Ezrin, Radixin And Moesin Proteins Research Articles

Store-operated calcium entry enhances the polarization and chemotaxis of neutrophils in the peripheral venous blood of patients with bronchial asthma by upregulating ERM protein.

Neutrophils can be rapidly recruited and are largely abundant in the airways of patients with asthma. However, whether the polarization and chemotaxis of neutrophils in patients with asthma are abnormal, and the underlying mechanisms, have not been clarified. Pseudopods formation is the initial step of neutrophils' polarization, ezrin, radixin and moesin (ERM) play an important role in the polarization of neutrophils. As an important signaling molecule in cell physiological processes, Ca2+ has been shown to be involved in the polarity changes of neutrophils. This study thus aimed to explore polarization and chemotaxis of neutrophils in patients with asthma and the underlying mechanism. Fresh neutrophils were isolated using standard separation protocols. The polarization and chemotactic activity of neutrophils were observed using Zigmond chamber and Transwell migration assay under linear concentration gradients of N-formyl-methionine-leucine-phenylalanine (fMLP) or interleukin (IL)-8. The distribution of calcium, ERMs and F-actin in neutrophils were observed by confocal laser scanning microscope. The expression of the main components of ERMs (moesin and ezrin) was detected with reverse transcription-polymerase chain reaction (RT-PCR). Compared with those in the healthy control group, the polarization and chemotaxis of neutrophils in the venous blood of patients with asthma were significantly increased, and the expression and distribution of cytoskeletal proteins F-actin and ezrin were abnormal. The expression and function of key components of store-operated calcium entry (SOCE), stromal interaction molecule 1 (STIM1), STIM2, and Orai1 of neutrophils in patients with asthma were significantly increased. The polarization and chemotaxis of neutrophils in the venous blood of patients with asthma are increased. This may be due to the abnormal expression and distribution of ERM and F-actin as a result of abnormal SOCE function.

Examination of the Interplay between Membrane Tension and the Tight Junction Protein, ZO‐1

Zonula occluden‐1 (ZO‐1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO‐1 knocked‐down (ZO‐1 KD) or knocked‐out (ZO‐1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO‐1 KO, which may explain the phenotypic changes. The function of ZO‐1 as a regulator of myosin II was confirmed by exposing the ZO‐1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild‐type MDCK II cells (WT) and the curvature of the junctions. Flipper‐TR, which is a live cell fluorescent membrane tension probe, along with fluorescence‐lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO‐1 KO cells compared to WT cells. The Flipper‐TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO‐1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO‐1 KO cells. To measure the response of ZO‐1 KO cells to increased tension, the expression of tension‐related genes is being measured using quantitative real‐time polymerase chain reaction (qRT‐PCR). These tension‐related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO‐1 loss and regulation of membrane tension.

The interaction of ATP11C-b with ezrin contributes to its polarized localization

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.

Moesin is involved in microglial activation accompanying morphological changes and reorganization of the actin cytoskeleton

Moesin is a member of the ezrin, radixin and moesin (ERM) proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia, immune cells in the central nervous system, show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. In microglia, moesin is the predominant ERM protein. Here, we show that microglial activation after systemic lipopolysaccharide application is partly inhibited in moesin knockout (Msn-KO) mice. We prepared primary microglia from wild-type and Msn-KO mice, and studied them to compare their phenotypes accompanying morphological changes and reorganization of the actin cytoskeleton induced by UDP-stimulated phagocytosis and ADP-stimulated migration. The Msn-KO microglia showed higher phagocytotic activity in the absence of UDP, which was not further increased by the treatment with UDP. They also exhibited decreased ADP-stimulated migration activities compared with the wild-type microglia. However, the Msn-KO microglia retained their ability to secrete tumor necrosis factor α and nitric oxide in response to lipopolysaccharide.

The ERM Complex: A New Player Involved in Diabetes-induced Vascular Leakage.

