Role of Ezrin in Asthma-Related Airway Inflammation and Remodeling

Ezrin is an actin binding protein connecting the cell membrane and the cytoskeleton, which is crucial to maintaining cell morphology, intercellular adhesion, and cytoskeleton remodeling. Asthma involves dysfunction of inflammatory cells, cytokines, and airway structural cells. Recent studies have shown that ezrin, whose function is affected by extensive phosphorylation and protein interactions, is closely associated with asthma, may be a therapeutic target for asthma treatment. In this review, we summarize studies on ezrin and discuss its role in asthma-related airway inflammation and remodeling.


Introduction
Global Initiative for Asthma (GINA) guidelines suggest that asthma is a serious global health problem, affecting patients of all age groups [1]. Its prevalence is increasing globally, with the number of affected patients estimated to increase to 400 million by the year 2025 [2]. It is a heterogeneous disorder characterized by chronic airway inflammation and remodeling [3][4][5][6]. Since asthma occurs with chronic airway inflammation, corticosteroids are the most widely prescribed antiinflammatory agents for it [7]. Type 2 innate lymphoid cells (ILC2), type 2 T-helper (Th2) cells, and related cytokines (such as IL-4, IL-5, and IL-13) are major players in the pathological changes and clinical symptoms of asthma, and increasing attention has been paid to anti-IL-5, anti-IL-5Rα, and anti-IL-4Rα therapy [8]. However, asthma remains poorly controlled in about 53% of patients across Western Europe and Asia [9,10], and patients with severe disease respond poorly to corticosteroids [11]. Moreover, about 30-60% of patients with refractory disease have fixed airway obstruction, attributed to airway remodeling, including increased smooth muscle mass, thickened reticular basement membrane, subepithelial collagen deposition, epithelial desquamation, goblet cell hyperplasia, and angiogenesis [3,12,13]. Airway remodeling might be a consequence of inflammation, or vice versa, therefore it is necessary to develop an approach to understanding the complex interplay between inflammation and airway structure.
Ezrin is a plasma membrane and cytoskeleton linking protein that is concentrated in actin-rich surface structures [14]. The plasma membrane-cytoskeleton complex is involved in physiological and pathological states of cell regulation, such as cell migration, cell proliferation, cell adhesion, and cytoskeletal remodeling [15]. Research demonstrates that ezrin may have a role in the pathogenesis of asthma by affecting both bronchial epithelial repair and contraction of airway smooth muscle cells. Moreover, it regulates the rhoassociated protein kinase (ROCK), NF-κB, and cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signal transduction pathways [16,17]. In this review, we summarize studies on the structure, distribution, and regulation of ezrin, and discuss its role in airway inflammation and remodeling in asthma.

What Is Ezrin?
2.1. Gene and Structure. Ezrin is a principal member of the ezrin-radixin-moesin (ERM) family of proteins, and shares about 75% of the amino acid identity [18][19][20]. It was originally isolated from chicken intestinal brush border epithelium as a component of the microvilli in 1983 and named after Ezra Cornell, cofounder of Cornell University, where it was discovered [21][22][23]. Villin 2 (now called ezrin), the gene-encoding ezrin, is located on chromosome 6q25-6q26, contains 13 exons, is 24 kb long, and has an apparent molecular weight of 81 kDa [24,25]. In 1988, ezrin was isolated from the microvillar membranes of human choriocarcinoma cells as a 75 kDa protein and named cytovillin 2 [26]. The difference in apparent molecular weight could be due to the structural changes caused by phosphorylation, which affects its electrophoretic mobility [16].
Ezrin is the most studied member of the ERM family, and its structural composition has been highly conserved in the evolutionary process. The protein contains 586 amino acids and is mainly composed of three domains, the Nterminal FERM (F for 4.1 protein, E for ezrin, R for radixin, and M for moesin) domain, a central α-helical domain, and the C-terminal ERM association domain (C-ERMAD) [27][28][29]. The FERM domain is a 30 kDa composite domain (residues 1-296), which comprises three subdomains: F1, a ubiquitin-like domain; F2, with four α-helices; and F3, a pleckstrin homology domain [23]. These subdomains form a trilobed, cloverleaf structure with a rough resolution of 70 Å by 70 Å across and 40 Å deep [30][31][32][33]. The C-ERMAD domain (residues 470-586) contains the F-actin binding site that associates with the FERM domain of the N-terminal or actin [34,35]. A central α-helical domain (residues 297-469) lies between the FERM domain and the C-ERMAD domain, which is composed of three helices: α1H, α2H, and α3H, forming a coiled-coil in the monomer structure [36]. A linker region is shown after the α-helical domain [37] (Figure 1).
Ezrin can have monomer and dimer states, with the former being a stable configuration that can function independently. It has an active open form localized at the plasma membrane and a dormant closed form localized in the cytoplasm [38]. In the dormant closed form, the coiled-coil in the α-helical domain folds back on itself and the C-ERMAD associates with the FERM domain of its own or other ERM family members to form monomers or homodimers/heterodimers, with actin and other membrane receptor binding sites wrapped inside [39,40]. In this form, ezrin fails to cross-link the plasma membrane and the cytoskeleton. FERM is a labile domain [14], and ezrin is activated in the open conformation when phosphatidylinositol 4, 5bisphosphate (PIP2) binds to the FERM domain, exposing C-ERMAD sites such as threonine and tyrosine for phosphorylation [15].

