Ezrin/radixin/moesin Research Articles

Role of Ezrin/Radixin/Moesin in the Surface Localization of Programmed Cell Death Ligand-1 in Human Colon Adenocarcinoma LS180 Cells.

Programmed cell death ligand-1 (PD-L1), an immune checkpoint protein highly expressed on the cell surface in various cancer cell types, binds to programmed cell death-1 (PD-1), leading to T-cell dysfunction and tumor survival. Despite clinical successes of PD-1/PD-L1 blockade therapies, patients with colorectal cancer (CRC) receive little benefit because most cases respond poorly. Because high PD-L1 expression is associated with immune evasion and poor prognosis in CRC patients, identifying potential modulators for the plasma membrane localization of PD-L1 may represent a novel therapeutic strategy for enhancing the efficacy of PD-1/PD-L1 blockade therapies. Here, we investigated whether PD-L1 expression in human colorectal adenocarcinoma cells (LS180) is affected by ezrin/radixin/moesin (ERM), functioning as scaffold proteins that crosslink plasma membrane proteins with the actin cytoskeleton. We observed colocalization of PD-L1 with all three ERM proteins in the plasma membrane and detected interactions involving PD-L1, the three ERM proteins, and the actin cytoskeleton. Furthermore, gene silencing of ezrin and radixin, but not of moesin, substantially decreased the expression of PD-L1 on the cell surface without affecting its mRNA level. Thus, in LS180 cells, ezrin and radixin may function as scaffold proteins mediating the plasma membrane localization of PD-L1, possibly by post-translational modification.

Mechanical stiffness softening and cell adhesion are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Phosphoinositide Signal Transduction Pathway and Osteosarcoma Metastases

: Metastasis spreading confers a worse prognosis to the clinical outcome among patients suffering from osteosarcoma, the most common primary bone tumor in childhood and adolescence. The detection of molecules involved in metastasis spreading might contribute to understanding tumor dissemination mechanisms, thereby opening the way to novel therapeutic strategies. The Ezrin-radixin-moesin (ERM) family proteins are activated after interacting with molecules belonging to the phosphoinositide signal transduction pathway. The phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule in the PI pathway, stabilizes the ERM proteins or a more efficient receptor binding. The PIP2 levels in the pathway are a critical element for regulating several cell events. The PIP2 levels are regulated using enzymes, including the PI-specific Phospholipase C family. A decrease in the PIP2 levels induces the dissociation of the ERM protein from the membrane. In this regard, the PI-PLC enzymes regulate this event. In this paper, the role of the PI signal transduction molecules in osteosarcoma metastases is discussed.

STK10 knockout inhibits cell migration and promotes cell proliferation via modulating the activity of ERM and p38 MAPK in prostate cancer cells.

Prostate cancer (PCa) is one of the most common types of cancer and is a serious threat to men's health due to the high rate of incidence and metastasis. However, the exact underlying pathology of this malignant disease has yet to be fully elucidated. The ezrin-radixin-moesin (ERM) family of proteins are associated with the development and metastasis of various types of cancer. Serine threonine kinase 10 (STK10) is an ERM kinase that is involved in the activation of ERM proteins and serves essential roles in the aggregation and adhesion of lymphocytes. To evaluate the functional roles of STK10 in the pathogenesis of PCa, a STK10-knockout (KO) DU145 PCa cell line was generated using the CRISPR-Cas9 gene editing system, and the effects of STK10 deletion on tumor biological behaviors were further analyzed. The present data suggested that STK10 KO promoted PCa cell proliferation by inhibiting p38 MAPK activation and suppressed migration primarily via the inhibition of p38 MAPK signaling and ERM protein activation. To the best of our knowledge, this is the first study to provide evidence that STK10 plays important roles in the proliferation and migration of PCa cells, which will be useful for further investigation into the pathogenesis of this disease.

Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells.

