ALI and its severe form, ARDS, are potentially fatal complications of sepsis and important causes of high mortality in critically ill patients . Several recent studies have linked ERM to LPS-induced lung injury [4–6]. However, the specific role of ezrin during this process has yet to be elucidated. In this paper, we describe a critical function for ezrin in LPS-induced pro-inflammatory cytokine production.
Our results demonstrate that LPS induced the phosphorylation of ezrin in a concentration- and time- dependent manner. LPS up-regulated RhoA activity and ROCK expression. The blockade of the RhoA/ROCK signaling pathway inhibited LPS- induced ezrin phosphorylation and its translocation from cytoplasm to cell membrane. Interestingly, ezrin interacted with Syk in a constitutive manner; in contrast, ezrin only associated with MyD88/IRAK1 under LPS challenge. Further analysis demonstrated that LPS-induced p38 and NF-κB activation was ezrin-dependent. More importantly, the suppression of ezrin by siRNA, or the blockade of ROCK activation with Y-27632, led to a reduction in the production of TNF-α, IL-1β and HMGB1 in response to LPS. These findings provide evidence that ezrin is upregulated in a RhoA/ROCK-dependent manner by LPS and that ezrin acts as an upstream regulator of p38 and NF-κB activation and the production of pro-inflammatory cytokines.
As a member of the ERM protein family, ezrin is considered not only as a cross-linker between the cytoskeleton and plasma membrane, but also an important signal transducer that participates in cell adhesion and motility . For these cellular functions, ezrin needs to be activated. When subjected to stimuli, ezrin binds to phosphatidylinositol 4,5-bisphosphate (PIP2) in the cell membrane and subsequently the threonine residue (ezrinT567) at the C-terminal is phosphorylated, thus leading to dissociation between the N-terminal and C-terminal domains; this unmasks binding sites for other molecules . ERMs are phosphorylated by TNF-α, advanced glycation end products (AGEs), 2-Methoxyestradiol, and thrombin, and are known to modulate endothelial hyperpermeability [24–27]. Here, we report the phosphorylation of ezrin induced by LPS, as well as its translocation from the cytoplasm to the cell membrane in manner that is concomitant with the reorganization of the F-actin cytoskeleton. It has been shown that ERMs are translocated to the plasma membrane as a result of interactions with the cytoplasmic domain of integral membrane proteins, such as CD44, thus providing a site for association with actin filaments . Thus, LPS-induced ezrin phosphorylation was accompanied by cytoskeleton reorganization, indicating that LPS-induced conserved threonine residue phosphorylation might contribute to the formation of actin filaments and the reorganization of the actin cytoskeleton. These results are consistent with previous findings in pulmonary endothelial cells that were stimulated by TNF-α , 2-Methoxyestradiol , and thrombin , thus suggesting that the activation of ezrin is part of a general response to inflammatory stress .
Several protein kinases, including Rho/ROCK and PKC, are known to phosphorylate ERMs on the C-terminal threonine [30, 31]. ERMs can be directly phosphorylated by ROCK to enhance its binding to membrane proteins and F-actin, thus regulating the reorganization of actin filaments to participate in a range of cellular functions [32, 33]. In the present study, pre-treatment with Y-27632 prevented LPS- induced ezrin phosphorylation and its subsequent translocation from the cytosol to the cell membrane, as well as F-actin reorganization, thus suggesting a critical role for ROCK in the activation of ezrin. Interestingly, ERMs can play a dual role in the Rho/ROCK signaling pathway by acting both upstream and downstream of ROCK. ERMs can be activated by ROCK; once activated, ERMs dissociate Rho-GDI (a GDP dissociation inhibitor) from Rho and thereby activates Rho/ROCK [32, 33]. Thus, the rapid activation of ezrin and Rho/ROCK observed in the present study suggests that the levels of phosphorylated ezrin may act as a limiting factor for signaling pathways involving ezrin and that the marked upregulation of ezrin in response to LPS could be an important feed-forward mechanism.
Syk was originally identified in hemopoietic cells where it plays an important role in the regulation of the innate immune response . Syk also acts as a key molecule in signaling processes that are initiated by pattern recognition receptors (PRRs); the role of this molecule in inflammation has been described in non-immune cells, such as the airway epithelium . In a previous study, Urzainqui et al. showed that phosphorylation of the ITAM-like motif on ERMs is critical for recruiting Syk molecules, thus triggering cellular responses . Zawawi et al. reported that cells stimulated with LPS showed clear phosphorylation of IRAK, although cells that were pre-incubated with an anti-moesin antibody exhibited total inhibition of IRAK phosphorylation and associations with IRAK MyD88 . Therefore, it appears that ERMs act as an adapter protein that links Syk to MyD88/IRAK1. To decipher the likely associations between ezrin, Syk, and MyD88/IRAK1, in the presence of LPS, we next performed co-immunoprecipitation experiments. We found that the expression of Syk corresponded with that of ezrin, thus indicating that Syk interacts with ezrin in a constitutive manner. However, MyD88 and IRAK1 only immunoprecipitated with ezrin upon LPS challenge. These results demonstrate that ezrin interacts with MyD88/IRAK1 in a LPS stimulation-dependent manner and suggest that ezrin links Syk to MyD88/IRAK1 following LPS stimulation. ERMs play an important role in LPS- induced inflammation; however, the mechanisms involved remain unclear. Thus, our results suggest that the upregulation of ezrin, and its interaction with Syk/MyD88/IRAK1, can initiate Rho/ROCK-MyD88/IRAK1-ezrin/Syk signaling, and that this is one of the main mechanisms involved in the LPS.
To further explore the functional relevance of the ROCK-ezrin pathway in LPS- induced responses, we next used Y-27632 to inhibit ROCK activity and siRNA to suppress ezrin. We demonstrated that LPS induced significant reductions in the production of TNF-α, IL-1β, and HMGB1. The observed reduction in cytokine production was associated with the inhibition of NF-κB and p38 MAPK activation. Interestingly, Zawawi et al  previously reported that the blockade of moesin function inhibited the LPS-induced activation of MyD88, IRAK and TRAF6, as well as subsequent MAPK activation and NF-κB translocation to the nucleus. Weng et al.  also confirmed that the phosphorylation of ezrin triggered MAPK signal transduction in tumor progression. Thus, our results are consistent with a specific role for the ERMs family in the activation of p38 and NF-κB activation.