Expression and Subcellular Distribution.
Ezrin is expressed in the lungs, intestines, stomach, and kidneys, and it exhibits clear tissue specificity [41]. It is mainly located in the epidermal rather than muscle cells [42,43]. In the small intestine, ezrin expression is limited to the absorptive epithelium and the visceral mesothelium and is absent in the endothelium and smooth muscle [44]. It is mainly distributed on the apical aspect of the plasma membrane which is rich in actin, such as the microvilli, filopodia, ruffling membranes, and cell adhesion sites [24,45]. Of note, ezrin remains inactive in the cytoplasm and exerts its key regulatory role in plasma membrane-based processes via direct interactions with other membrane proteins [46].

Posttranslational Modifications
3.1. Phosphorylation. Protein functions can be regulated by various posttranslational modifications (PTMs), like phosphorylation [47]. Ezrin is mainly subject to protein phosphorylation, which allows it to exert its crucial regulatory role. Ezrin is activated by recruitment to the plasma membrane by PIP2 and subsequent phosphorylation [48][49][50]. Thr567 in C-ERMAD is the most recognized target for phosphorylation-mediated ezrin activation [23]. It is phosphorylated by Nck-interacting kinase (NIK) and G proteincoupled receptor kinase 2 (GRK2), exposing the active sites through uncoupling of FERM and C-terminal domains. Thus, the transition of ezrin from inactive oligomers into active monomers occurs [51][52][53]. Phospho-deficient mutants, generated through replacement of Thr567 with alanine, exhibited increased apoptosis, demonstrating the necessity of Thr567 for ezrin activation [54]. Thr235 is phosphorylated by cyclindependent kinase 5 (CDK5) and cooperates with Thr576 for full activation of ezrin [23,55] (Figure 1, Table 1).
Ezrin can undergo tyrosine (Tyr) phosphorylation through stimulation by platelet-derived and hepatocyte growth factors [41]. Tyr145 and Tyr146 can be phosphorylated by tyrosine specific Src kinases [56,57]. Tyr190 may act as a docking point for the binding of Src Homology-2 (SH2) to ezrin, and that becomes the premise for phosphorylation of Tyr145 [24]. Tyr270 is on the β strand of the outer β sheet in subdomain F3, and its phosphorylation may affect FERM-membrane interaction [30]. Tyr291 phosphorylation might weaken FERM and C-terminal interaction and keep ezrin in an open state [30]. Tyr353 phosphorylation is regulated through the phosphoinositide 3-kinase (PI3K)/Akt pathway and mediated by CD81; leading to reorganization of the actin cytoskeleton and activation of B cells [58][59][60]. Tyr354 phosphorylation affects ezrin's link to mobility and cellular adhesion through the modulation of cellular tubulogenesis [57,61]. Tyr477 phosphorylation promotes cell motility and invasion induced by Src kinase, which are required for the intrinsic Tyr kinase activity of growth factor receptors for epidermal growth factor (EGF) [62,63] ( Figure 1, Table 1).
Ser66 of subdomain F1, is a substrate for PKA, and its phosphorylation plays an important role in mediating remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells [64]. Ser535 is essential 2 Mediators of Inflammation for inhibiting the nuclear accumulation of Bach2 and its repressor activity in B cells, while Ser535 and Ser536 phosphorylation might weaken FERM and C-terminal interaction [30,65] (Figure 1, Table 1).

Dephosphorylation and Other
PTMs. Ezrin is also regulated through dephosphorylation and hydrolysis of PIP2. Dephosphorylation is the process of removing phosphate groups from an organic compound (such as ATP) by hydrolysis. During cell division, a kinetochore-localized PP1-sds22 complex induces dephosphorylation and inactivation of polar ezrin [66]. The dephosphorylation of ezrin induced by T-cell receptor is related to the redistribution of CD43 and the formation of an immunological synapse. Activation of stromal cell-derived factor-1 (SDF-1) and its downstream phospholipase C (PLC) rapidly reduced PIP2 levels and induced ezrin dephosphorylation and disassociation from the cell membrane in lymphocytes. [ [41,70]. Arg579 and Glu244 form a tight salt bridge, which is important for the affinity between FERM and the C-terminal [30,71]. Lys60 acetylation has also been reported, and it may impair the phosphorylation-dependent interactions of ezrin [72] (Figure 1, Table 1).