Microvascular complications remain an important cause of morbidity in diabetic patients, and they are associated with a significant economic burden for healthcare systems. Vascular leakage is one of the earlier hallmarks in diabetic microvascular complications. Ezrin, Radixin and Moesin (ERM) proteins have recently been involved in vascular dysfunction under the effect of molecular mediators of diabetes complications. In this review, we will present the available evidence regarding the role of these proteins in vascular leakage and their putative implication in diabetic microvascular complications. A comprehensive literature search of the electronic MEDLINE database was performed between November 2017 and January 2018. As a result, 36 articles have been reviewed and discussed. ERM proteins are cytoskeleton-membrane linkers, and when activated in endothelial cells are able to induce cytoskeleton reorganization in stress fibers leading to the disassembly of focal adhesions and the formation of paracellular gaps which result in an increase of vascular permeability. The activation of these proteins is induced by mediators involved in diabetic complications such as PKC activation, TNF-α, AGEs and oxidative stress. In conclusion, ERMs play an essential role in endothelium homeostasis and can be envisaged as a new therapeutic molecular target for preventing or arresting diabetes-induced vascular leakage.

Inactivation of Cyclin-Dependent Kinase 5 in Hair Cells Causes Hearing Loss in Mice.

Cyclin-dependent kinase 5 (CDK5) is abundantly expressed in post-mitotic cells including neurons. It is involved in multiple cellular events, such as cytoskeletal dynamics, signaling cascades, gene expression, and cell survival, et al. Dysfunction of CDK5 has been associated with a number of neurological disorders. Here we show that CDK5 is expressed in mouse cochlear hair cells, and CDK5 inactivation in hair cells causes hearing loss in mice. CDK5 inactivation has no effect on stereocilia development in the cochlear hair cells. However, it affects stereocilia maintenance, resulting in stereocilia disorganization and eventually stereocilia loss. Consistently, hair cell loss was significantly elevated by CDK5 inactivation. Despite that CDK5 has been shown to play important roles in synapse development and/or function, CDK5 inactivation does not affect the formation of ribbon synapses of cochlear hair cells. Further investigation showed that CDK5 inactivation causes reduced phosphorylation of ERM (ezrin, radixin, and moesin) proteins, which might contribute to the stereocilia deficits. Taken together, our data suggest that CDK5 plays pivotal roles in auditory hair cells, and CDK5 inactivation causes hearing loss in mice.

Moesin expression by tumor cells is an unfavorable prognostic biomarker for oral cancer

BackgroundMoesin is a member of the ERM (ezrin, radixin and moesin) proteins that participate in cell migration and tumor invasion through transductional signals sent to actin filaments by glycoproteins, such as podoplanin.MethodsThis study aimed to evaluate the participation of moesin and podoplanin in the invasive tumor front of oral squamous cell carcinomas, and their influence on patients’ prognosis. Podoplanin and moesin immunoexpressions were evaluated by a semi-quantitative score method, based on the capture of 10 microscopic fields, at 400X magnification, in the invasive tumor front of oral squamous cell carcinomas. The association of moesin and podoplanin expression with clinicopathological variables was analyzed by the chi-square, or Fisher’s exact test. The 5 and 10 years survival rates were calculated by the Kaplan-Meier method and the survival curves were compared by using the log-rank test.ResultsThe immunohistochemical expression of moesin in the invasive front of oral squamous cell carcinomas was predominantly strong, homogenously distributed on the membrane and in the cytoplasm of tumor cells. The expression of moesin was not associated with clinical, demographic and microscopic features of the patients. Otherwise, podoplanin expression by malignant epithelial cells was predominantly strong and significantly associated with radiotherapy (p = 0.004), muscular invasion (p = 0.006) and lymph node involvement (p = 0.013). Strong moesin expression was considered an unfavorable prognostic factor for patients with oral squamous cell carcinomas, clinical stage II and III (p = 0.024).ConclusionsThese results suggested that strong moesin expression by malignant cells may help to determine patients with oral squamous cell carcinoma and poor prognosis.

The Role of CD44 and ERM Proteins in Expression and Functionality of P-glycoprotein in Breast Cancer Cells.

Multidrug resistance (MDR) is often attributed to the over-expression of P-glycoprotein (P-gp), which prevents the accumulation of anticancer drugs within cells by virtue of its active drug efflux capacity. We have previously described the intercellular transfer of P-gp via extracellular vesicles (EVs) and proposed the involvement of a unique protein complex in regulating this process. In this paper, we investigate the role of these mediators in the regulation of P-gp functionality and hence the acquisition of MDR following cell to cell transfer. By sequentially silencing the FERM domain-binding proteins, Ezrin, Radixin and Moesin (ERM), as well as CD44, which we also report a selective packaging in breast cancer derived EVs, we have established a role for these proteins, in particular Radixin and CD44, in influencing the P-gp-mediated MDR in whole cells. We also report for the first time the role of ERM proteins in the vesicular transfer of functional P-gp. Specifically, we demonstrate that intercellular membrane insertion is dependent on Ezrin and Moesin, whilst P-gp functionality is governed by the integrity of all ERM proteins in the recipient cell. This study identifies these candidate proteins as potential new therapeutic targets in circumventing MDR clinically.

