USP5 Deletion Inhibits KD Serum Induced-Human Coronary Artery Endothelial Cell Dysfunction by Regulating the NFATC1/TLR4-Mediated NF-κB Signaling Pathway in Kawasaki Disease.

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.