Protein Interactions
4.1. Interaction of Ezrin with PIP2. PIP2 constitutes only 1% of the total cellular acidic lipid; however, it is the most abundant phosphoinositide that recruits ezrin to the plasma membrane [73][74][75]. A binding site, specific for PIP2 that uncouples FERM from the C-terminal plays an essential role in the activation of ezrin [76][77][78]. The lysines located in subdomains F1 and F3 have a strong effect on the binding of ezrin to PIP2 [14,73]. Priming of ezrin for phosphorylation requires an appropriate charge distribution provided by phosphate groups in the head group of PIP2, and a high local concentration of the head group can only be achieved in vitro using micelles or liposomes. Moreover, the central α-helical coiled-coil hairpin is necessary for the priming of ezrin for phosphorylation, which counteracts the strong association between FERM and C-ERMAD. Upon binding with PIP2, this effect was enhanced [79]. Additionally, PIP2 binding increased the number of weak bonds between ezrin and F-actin, thereby upregulating the adhesion energy and membrane tension to regulate cell adhesion, migration, and morphology [80].

Interaction of Ezrin with Other
Proteins. Several proteins can directly or indirectly interact with the FERM domain. PIP2 binds to cytoplasmic tails of intracellular adhesion molecule-1, -2 (ICAM-1, -2), inducing an interaction between ICAM-1, -2 and ezrin, thus participating in the phosphoinositide signaling pathways [81]. Meanwhile, a complex between the cytoplasmic tail of CD44 and FERM or the entire length of ezrin forms, and coordinates the spatial and temporal localization of PIP2-CD44-ezrin complex-mediated signaling events [82]. CD43 regulates T-cell trafficking through Ser76 in its cytoplasmic tail, while its binding to ezrin is crucial for Ser76 phosphorylation [83]. Ezrin regulates E-cadherindependent adherens junction assembly by activating the GTPase Rac1 rather than RhoA or Cdc42 [84]. The Na + /H + exchanger 1 (NHE1) activates and recruits ezrin to specific plasma membrane, thus regulating cell survival as a scaffold for the recruitment of a signal complex that consists of PI3K and Akt [85,86]. Ezrin can anchor to the plasma membrane using postsynaptic density proteins (PDZs), such as the Na + -H + exchanger regulatory factor (NHERF1), also known as the ERM-binding phosphoprotein 50 (EBP50), and regulatory proteins such as NHE3 kinase (E3KARP, also known as NHERF2) [23,87]. PKA is linked physically and functionally to cystic fibrosis transmembrane conductance regulator (CFTR) by an A-kinase anchoring protein (AKAP) interaction, suggesting that ezrin serves as an AKAP for PKAmediated phosphorylation of CFTR [88], and as the specific binding site, ezrin binds to PKA at the α-helical domain (amino acids 363-470) [89,90]. (Table 2).

Role of Ezrin in Asthma-Related Airway
Inflammation and Remodeling 5.1. Role of Ezrin in Asthma-Related Airway Inflammation. As is known to all, the pathological mechanism of asthma is type 2 airway inflammation involving type 2 innate lymphoid cells (ILC2) and type 2 T-helper (Th2) cells. Allergens are the main triggers [91]. Airway epithelial cells are the first line of defense, releasing alarmins interleukin-25 (IL-25), thymic stromal lymphopoietin (TSLP), ezrin, and initiating the immune cascade, leading to a release of interleukin-4 (IL-4) and interleukin-13 (IL-13) [92]. IL-4 and IL-13 are called prototypical type 2 cytokines, which can cause immunoglobulin E (IgE) production and eosinophils can transport to the airways [91], thus triggering an asthma attacks. Furthermore, IL-13 induces the phosphorylation of Janus Kinase 2 (JAK2), followed by increased nuclear translocation of phosphorylated signal transducers and activators of transcription 6 (STAT6), leading to downregulated ezrin expression, which is related to IL-13-induced epithelial injury [93]. Ezrin may be a potential biomarker of epithelial damage in asthma ( Figure 2). Mast cells are crucial in asthma; they secrete various proinflammatory mediators [94]. IgE-mediated crosslinking of the high-affinity receptor for IgE (FcεRI) with allergens induces the p21-activated kinase 1 (Pak1) binding and activation to PP2A. PP2A then dephosphorylates ezrin at threonine 567 to promote the degranulation of mast cells [95], leading to the asthmatic response ( Figure 2).
As an anti-inflammatory mediator, IL-10 is expressed by B cells and plays a role in suppressing the immune response of asthma [106]. The research shows that the genetic deletion of ezrin in B cells amplifies B-cell receptor (BCR) signaling, and ezrin phosphorylation limits the production of IL-10 in B cells [107]. Interestingly, ezrin peptide therapy involving human ezrin peptide one (HEP1) and regulatory ezrin peptide glycine 3 (RepG3) may switch the "receptor" form of ezrin to the submembrane active form, organizing cell signaling complexes and quickly engaging a cellsurface receptor, to enhance B cell activation and IL-10 production and suppress inflammation [108]. Hence the regulation of ezrin-multiprotein complexes is rather complicated.
The severity of asthma is closely related to airway inflammation; thus, reducing airway inflammation is vital in asthma treatment. Ezrin is overexpressed in the bronchi of humans with chronic airway diseases [105], involving ILC2, Th2, mast cells, neutrophils, B cells, IL-4, IL-13, IL-8, IL-1β, TNF-α, and other cytokines. All these findings will contribute to finding new targets for asthma treatment.

