR, Western blot, ELISA, and Co-immunoprecipitation (CoIP).
- Cell viability, proliferation, and apoptosis were assessed using CCK-8, EdU assays, and flow cytometry.
Main Results:
- NFATC1 expression was upregulated in KD patients and KD serum-treated HCAECs.
- USP5 deubiquitinated NFATC1, preventing its degradation and facilitating its interaction with TLR4, thereby activating the NF-κB pathway.
- Blocking NFATC1 or USP5 deficiency ameliorated KD serum-induced HCAEC injury, inflammation, and oxidative stress.
Conclusions:
- The USP5/NFATC1/TLR4 axis plays a critical role in KD-induced endothelial cell injury by activating NF-κB signaling.
- Targeting USP5 or NFATC1 presents a potential therapeutic strategy for mitigating Kawasaki disease vasculitis.
Abstract:
Kawasaki disease (KD) is an acute febrile illness characterized by systemic vasculitis, especially in coronary arteries. Previous studies have indicated that nuclear factor of activated T cells, cytoplasmic 1 (NFATC1, also known as NFAT2) plays a crucial role in the pathogenesis of KD. However, the molecular mechanism of NFATC1 involved in KD is poorly defined. Human coronary artery endothelial cells (HCAECs) were treated with 15% serum from KD patients to mimic the inflammatory injury model in vitro. NFATC1 mRNA level was determined using real-time quantitative polymerase chain reaction (RT-qPCR). NFATC1, Bax, Bcl-2, Ubiquitin-specific peptidase 5 (USP5), Toll-like receptor 4 (TLR4), p-P65, P65, p-IκBα, and IκBα protein levels were determined by Western blot. Cell viability, proliferation, and apoptosis were assessed using the Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and flow cytometry. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) levels were analyzed using ELISA. ROS and SOD levels were detected using special assay kits. After ubibrowser database analysis, the interaction between USP5 and NFATC1 was verified using Co-immunoprecipitation (CoIP) assay. Meanwhile, the possible interaction between NFATC1 and TLR4 was predicted by STRING databases and identified using CoIP assay. NFATC1 expression was increased in KD patients and KD serum-treated HCAECs. KD serum-mediated HCAEC viability and proliferation inhibition, apoptosis, inflammatory response, and oxidative stress promotion. Furthermore, blocking NFATC1 relieved KD serum-evoked HCAEC injury in vitro. Mechanistically, USP5 triggered the deubiquitination of NFATC1 and prevented its degradation. NFATC1 interacted with TLR4 to regulate its expression in HCAECs. Besides, KD serum activated the nuclear factor kappa-B (NF-κB) signaling pathway by regulating the USP5/NFATC1/TLR4 axis in HCAECs. USP5 deficiency mitigated KD serum-induced inflammation and injury in HCAECs through targeting NFATC1 and TLR4-mediated NF-κB signaling, providing a possible therapeutic target for KD treatment.