Chondrocyte dedifferentiation increases cell stiffness by strengthening membrane-actin adhesion

Chondrocyte dedifferentiation is known to influence cell mechanics leading to alterations in cell function. This study examined the influence of chondrocyte dedifferentiation in monolayer on cell viscoelastic properties and associated changes in actin organisation, bleb formation and membrane-actin cortex interaction. Micropipette aspiration was used to estimate the viscoelastic properties of freshly isolated articular chondrocytes and the same cells after passage in monolayer. Studies quantified the cell membrane-actin cortex adhesion by measuring the critical pressure required for membrane detachment and bleb formation. We then examined the expression of ezrin, radixin and moesin (ERM) proteins which are involved in linking the membrane and actin cortex and combined this with theoretical modelling of bleb dynamics. Dedifferentiated chondrocytes at passage 1 (P1) were found to be stiffer compared to freshly isolated chondrocytes (P0), with equilibrium modulus values of 0.40 and 0.16 kPa respectively. The critical pressure increased from 0.59 kPa at P0 to 0.74 kPa at P1. Dedifferentiated cells at P1 exhibited increased cortical F-actin organisation and increased expression of total and phosphorylated ERM proteins compared to cells at P0. Theoretical modelling confirmed the importance of membrane-actin cortex adhesion in regulating bleb formation and effective cellular elastic modulus. This study demonstrates that chondrocyte dedifferentiation in monolayer strengthens membrane-actin cortex adhesion associated with increased F-actin organisation and up-regulation of ERM protein expression. Thus dedifferentiated cells have reduced susceptibility to bleb formation which increases cell modulus and may also regulate other fundamental aspects of cell function such as mechanotransduction and migration.

Therapeutic Potential of Beta-Nicotinamide Adenine Dinucleotide to Reverse Endothelial Barrier Dysfunction in Sickle Cell Mouse Model