The ezrin/radixin/moesin (ERM) family proteins act as linkers between the actin cytoskeleton and P-glycoprotein (P-gp) and regulate the plasma membrane localization and functionality of the latter in various cancer cells. Notably, P-gp overexpression in the plasma membrane of cancer cells is a principal factor responsible for multidrug resistance and drug-induced mutagenesis. However, it remains unknown whether the ERM proteins contribute to the plasma membrane localization and transport function of P-gp in human colorectal cancer cells in which the subcellular localization of ERM has yet to be determined. This study aimed to determine the gene expression patterns and subcellular localization of ERM and P-gp and investigate the role of ERM proteins in the plasma membrane localization and transport function of P-gp using the human colon adenocarcinoma cell line LS180. Using real-time reverse transcription polymerase chain reaction and immunofluorescence analyses, we showed higher levels of ezrin and moesin mRNAs than those of radixin mRNA in these cells and preferential distribution of all three ERM proteins on the plasma membrane. The ERM proteins were highly colocalized with P-gp. Additionally, we show that the knockdown of ezrin, but not of radixin and moesin, by RNA interference significantly decreased the cell surface expression of P-gp in LS180 cells without affecting the mRNA expression of P-gp. Furthermore, gene silencing of ezrin substantially increased the intracellular accumulation of rhodamine123, a typical P-gp substrate, with no alterations in the plasma membrane permeability of Evans blue, a passive transport marker. In conclusion, ezrin may primarily regulate the cell surface localization and transport function of P-gp as a scaffold protein without influencing the transcriptional activity of P-gp in LS180 cells. These findings should be relevant for treating colorectal cancer, which is the second leading cause of cancer-related deaths in males and females combined.

A Direct CLIC5A/Ezrin Interaction Enables CLIC5A‐Dependent Rac1 Activation

Introduction The CLIC (Chloride Intracellular Channel) protein CLIC5A is a component of the ezrin/podocalyxin/actin complex in kidney podocytes, where it stabilizes actin-based projections (foot processes). Similarly, in inner ear hair cells CLIC5A in the radixin complex stabilizes sensory stereocilia, also actin-based projections. ERM (ezrin, radixin, moesin) proteins help organize the cortical actin cytoskeleton, linking membrane-spanning proteins to actin. We have reported that CLIC5A stimulates Rac1-GTP-dependent phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] generation, causing ezrin activation and phosphorylation, thus coupling podocalyxin, via ezrin, to the cytoskeleton (J. Cell Sci. 127:5164, 2014 & Kidney Int. 89:833, 2016). Hypothesis Direct interaction(s) of CLIC5A with Rac1 and/or ezrin enable CLIC5A-dependent Rac1 activation. Methods Rac1 activity was determined by Rac1-GTP specific PAK-PBD pull-down from CLIC5A or vector transfected cells and by Rac1-GTP G-LISA® (Cytoskeleton Inc). Association of CLIC5A with Rac1 was determined by GST-CLIC5A pull-down of exogenously expressed wild-type GFP-Rac1(WT), constitutively active GFP-Rac1 (Q61L) and dominant negative GFP-Rac1 (D17N) from COS7 cell lysates and by in vitro GST-CLIC5A pull-down of GTPγS- or GDP-loaded recombinant His-Rac1. Direct protein-protein interactions were evaluated by Yeast two-Hybrid (Y2H) screening of a mouse kidney protein domain library, and Y2H mapping of interacting domains in the related ERM protein ezrin, using CLIC5A as the bait (Fig 1A). The CLIC5A/ezrin association was studied by co-immunoprecipitation and GST-CLIC5A pull-down. Results Exogenous CLIC5A expression activated Rac1 and CLIC5A was co-precipitated with Rac1-GTP and ezrin by PAK-PBD affinity beads. GST-CLIC5A pulled GFP-Rac1 WT, -Q61L, and -D17N equally from cell lysates, but recombinant GTPγS- or GDP-loaded His-Rac1 did not directly bind GST-CLIC5A, suggesting that the CLIC5A/Rac1 interaction is indirect. The Y2H screen revealed that CLIC5A interacts directly with the C-terminal domain of radixin, and Y2H mapping (Fig. 1A) showed that CLIC5A similarly interacts directly with the ezrin C-terminal domain (432-586). Direct interactions with full-length ezrin (1-586) and the C-terminally deleted ezrin (432-570) domain were not detected. Furthermore, ezrin (432-586), but not full-length ezrin (1-586) or ezrin (432-570) was pulled down by GST-CLIC5A and co-immunoprecipitated with CLIC5A. Finally, siRNA silencing of endogenous ezrin significantly reduced CLIC5A-stimulated Rac1 activation (Fig. 1B). Conclusion Although CLIC5A activates Rac1 and is part of the Rac1-GTP complex, the CLIC5A/Rac1 interaction appears to be indirect. CLIC5A interacts directly with ezrin (and radixin), and Rac1 activation by CLIC5A is ezrin-dependent. The finding that a direct interaction of CLIC5A with full-length ezrin was not detected suggests that only the active, open ezrin conformation, freeing the C-terminus, has a high affinity for CLIC5A. The data suggest that the direct CLIC5A/ezrin interaction may enhance localized Rac1 activation and thus Rac1-GTP-dependent PI(4,5)P2 generation.

