Manifold reduction of moesin in fetal Down syndrome brain

Moesin is a member of the ERM family and is involved in plasma membrane-actin cytoskeleton cross-linking, resulting cell adhesion, shape, and motility. Because moesin was shown to be highly expressed in growth cones and moesin/radixin suppression led to impaired structure and function of this key element in brain development, we tested the ERM family, ezrin, radixin, and moesin, in fetal Down syndrome (DS) cortex at the early second trimester. We applied two-dimensional gel electrophoresis with subsequent MALDI detection and identification of protein spots followed by quantification with specific software. Moesin was shown to be significantly and manifold reduced in fetal DS brain, whereas reduction of ezrin and radixin did not reach statistical significance. We therefore propose the involvement of moesin in developmental impairment of DS brain, including deteriorated arborisation, neuritic outgrowth, and neuronal migration. Furthermore, decreased moesin is the second F-actin bundling protein, besides drebrin, that is manifold reduced in fetal DS brain.     

Moesin‐dependent cytoskeleton remodelling is associated with an anaplastic phenotype of pancreatic cancer

Cell motility is controlled by the dynamic cytoskeleton and its related proteins, such as members of the ezrin/radixin/moesin (ERM) family, which act as signalling molecules inducing cytoskeleton remodelling. Although ERM proteins have been identified as important factors in various malignancies, functional redundancy between these proteins has hindered the dissection of their individual contribution. The aim of the present study was to analyse the functional role of moesin in pancreatic malignancies. Cancer cells of different malignant lesions of human and transgenic mice pancreata were evaluated by immunohistochemistry. For functional analysis, cell growth, adhesion and invasion assays were carried out after transient and stable knock‐down of moesin expression in pancreatic cancer cells. In vivo tumourigenicity was determined using orthotopic and metastatic mouse tumour models. We now show that moesin knock‐down increases migration, invasion and metastasis and influences extracellular matrix organization of pancreatic cancer. Moesin‐regulated migratory activities of pancreatic cancer cells were in part promoted through cellular translocation of β‐catenin, and re‐distribution and organization of the cytoskeleton. Analysis of human and different transgenic mouse pancreatic cancers demonstrated that moesin is a phenotypic marker for anaplastic carcinoma, suggesting that this ERM protein plays a specific role in pancreatic carcinogenesis.     

Dysregulation of lung injury and repair in moesin-deficient mice treated with intratracheal bleomycin

Moesin belongs to the ezrin/radixin/moesin (ERM) protein family and participates in cellular functions, such as morphogenesis and motility, by cross-linking between the actin cytoskeleton and plasma membranes. Although moesin seems necessary for tissue construction and repair, its function at the whole body level remains elusive, perhaps because of redundancy among ERM proteins. To determine the role played by moesin in the modulation of pulmonary alveolar structure associated with lung injury and repair, we examined the morphological changes in the lung and the effect of bleomycin-induced lung injury and fibrosis in moesin-deficient (Msn(-/Y)) and control wild-type mice (Msn(+/Y)). Immunohistochemical analysis revealed that moesin was specifically localized in the distal lung epithelium, where ezrin and radixin were faintly detectable in Msn(+/Y) mice. Compared with Msn(+/Y) mice, Msn(-/Y) mice displayed abnormalities of alveolar architecture and, when treated with bleomycin, developed more prominent lung injury and fibrosis and lower body weight and survival rate. Furthermore, Msn(-/Y) mice had abnormal cytokine and chemokine gene expression as shown by real-time PCR. This is the first report of a functional involvement of moesin in the regulation of lung inflammation and repair. Our observations show that moesin critically regulates the preservation of alveolar structure and lung homeostasis.     

Ezrin Is Highly Expressed in Early Thymocytes,but Dispensable for T Cell Development in Mice

Background

Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.

Methodology/Principal Findings

We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin^−/− mice likely arise as a consequence of nutritional stress.

