The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells

Abstract

Background Ezrin/radixin/moesin (ERM) proteins are members of the protein 4.1 superfamily and function as linkers that connect the actin cytoskeleton to the plasma membrane of cells. ERM also play critical role in the Lipopolysaccharide (LPS)-induced inflammatory response. However, the signaling mechanisms involved in this process remain unclear. In this study, we aimed to investigate the potential role of the rho-associated coiled-coil containing protein kinase (ROCK) pathway in LPS-induced ezrin phosphorylation and cytokine production in pulmonary alveolar epithelial cells. Methods Cultured A549 and HPAEpiC cells were treated with LPS. The expression and localization of ezrin in A549 and HPAEpiC cells were then analyzed by western blotting and immunoflurescence. Activation of RhoA/ROCK was assessed by western blotting and RhoA activity assays. The interaction of ezrin with Syk and myeloid differentiation factor 88 (MyD88)/IL-1R-associated kinase 1 (IRAK-1) was investigated by co-immunoprecipitation. The activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) was measured with electrophoretic mobility shift assays and by western blotting. ELISA and western blotting were performed to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and high mobility group box 1 protein (HMGB1) release into the culture supernatant, and cellular HMGB1 levels. Results LPS induced ezrin phosphorylation in a concentration- and time-dependent manner. The blockade of RhoA/ROCK inhibited LPS-induced ezrin phosphorylation and its translocation from the cytoplasm to the cell membrane. Co-immunoprecipitation assays further revealed that ezrin associated with Syk constitutively, but only associated with MyD88/IRAK-1 upon LPS challenge. Moreover, LPS-induced p38 and nuclear NF-κB activation was found to be ezrin dependent. The suppression of ezrin by siRNA or the blockade of ROCK activation with Y-27632 reduced the production of TNF-α, IL-1β, and HMGB1 in response to LPS. Conclusions Our findings reveal a novel regulatory mechanism involving ezrin in the LPS-induced production of pro-inflammatory cytokines, and highlight the importance of the RhoA/ROCK-ezrin/Syk-MyD88/IRAK1 axis. Data presented in this manuscript provide novel insights into the signaling pathways activated in pulmonary alveolar epithelial cells by LPS.

Transcriptome analysis reveals the impact of NETs activation on airway epithelial cell EMT and inflammation in bronchiolitis obliterans

“…LPS is a type of endotoxin-like biomolecule that exists in the outer membrane of many bacteria, which had been found to induce plethora of immune and inflammatory reactions 42 , 43 . The production of various cytokines, such as TNF, IL-1, IL-6, and other inflammatory factors stemmed from the interaction between LPS and host cells may inevitably leads to prolonged inflammation and cell damage 44 , 45 . Hence, we infer that NETs not only function to eliminate LPS invasion, but may also play a role in protecting the host from pathogenic bacterial invasion through activating and amplifying the response of human airway cells to LPS.…”

Ezrin expression in female reproductive tissues: A review of regulation and pathophysiological implications

“…Ezrin phosphorylation sites can be regulated by several signaling pathways and perform different physiological functions upon activation and phosphorylation ( Yin and Schnoor, 2022 ). Initial studies demonstrated that the critical T567 on C-ERMAD could be phosphorylated by Rho-kinase (ROCK) ( Ding et al, 2022 ), STE20-like protein kinase (SLK) ( Garland et al, 2021 ), lymphocyte-oriented kinase (LOK) ( Senju and Tsai, 2022 ) and protein kinase C(PKC) ( Khan et al, 2022 ) both in the laboratory and in living organisms. Ezrin Y145, Y353 and Y477 can be modified by phosphorylation through the action of Src kinases and the intrinsic Tyr kinase activity of receptors for epidermal growth factor, hepatocyte growth factor and Platelet-derived growth factor ( Huang et al, 2018 ; Derouiche and Geiger, 2019 ; Rainey et al, 2020 ).…”

Ez-Metastasizing: The Crucial Roles of Ezrin in Metastasis

“…ROCK1 has been shown to phosphorylate Ezrin at Thr567 [ 148 ]. Interestingly, Ezrin can act upstream and downstream of ROCK1 in the RhoA/ROCK signaling pathway, where ROCK1 activates Ezrin, dissociates RhoGDI from RhoA, and subsequently activates RhoA/ROCK [ 149 ]. In Jurkat cells, Fas-mediated apoptosis is regulated by the Rho/ROCK-dependent phosphorylation of Ezrin and Moesin, where Rho or ROCK inhibition prevents their phosphorylation, disrupting the formation of Fas aggregates and subsequent apoptosis induction [ 150 ].…”