Lack of ER Stress in NHERF1‐Deficient Proximal Tubule Cells Exhibiting Aberrant Npt2a Localization

Background NHERF1 (Na+-hydrogen exchanger regulatory factor isoform 1) anchors Npt2a (Na+-dependent phosphate cotransporter type IIa) in the brush border membrane (BBM) of proximal tubules through its cross-linking of Npt2a with the actin-filament binding protein, Ezrin. In the absence of NHERF1, Npt2a accumulates aberrantly in a subapical compartment. We have demonstrated that Npt2a associates with NHERF1 in the ER/Golgi as well as the BBM, suggesting a critical role for NHERF1 in the forward trafficking of Npt2a from the ER/Golgi to the BBM. Because increased levels of ER stress have been associated with intracellular accumulation of mis-localized proteins, we hypothesized that NHERF1 deficient proximal tubule cells may experience chronic ER stress associated with mis-localization of Npt2a. Methods We measured protein expression of four ER stress markers (phosphorylated eIF2α, GRP78, GRP94, and p58IPK) in kidney cortex isolated from C57BL/6J wild type (WT) mice and their NHERF1 knock-out (KO) littermates by immunoblot and mRNA levels by RNAseq. We expressed a fluorescently labeled WT Npt2a and NHERF1 or a Npt2a/NHERF1 chimera substituting the C terminal PDZ binding motif of Npt2a with the ERM (Ezrin-Radixin-Moesin) binding domain of NHERF1 in human embryonic kidney (HEK), opossum kidney (OK), and NHERF1-deficient opossum kidney (OKH) cells. We compared WT vs chimera protein expression by confocal microscopy and protein function by radiolabeled phosphate uptake. Results We found no differences in kidney cortex protein or mRNA expression of these ER stress markers between WT and KO animals. Confocal microscopy of the Npt2a chimera expressed in HEK, OK, and OKH cells exhibited enhanced apical membrane localization when compared to WT Npt2a in all three cell types. However, no increase in radiolabeled phosphate uptake was observed between cells expressing the Npt2a chimera or wild type Npt2a. Conclusions The lack of an ER stress response suggests that despite association between NHERF1 and Npt2a in the ER/Golgi, NHERF1 is not required for normal Npt2a folding. Enhanced apical expression of the Npt2a chimera confirms the essential role of Ezrin in BBM localization of Npt2a, however, appropriate Npt2a function requires the presence of more than the ERM binding domain of NHERF1, possibly involving the association of other unidentified proteins.