Conclusions/Significance

We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.     

Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS

Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin. The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity. The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation. Infection of HeLa cells with P. aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes. Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation. Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins. ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras. Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS. Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells. The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.     

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.     

Cloning and expression profile of chicken radixin

Radixin is a member of the ERM (ezrin/radixin/moesin) family of cytoskeletal linkers. We have cloned chicken radixin as a 4.3 kb cDNA, which encodes an 80 kDa protein that is more than 98% identical to radixin from evolutionarily diverse species. High sequence homology (70-80%) also extends into the 3'-untranslated region (UTR) of the radixin gene. The 3'-UTR of moesin, but not ezrin, was also conserved, suggesting an essential, and possibly specific, regulatory function. A distinct pattern of radixin expression is seen in chicken tissues, suggesting a cell-type-specific function.     

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: a comparative study of the affinity constants and conformational changes

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP(2)-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP(2)-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.     

Distinct cell type-specific expression of scaffolding proteins EBP50 and E3KARP: EBP50 is generally expressed with ezrin in specific epithelia, whereas E3KARP is not

The ezrin/radixin/moesin (ERM) proteins are regulated microfilament membrane linking proteins. Previous tissue localization studies have revealed that the three related proteins show distinct tissue distributions, with ezrin being found predominantly in polarized epithelial cells, whereas moesin is enriched in endothelial cells and lymphocytes. EBP50 and E3KARP are two related scaffolding proteins that bind to the activated form of ERM proteins in vitro, and through their PDZ domains to the cytoplasmic domains of specific membrane proteins, including the Na+/H+ exchanger isoform (NHE3) present in kidney proximal tubules and the beta2-adrenergic receptor. Using specific antibodies to EBP50 and E3KARP for localization in murine tissues, we find that the cellular distribution of EBP50 and E3KARP is mutually exclusive. Epithelial cells expressing ezrin generally co-express EBP50, such as intestinal epithelial cells, gastric parietal cells, the epithelial cells of the kidney proximal tubule, the terminal bronchiole of the lung, and in mesothelia. This correlation is not absolute as cells of the mucous epithelium of the stomach and in the renal corpuscle, express ezrin but no detectable EBP50, whereas the bile canaliculi of hepatocytes express EBP50 and not ezrin. E3KARP has a restricted tissue distribution with the highest expression being found in lung. It is largely colocalized with moesin and radixin, especially in the alveoli of the lung, as well as being highly enriched in the renal corpuscle. These results document a preference for co-expression of EBP50, but not E3KARP, with ezrin in polarized epithelia. These results place constraints on the physiological roles that can be proposed for these scaffolding molecules.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.     

Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.     

The 2.7 A crystal structure of the activated FERM domain of moesin: an analysis of structural changes on activation

Moesin binds to a large range of proteins through its N terminal FERM (band 4.1, ezrin, radixin, moesin) domain. In full-length moesin isolated from cells, this binding is masked by binding to the C-terminal domain of moesin (C-ERMAD). Activation takes place by phosphorylation of Thr 558 in the C-ERMAD, which releases the C-ERMAD. A recently determined crystal structure of a noncovalent complex of the FERM and C-ERMAD domains showed for the first time that the structure of the FERM domain consists of three subdomains, each of which is similar to known structures. The structure reported here also contains a unique 47-residue helix pointing away from the FERM domain at the start of the alpha domain, in agreement with secondary structure predictions. Removal of the C-ERMAD does not result in a huge rearrangement of the FERM domain, but comparison with the activated radixin structure shows a consistent set of small changes. Not surprisingly, the exposed C-ERMAD binding area interacts in crystal contacts. More interestingly, a negatively charged peptide binds to the inositol site in a crystal contact and causes a greater conformational change in the structure than inositol.     

Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression

Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.     

Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.     

Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2^kd/kd) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2^+/+) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2^kd/kd mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2^kd/kd neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2^kd/kd neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.