Background: Thepulmonary endothelium is a metabolically active monolayer on the luminal surface of the lung vasculature. It regulates a variety of functions including the passage of macromolecules and fluid between the blood and lung interstitial tissue. Inflammatory agents such as bacterial endotoxin lipopolysaccharide (LPS) cause lung endothelial cells (EC) dysfunction leading to pulmonary edema predisposing to acute chest syndrome, a leading cause of death in sickle cell disease (SCD); therefore, a drug which repairs the damaged EC barrier function might be of therapeutic potential. Recently we demonstrated that β-nicotinamide adenine dinucleotide (β-NAD) reverses LPS-induced EC barrier dysfunction in a model of acute lung injury (Exp Lung Res 2012, 38, 223-32). In this study, we investigated the protective effect of β-NAD against the damaging effects of LPS using primary ECs from human and sickle cell mouse lungs as well as survival studies using the preclinical sickle cell mouse model.Methods: Early passaged human lung microvascular EC (HLMVEC) were obtained from Lonza and mouse lung microvascular EC (MLMVEC) were isolated from the Townes knock-in homozygous sickle cell (SS) and heterozygous sickle trait (AS) transgenic mice. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) technique. Western blot was done to measure phosphorylation of proteins involved in the regulation of EC barrier function. Survival studies were performed in SS mice treated with intraperitoneal (IP) LPS (0.05 mg/kg) alone or combined with β-NAD (11 mg/kg); normal saline served as a control. Lungs were harvested at 10-12 hrs from the LPS group and at 24 hrs from saline or LPS/β-NAD groups and stained with Hematoxylin and Eosin for to assess interstitial edema and myeloperoxidase as a marker of neutrophil infiltration.Results: Barrier function measurements using ECIS of HLMVEC indicate that β-NAD (100 µM) enhanced basal EC barrier function by 50-60%. In addition, β-NAD attenuated LPS-induced EC gap formation and actin stress fibers. In human lung endothelium, myosin light chain phosphatase (MLCP) plays a crucial role in the maintenance of the EC barrier integrity. Furthermore, LPS mediates MLCP inactivation by phosphorylating the MLCP regulatory subunit MYPT1 and cytoskeletal targets ERM (Ezrin, Radixin, and Moesin) and CaD (Caldesmon). Western blot analysis demonstrated that β-NAD reversed LPS-induced phosphorylation of MYPT1, ERM and CaD proteins. To determine a functional role, we performed studies using 10 nM MYPT1 siRNA treatment followed by β-NAD (100 μM) for 5 hrs. We observed a 50% attenuation of β-NAD enhanced barrier function suggesting MYPT1 is involved in the mechanism of action for β-NAD. Subsequent studies with SS-MLMVEC demonstrated β-NAD mediated a 40% improvement in barrier function which was comparable to HLMVEC. Further, LPS treatment for 16 hrs produced a 25% decrease in barrier function in SS-MLMVEC compared to a 7% decrease in AS-MLMVEC suggesting impaired EC barrier function in SS lungs. Finally we performed survival studies to determine the ability of β-NAD to protect against LPS-induced mortality in SS mice. In dose optimization studies of LPS (0.1-1.0 mg/kg/dose), SS mice survived for 10-12 hrs with the 0.05 mg/kg LPS dose. By contrary, all LPS doses tested had no effect on AS mice survival. Based on this result, SS mice were given IP injections of saline (3 mice), LPS (4 mice) or LPS/β-NAD (4 mice) and survival rates were monitored. The saline or LPS/β-NAD treated mice survived for 24 hrs (100%) compared with LPS mice which died by 10-12 hrs (p=0.01) supporting a protective effect of β-NAD. Histological assessment of the lung tissue showed increased interstitial edema by Hematoxylin and Eosin stain and neutrophil infiltration by myeloperoxidase in the LPS-treated lungs; these changes were much less prominent in the lungs from LPS/β-NAD treated SS mice.Conclusions: Our data support a role of MLCP and its cytoskeletal targets in the protective effects of β-NAD against LPS-induced HLMVEC barrier dysfunction. Studies using SS-MLMVEC suggest that SS mice have impaired barrier function and greater susceptibility to LPS toxicity. However, β-NAD treatment improved survival of SS mice supporting the therapeutic potential of this agent to treat the complication of acute chest syndrome in SCD. DisclosuresMakala:Georgia Regents University: Employment.

Moesin and myosin phosphatase confine neutrophil orientation in a chemotactic gradient.

Neutrophils respond to invading bacteria by adopting a polarized morphology, migrating in the correct direction, and engulfing the bacteria. How neutrophils establish and precisely orient this polarity toward pathogens remains unclear. Here we report that in resting neutrophils, the ERM (ezrin, radixin, and moesin) protein moesin in its active form (phosphorylated and membrane bound) prevented cell polarization by inhibiting the small GTPases Rac, Rho, and Cdc42. Attractant-induced activation of myosin phosphatase deactivated moesin at the prospective leading edge to break symmetry and establish polarity. Subsequent translocation of moesin to the trailing edge confined the formation of a prominent pseudopod directed toward pathogens and prevented secondary pseudopod formation in other directions. Therefore, both moesin-mediated inhibition and its localized deactivation by myosin phosphatase are essential for neutrophil polarization and effective neutrophil tracking of pathogens.

ERM proteins colocalize with the Na+/H+ exchanger NHE1 in MCF‐7 breast cancer cell invadopodia and affect invadopodia number

Invadopodia are actin‐rich membrane protrusions which have been assigned essential roles in cancer cell invasiveness. Here, we investigate the roles of ezrin, radixin and moesin (ERM), three closely related F‐actin‐plasma membrane linker proteins implicated in metastasis, and their binding partner the Na+/H+ exchanger isoform 1 (NHE1), in the formation and function of invadopodial rosettes in MCF‐7 human breast cancer cells. ERM proteins colocalized strongly with NHE1 in invadopodia in MCF‐7 cells +/− constitutively active, N‐terminally truncated ErbB2 (ΔNErbB2). In contrast, the tumor suppressor Merlin which is more distantly related to the ERM proteins, did not. siRNA‐mediated knockdown of NHE1 increased the average number of invadopodia per cell in ΔNErbB2‐expressing cells but not in vector controls. Overexpression of either dominant negative (DN) or constitutively active (CA) ezrin decreased the number of invadopodia per cell, in vector control cells only. Average invadopodial rosette diameter was unaffected by knockdown of ezrin or NHE1, or by expression of DN‐ or CA‐ezrin. Thus, in MCF‐7 cells, NHE1 colocalizes strongly with ERM proteins in invadopodial rosettes and both NHE1 and ezrin appear to affect their formation. Studies are ongoing to determine the generality and roles of these interactions in cancer invasiveness.Funding: Danish Cancer Society, Danish Research Council

Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs

T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs.

