ISGylation Inhibits an LPS-Induced Inflammatory Response via the TLR4/NF-κB Signaling Pathway in Goat Endometrial Epithelial Cells

Abstract

Endometritis is a common and important reproductive disease of domestic animals. The principal factors responsible for the disease are infection with Gram-negative bacteria, the release of Lipopolysaccharides (LPS) and activation of the TLR4/NF-κB signaling pathway. However, we do not fully understand the interaction between endometrial immunity and bacterial infection in the disease etiology. The ubiquitin-like protein ISG15 can regulate the TLR4/NF-κB signaling pathway via the ISGylation modification system, modulating the inflammatory response. In the present study, we found that ISG15 protein was expressed mainly in the cytoplasm of goat endometrial epithelial cells (gEECs) and that the expression of key genes and proteins of ISGylation increased in LPS-induced gEECs. Overexpression and silencing of the ISG15 gene demonstrated that ISGylation inhibited an LPS-induced inflammatory response via the TLR4/NF-κB signaling pathway in gEECs. Here, we provide the experimental basis for further exploration of the role of the ISGylation modification system in the inflammatory response of endometrium and a potential method for the treatment of endometritis.

ISGylation of NF-κBp65 by SCF FBXL19 E3 Ligase Diminishes Endothelial Inflammation

“…43 NF-κBp65 undergoes several PTMs that regulate p65 stability, localization, and transcriptional activation. 41 The effect of ISGylation on NF-κB activation has been reported [32][33][34]36 ; however, the ISGylation of p65 has not been identified. This study reveals that p65 can be ISGylated and that the ISGylation is regulated by E3 ligase SCF FBXL19 and de-ISGylation enzyme USP18.…”

“…23,24,26,28 Recent studies suggest that ISGylation plays an anti-inflammatory role in bacterial infection. 34,44 NF-κB activation contributes to inflammatory responses including chemokine, ICAM1, and VCAM1 production in ECs. 43 NF-κBp65 undergoes several PTMs that regulate p65 stability, localization, and transcriptional activation.…”

“…30,31 The functional consequences of ISGylation are largely unexplored even though it has been shown to regulate protein-protein interaction, enzyme activity, and protein translation. Recent studies indicate that ISG15 regulates TRAF6 (TNF recetpor-associated factor 6)/ TAK1 (transforming growth factor-beta-associated kinase 1), IKKβ, NEMO (NF-kappa B essential modulator) protein levels and p65 mRNA expression [32][33][34][35][36] ; however, ISGylation of NF-ĸBp65 and the role of protein ISGylation in EC inflammation have not been reported.…”

Scavenger receptor B1 facilitates the endocytosis of Escherichia coli via TLR4 signaling in mammary gland infection

“…Real-time quantitative RT-qPCR was performed using SYBR Green PCR Master Mix (KAPA™ SYBR® qPCR Kit, KapaBiosystems, Woburn, MA) according to the manufacturer instructions. The primers used for expression were already reported ( GAPDH, β-Actin, CSN-2, TLR4, TRIF, MyD88, IRF3, TRAF3, MAPK1, TNF-α, IL-8, NF-kB, TRAF6 and INF-β) (28, 50, 51, 52, 53, 54). SCARB1 primers were designed using Primer3 Plus software.…”

Baicalin alleviates endometrial inflammatory injury through regulation of tight junction proteins

“…To further explore the potential mechanism by which baicalin prevents endometrial barrier injury, we employed immortalized goat endometrial epithelial cells (EECs) as an in vitro model of the endometrial barrier; these cells are frequently used as an endometrial injury model. [15][16][17] 2 Materials and methods…”