Role of Ezrin in Asthma-Related Airway
Remodeling. Airway remodeling, based on airway inflammation, is one of the pathological characteristics of asthma, including epithelial damage, goblet cell hyperplasia, abnormal deposition of extracellular matrix (ECM), airway smooth muscle (ASM) cell proliferation, fibroblast activation, and angiogenesis [3,109].

Ezrin and Epithelial
Integrity. The major defense mechanism of the airway epithelium is mucociliary clear-ance conducted by airway ciliary cells cooperating with the protective mucous layer [110]. Ezrin via NHERF1 regulates the beating of airway ciliary cells by promoting the apical surface localization of β2 adrenergic receptor (β2AR) [110]. IL-13 directly acts on airway epithelial cells, interferes with the apical localization of ezrin, affects the differentiation of ciliary cells, reduces the ciliary beat frequency of ciliated epithelial cells, and increases mucus production [111]. Research also suggests that forkhead box protein J1 (foxj1) is required for apical localization of ezrin in airway epithelial cells [112]. IL-13 phosphorylates STAT-6 and transfers it to the nucleus to bind the foxj1 promoter to inhibit the expression of foxj1. Ezrin was lost from the apical cell compartment, leading to cilia loss and goblet cell hyperplasia [113]. Besides, in airway epithelial cells, the Ezrin-E3KARP-PKA complex stimulates CFTR Cl-channels through cAMP to regulate water and salt balance [114][115][116][117]. Clearly, ezrin plays an important role in maintaining epithelial integrity (Figure 3).

Ezrin and Epithelial-Mesenchymal Transition (EMT).
EMT refers to the phenotypic change from epithelial to mesenchymal morphology, a key step of airway remodeling in asthma [118]. Among them, collagen I, α-smooth muscle actin (α-SMA), and fibronectin are mesenchymal markers [119]. Transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF) are well-known inducers of EMT [120]. TGF-β1 binds to its receptor, increasing collagen I via the Smad-dependent pathway. Ezrin is required for TGF-β1-induced EMT in airway epithelial cells [121]. TGF-β1 also induces the expression of CD44, and ezrin activated by ROCK forms a complex, leading to EMT induction   Mediators of Inflammation in alveolar epithelial cells [122]. Another inducer EGF induces phosphorylation of Ezrin Tyr353 and Thr567, causing EMT. It may be related to the NF-κB pathway [123,124].

Ezrin, ASM Cells, and Fibroblasts. ASM cells and fibro-
blasts are key cells driving airway remodeling in asthma [3]. The ASM cells are considered to be part of the contractile or relaxant apparatus of the airways [125]. As an AKAP, ezrin combines with EBP50 to form a complex with PKA, activates cAMP signaling in ASM cells, and regulates the contraction and relaxation of ASM cells. This signaling can also drive CCAAT-enhancer-binding protein β (c/EBPβ) to cause IL-6 gene expression and cytokine production, thus promoting the inflammatory process and airway remodeling [126,127].
Fibroblasts are the major producer of ECM in asthmatic airways [3]. For one thing, ezrin may plays role in maintenance of fibroblast size and mechanical force by organizing actin filaments and regulating cortical stiffness. For another, ezrin promotes the proliferation of fibroblasts and collagen deposition through YAP (the cascade effector of the Hippo signaling pathway), causing ECM and fibrosis [128,129].

Discussion
Increasing attention has been paid to ezrin in asthma pathophysiology. We summarized that ezrin regulates airway inflammation through type 2 immune cells, mast cells, neutrophils, and B cells and participates in airway remodeling via epithelial cells, EMT, ASM cells, and fibroblasts in asthma. Ezrin has various phosphorylation sites and binding partners and plays pivotal roles depending mainly on the form of the multi-protein complex. However, the related biological and clinical significance requires more study to explore its role as a potential biomarker and therapeutic target in asthma treatment.

Conflicts of Interest
The authors declare no conflict of interest.