Cadherin adhesome at a glance

This article is part of a Minifocus on Adhesion. For further reading, please see related articles: ‘Cycling around cell–cell adhesion with Rho GTPase regulators’ by Jessica McCormack et al. ( J. Cell Sci. 126 , 379-391). ‘E-cadherin–integrin crosstalk in cancer invasion and metastasis’

Phosphorylation of moesin by c-Jun N-terminal kinase is important for podosome rosette formation in Src-transformed fibroblasts

Podosomes are actin-based membrane protrusions that facilitate extracellular matrix degradation and motility of invasive cells. Podosomes can self-organize into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and some highly invasive cancer cells. However, the mechanism of this assembly remains obscure. In this study, we show that the suppression of Jun N-terminal kinase (JNK) by the JNK inhibitor SP600125 or short-hairpin RNA inhibited podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. In addition, SrcY527F was less able to induce podosome rosettes in JNK1-null or JNK2-null mouse embryo fibroblasts than in wild-type counterparts. The kinase activity of JNK was essential for promoting podosome rosette formation but not for its localization to podosome rosettes. Moesin, a member of the ERM (ezrin, radixin and moesin) protein family, was identified as a substrate of JNK. We show that the phosphorylation of moesin at Thr558 by JNK was important for podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. Taken together, our results unveil a novel role of JNK in podosome rosette formation through the phosphorylation of moesin.

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin.

Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Recent reviews extensively discuss their biological functions [1], [2], [3], [4]. In this review, we will first remind the main features of this family of proteins, which are known as linkers and regulators of plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments.

Protein Phosphatase 1α Mediates Ceramide-induced ERM Protein Dephosphorylation: A NOVEL MECHANISM INDEPENDENT OF PHOSPHATIDYLINOSITOL 4, 5-BIPHOSPHATE (PIP2) AND MYOSIN/ERM PHOSPHATASE

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP(2)) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP(2) hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP(2) and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1α, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1α acted as dominant negative of the endogenous PP1α. Additional results showed that the ceramide mechanism of PP1α activation is largely independent of PIP(2) hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1α and plasma membrane ceramide.

Tumor suppressive microRNA-133a regulates novel targets: Moesin contributes to cancer cell proliferation and invasion in head and neck squamous cell carcinoma

Recently, many studies suggest that microRNAs (miRNAs) contribute to the development, invasion and metastasis of various types of human cancers. Our recent study revealed that expression of microRNA-133a (miR-133a) was significantly reduced in head and neck squamous cell carcinoma (HNSCC) and that restoration of miR-133a inhibited cell proliferation, migration and invasion in HNSCC cell lines, suggesting that miR-133a function as a tumor suppressor. Genome-wide gene expression analysis of miR-133a transfectants and TargetScan database showed that moesin (MSN) was a promising candidate of miR-133a target gene. MSN is a member of the ERM (ezrin, radixin and moesin) protein family and ERM function as cross-linkers between plasma membrane and actin-based cytoskeleton. The functions of MSN in cancers are controversial in previous reports. In this study, we focused on MSN and investigated whether MSN was regulated by tumor suppressive miR-133a and contributed to HNSCC oncogenesis. Restoration of miR-133a in HNSCC cell lines (FaDu, HSC3, IMC-3 and SAS) suppressed the MSN expression both in mRNA and protein level. Silencing study of MSN in HNSCC cell lines demonstrated significant inhibitions of cell proliferation, migration and invasion activities in si-MSN transfectants. In clinical specimen with HNSCC, the expression level of MSN was significantly up-regulated in cancer tissues compared to adjacent non-cancerous tissues. These data suggest that MSN may function as oncogene and is regulated by tumor suppressive miR-133a. Our analysis data of novel tumor-suppressive miR-133a-mediated cancer pathways could provide new insights into the potential mechanisms of HNSCC oncogenesis.

Two-State Conformations in the Hyaluronan-Binding Domain Regulate CD44 Adhesiveness under Flow Condition

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.