Roles of Ezrin in regulation of ciliary beating in lung multiciliated cell

The mucociliary clearance is the first line of defense in the lungs. The function is given by the beating cilia on the surface of ciliated cells cooperating with the protective mucous layer. The beating cilia plays removing inhaled particles and pathogens (viruses, bacteria and fungi). Thus, the beating cilia are the key apparatus to conduct the mucociliary clearance. For ciliary beating to function properly, localization of ciliary proteins that regulate ciliary beating and formation of epithelial cell polarity are important. Ezrin/Radixin/Moesin (ERM) proteins function as general cross-linkers between apical membrane proteins and the actin cytoskeleton and are involved in the functional expression of apical membrane proteins. Especially, ezrin may play the important role of apical localization of β2-adrenergic receptor (β2AR) and cAMP-dependent protein kinase (PKA) in multiciliated cells. Here we examined roles of ezrin in the regulation of ciliary beating in lung multiciliated cells by using ezrin-knockdown (Vil2kd/kd) mice. To evaluate ciliary function, we analyzed ciliary beat frequency (CBF) and ciliary bend distance (CBD, and index of amplitude) in lung multiciliated cells of mice. The multiciliated cells were isolated by an elastase treatment. Ciliary beatings of lung multiciliated cells were observed by the videomicroscopy equipped with high speed camera (500 fps) and CBF and CBD were measured by an image analysis program using recorded images. We performed immunofluorescence microscopy analysis to evaluate the localization of β2AR, Na+/H+ exchanger regulatory factor (NHERF) 1, which is a PSD-95/Discs-large/ZO-1 (PDZ) scaffold protein, and PKA. In addition, we also performed cell surface protein biotinylation assay. In the results, stimulation with 1 nM procaterol, which is selective β2AR agonist, increased the ratios of CBF and CBD by about 80% in WT multiciliated cells. However, in Vil2kd/kd, stimulation with 1 nM procaterol increased the ratios of CBF and CBD by only 40%. While it is no significant difference that the increase of CBF and CBD by stimulation with 100 μM IBMX, which is a broad-spectrum phosphodiesterase inhibitor, in WT and Vil2kd/kd. In addition, the cell surface localization of NHERF1 and β2AR is disturbed in Vil2kd/kd multiciliated cells. There were no apparent changes in the subcellular localizations of PKA RIα and RIIα, which are PKA regulatory subunits, in Vil2kd/kd multiciliated cells. These results suggest that ezrin regulates the ciliary beating in lung multiciliated cells by promoting the apical localization of β2AR.

Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis

The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain.

Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II-containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

Occurrence of a Hybrid Between Taenia saginata and Taenia asiatica Tapeworms in Cambodia

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also ‘pure’ T. asiatica in Cambodia.

NHERF1/EBP50 as a Target for Modulation of MRP Function in HepG2 Cells.

As increased expression and activities of efflux transporters (ETs) often cause drug resistance in cancers, we tried modulating ET activity in cancer cells, using scaffold proteins such as ezrin/radixin/moesin (ERM) proteins, and Na+/H+ exchanger regulatory factor-1 (NHERF1)/ERM-binding phosphoprotein of 50 kDa (EBP50). To see whether EBP50 modulated ET activities in human liver cancer HepG2 cells, we used EBP50 siRNA and a designed TAT-PDZ1 peptide. The EBP50 knockdown (EBP50KD) cells had significantly higher intracellular accumulations of Rho123 and carboxy-dichlorofluorescein (CDF), but not H33342 (i.e., the respective substrates of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP)), compared with control HepG2, suggesting that EBP50 knockdown in HepG2 cells decreased activity of P-gp and MRP but not BCRP. Treatment with TAT-PDZ1 peptide (>1 pM) resulted in significantly higher CDF accumulation in HepG2 cells, which persisted for ≥180 min after TAT-PDZ1 peptide treatment. These results imply that EBP50 can modulate ET activities. To our knowledge, this is the first report on using a competitive peptide to modulate interactions between MRP and EBP50.

The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila.

Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the invivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression ofprotein chaperones requires the nuclear functions of Moesin. DATABASE: GEO accession number: GSE155778.

Prolactin receptor expression and its role in trophoblast outgrowth in human embryos

Research questionWhat is the gene expression pattern of prolactin receptor (PRLR) in human pre-implantation embryos and what are its functions during the embryonic development and adhesion process? DesignA total of 405 discarded human vitrified oocytes and embryos donated for research by consenting couples were used in this study. The oocytes and embryos were used to analyse PRLR expression and to evaluate the influence of prolactin (PRL) supplementation in the embryo culture medium on embryo developmental competence and viability. The rates of blastocyst development and adhesion, outgrowth area, cytoskeletal reorganization and nascent adhesion formation were compared between groups. ResultsPRLR expression increased significantly after embryo compaction (P < 0.0001) and blastulation (P < 0.0001). Supplementation of the embryo culture medium with PRL did not improve the developmental rate and morphological grade. In contrast, blastocyst outgrowth was significantly increased in embryos cultured with PRL (P = 0.0004). Phosphorylation of JAK2, downstream of the prolactin receptor family, was markedly higher in the PRL-treated embryos than in embryos cultured without PRL. Furthermore, the expression of mRNAs encoding ezrin–radixin–moesin proteins and epithelial–mesenchymal transition-related genes was stimulated by the activation of PRL-JAK2 signalling. The PRL-treated embryos had higher mRNA expression of integrins than non-treated embryos, and transcriptional repression of cadherin 1 was observed after PRL treatment. More nascent adherent cells expressed focal adhesion kinase and paxillin in PRL-treated embryos than in non-treated embryos. ConclusionsHuman embryos express PRLR at the morula and blastocyst stages, and PRLR signalling stimulates blastocyst adhesion by promoting integrin-based focal adhesions and cytoskeletal organization during trophoblast outgrowth.

Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma.

In a previous study, we demonstrated that CHL1, the neuronal cell adhesion molecule close homolog of L1, acts as a tumor suppressor in human neuroblastoma (NB), a still highly lethal childhood malignancy, influencing its differentiation and proliferation degree. Here we found that ezrin, one of the ERM (ezrin, radixin, moesin) proteins involved in cytoskeleton organization, strongly interacts with CHL1. The low expression of EZRIN, as well as the low expression of CHL1 and of the neuronal differentiation marker MAP2, correlates with poor outcome in NB patients. Knock-down of ezrin in HTLA-230 cell line induces neurite retraction, enhances cell proliferation and migration, and triggers anchorage-independent growth, with effects very similar to those already obtained by CHL1 silencing. Furthermore, lack of ezrin inhibits the expression of MAP2 and of the oncosuppressor molecule p53, whereas it enhances MAPK activation, all typical features of tumor aggressiveness. As already described, CHL1 overexpression in IMR-32 cell line provokes an opposite trend, but the co-silencing of ezrin reduces these effects, confirming the hypothesis that CHL1 acts in close connection with ezrin. Overall, our data show that ezrin reinforces the differentiating and oncosuppressive functions of CHL1, identifying this ERM protein as a new targetable molecule for NB therapy.

Ezrin Mediates Invasion and Metastasis in Tumorigenesis: A Review.

Ezrin, as encoded by the EZR gene, is a member of the Ezrin/Radixin/Moesin (ERM) family. The ERM family includes three highly related actin filament binding proteins, Ezrin, Radixin, and Moesin. These three members share similar structural properties containing an N-terminal domain named FERM, a central helical linker region, and a C-terminal domain that mediates the interaction with F-actin. Ezrin protein is highly regulated through the conformational change between a closed, inactivate form and an open, active form. As a membrane-cytoskeleton linker protein, Ezrin facilitates numerous signal transductions in tumorigenesis and mediates diverse essential functions through interactions with a variety of growth factor receptors and adhesion molecules. Emerging evidence has demonstrated that Ezrin is an oncogene protein, as high levels of Ezrin are associated with metastatic behavior in various types of cancer. The diverse functions attributed to Ezrin and the understanding of how Ezrin drives the deadly process of metastasis are complex and often controversial. Here by reviewing recent findings across a wide spectrum of cancer types we will highlight the structures, protein interactions and oncogenic roles of Ezrin as well as the emerging therapeutic agents targeting Ezrin. This review provides a comprehensive framework to guide future studies of Ezrin and other ERM proteins in basic and clinical studies.

Importance of cholesterol-rich microdomains in the regulation of Nox isoforms and redox signaling in human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) function is regulated by Nox-derived reactive oxygen species (ROS) and redox-dependent signaling in discrete cellular compartments. Whether cholesterol-rich microdomains (lipid rafts/caveolae) are involved in these processes is unclear. Here we examined the sub-cellular compartmentalization of Nox isoforms in lipid rafts/caveolae and assessed the role of these microdomains in VSMC ROS production and pro-contractile and growth signaling. Intact small arteries and primary VSMCs from humans were studied. Vessels from Cav-1−/− mice were used to test proof of concept. Human VSMCs express Nox1, Nox4, Nox5 and Cav-1. Cell fractionation studies showed that Nox1 and Nox5 but not Nox4, localize in cholesterol-rich fractions in VSMCs. Angiotensin II (Ang II) stimulation induced trafficking into and out of lipid rafts/caveolae for Nox1 and Nox5 respectively. Co-immunoprecipitation studies showed interactions between Cav-1/Nox1 but not Cav-1/Nox5. Lipid raft/caveolae disruptors (methyl-β-cyclodextrin (MCD) and Nystatin) and Ang II stimulation variably increased O2− generation and phosphorylation of MLC20, Ezrin-Radixin-Moesin (ERM) and p53 but not ERK1/2, effects recapitulated in Cav-1 silenced (siRNA) VSMCs. Nox inhibition prevented Ang II-induced phosphorylation of signaling molecules, specifically, ERK1/2 phosphorylation was attenuated by mellitin (Nox5 inhibitor) and Nox5 siRNA, while p53 phosphorylation was inhibited by NoxA1ds (Nox1 inhibitor). Ang II increased oxidation of DJ1, dual anti-oxidant and signaling molecule, through lipid raft/caveolae-dependent processes. Vessels from Cav-1−/− mice exhibited increased O2− generation and phosphorylation of ERM. We identify an important role for lipid rafts/caveolae that act as signaling platforms for Nox1 and Nox5 but not Nox4, in human VSMCs. Disruption of these microdomains promotes oxidative stress and Nox isoform-specific redox signalling important in vascular dysfunction associated with cardiovascular diseases.

Biphasic Force-Regulated Phosphorylation Site Exposure and Unligation of ERM Bound with PSGL-1: A Novel Insight into PSGL-1 Signaling via Steered Molecular Dynamics Simulations.

The PSGL-1-actin cytoskeleton linker proteins ezrin/radixin/moesin (ERM), an adaptor between P-selectin glycoprotein ligand-1 (PSGL-1) and spleen tyrosine kinase (Syk), is a key player in PSGL-1 signal, which mediates the adhesion and recruitment of leukocytes to the activated endothelial cells in flow. Binding of PSGL-1 to ERM initials intracellular signaling through inducing phosphorylation of Syk, but effects of tensile force on unligation and phosphorylation site exposure of ERM bound with PSGL-1 remains unclear. To answer this question, we performed a series of so-called “ramp-clamp” steered molecular dynamics (SMD) simulations on the radixin protein FERM domain of ERM bound with intracellular juxtamembrane PSGL-1 peptide. The results showed that, the rupture force of complex pulled with constant velocity was over 250 pN, which prevented the complex from breaking in front of pull-induced exposure of phosphorylation site on immunoreceptor tyrosine activation motif (ITAM)-like motif of ERM; the stretched complex structure under constant tensile forces <100 pN maintained on a stable quasi-equilibrium state, showing a high mechano-stabilization of the clamped complex; and, in consistent with the force-induced allostery at clamped stage, increasing tensile force (<50 pN) would decrease the complex dissociation probability but facilitate the phosphorylation site exposure, suggesting a force-enhanced biophysical connectivity of PSGL-1 signaling. These force-enhanced characters in both phosphorylation and unligation of ERM bound with PSGL-1 should be mediated by a catch-slip bond transition mechanism, in which four residue interactions on binding site were involved. This study might provide a novel insight into the transmembrane PSGL-1 signal, its biophysical connectivity and molecular structural basis for cellular immune responses in mechano-microenvironment, and showed a rational SMD-based computer strategy for predicting structure-function relation of protein under loads.

Ezrin promotes hepatocellular carcinoma progression by modulating glycolytic reprogramming.

Ezrin, one of the ezrin‐radixin‐moesin (ERM) proteins, is involved in the formation of cell membrane processes and has been implicated in the promotion of cancer proliferation and metastasis. However, the possible role of ezrin in hepatocellular carcinoma (HCC) metastasis and glycolysis reprogramming has remained unclear. In this study, we found that ezrin was upregulated in HCC tissues, and its overexpression was linked with HCC patients’ aggressive tumor characteristics and poor prognosis. Functional experiments further revealed that ezrin overexpression promoted HCC cell proliferation, epithelial‐to‐mesenchymal transition (EMT) progression and angiogenesis. In addition, by measuring glucose consumption, lactate production, ATP levels and the expression of glucose metabolism‐related markers in HCC cells, we investigated whether ezrin regulated glucose metabolism. Moreover, 2‐deoxy‐D‐glucose (2‐DG) affected ezrin‐mediated proliferation, migration and EMT of HCC cells, which suggested that ezrin may, at least in part, promote HCC progression by regulating glycolysis reprogramming. Based on our results, we proposed that ezrin was involved in HCC progression and may be a valid prognostic marker.

Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma.

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.