Moesin Expression Research Articles

Elevated Expression of Moesin in Muscular Dystrophies

Fibrosis is the main complication of muscular dystrophies. We identified moesin, a member of the ezrin-radixin-moesin family, in dystrophic muscles of mice representing Duchenne and congenital muscular dystrophies (DMD and CMD, respectively) and dysferlinopathy, but not in the wild type. High levels of moesin were also observed in muscle biopsy specimens from DMD, Ullrich CMD, and merosin-deficient CMD patients, all of which present high levels of fibrosis. The myofibroblasts, responsible for extracellular matrix protein synthesis, and the macrophages infiltrating the dystrophic muscles were the source of moesin. Moesin-positive cells were embedded within the fibrotic areas between the myofibers adjacent to the collagen type I fibers. Radixin was also synthesized by the myofibroblasts, whereas ezrin colocalized with the myofiber membranes. In animal models and patients' muscles, part of the moesin was in its active phosphorylated form. Inhibition of fibrosis by halofuginone, an antifibrotic agent, resulted in a major decrease in moesin levels in the muscles of DMD and CMD mice. In summary, the results of this study may pave the way for exploiting moesin as a novel target for intervention in MDs, and as part of a battery of biomarkers to evaluate treatment success in preclinical studies and clinical trials.

MiR-200c Inhibits the Tumor Progression of Glioma via Targeting Moesin.

We attempt to demonstrate the regulatory role of miR-200c in glioma progression and its mechanisms behind. Here, we show that miR-200c expression was significantly reduced in the glioma tissues compared to paratumor tissues, especially in malignant glioma. Exogenous overexpression of miR-200c inhibited the proliferation and invasion of glioma cells. In addition, the in vivo mouse xenograft model showed that miR-200c inhibited glioma growth and liver metastasis, which is mainly regulated by targeting moesin (MSN). We demonstrated that the expression of MSN in glioma specimens were negatively correlated with miR-200c expression, and MSN overexpression rescued the phenotype about cell proliferation and invasion induced by miR-200c. Moreover, knockdown of MSN was able to mimic the effects induced by miR-200c in glioma cells. These results indicate that miR-200c plays an important role in the regulation of glioma through targeting MSN.

Expression and clinical significance of moesin and E-cadherin in invasive carcinoma of breast, no specific type

To investigate the correlation of moesin and E-cadherin with biological behavior of breast cancer and its mechanism by comparing expression of moesin and E-cadherin in breast invasive carcinoma of no specific type(BIC-NST), breast ductal carcinoma in situ(BDCIS) and normal breast tissues adjacent to carcinoma. Breast cancer cases of the Huizhou Municipal Center People Hospital were collected between Jan 2008 and Dec 2010, expression of moesin and E-cadherin in 104 cases of BIC-NST, 84 cases of BDCIS and 53 cases of normal breast tissues adjacent to carcinoma were detected by tissue-microarray and SP immunohistochemical staining. Western blot was used to detect moesin expression of 16 BIC-NST fresh tissues. Expression rate of moesin in BIC-NST and BDCIS were significantly higher than normal tissues(P<0.01), but the expression rate of E-cadherin in BIC-NST and BDCIS were significantly lower than those of normal tissues(P<0.01). Expression rate of moesin in BIC-NST grade Ⅲ group was significantly higher than that of the grade Ⅰ group.There was a significantly positive correlation between histological grade and moesin expression(P<0.05). However, E-cadherin expression rate in BIC-NST grade Ⅲ group was significantly lower than that in grade Ⅰ group , and there was a significantly negative correlation between histological grade and E-cadherin expression(P<0.05). Moreover, no significant correlation was observed between moesin and E-cadherin expression in BDCIS tissues. Expression of moesin in clinical stage Ⅱ + Ⅲ BIC-NST was significantly higher than that in stage Ⅰ(P<0.01) . Expression of moesin was significantly associated with lymph node metastasis (P<0.01). But no significant correlation was observed between moesin expression and age, tumor size and vascular invasion . However, expression of E-cadherin in clinical stage Ⅱ+ Ⅲ BIC-NST was significantly lower than that in stage Ⅰ(P<0.01). Expression of E-cadherin was significantly associated with lymph node metastasis and vascular invasion (P<0.01). But no significant correlation was observed between E-cadherin expression, age and tumor size. There was a negative correlation between expression of moesin and E-cadherin in BIC-NST(P=0.021)and BDCIS(P=0.032). Higher moesin and lower E-cadherin signal transduction is closely related to the recurrence and development of breast carcinoma, therefore moesin and E-cadherin might provide new targets for gene therapy in breast carcinoma.

Moesin is activated in cardiomyocytes in experimental autoimmune myocarditis and mediates cytoskeletal reorganization with protrusion formation

Acute myocarditis is a self-limiting disease. Most patients with myocarditis recover without cardiac dysfunction in spite of limited capacity of myocardial regeneration. Therefore, to address intrinsic reparative machinery of inflamed hearts, we investigated the cellular dynamics of cardiomyocytes in response to inflammation using experimental autoimmune myocarditis (EAM) model. EAM was induced by immunization of BALB/c mice with α-myosin heavy chain peptides twice. The inflammatory reaction was evoked with myocardial damage with the peak at 3 wk after the first immunization (EAM3w). Morphological and functional restoration started from EAM3w, when active protrusion formation, a critical process of myocardial healing, was observed in cardiomyocytes. Shotgun proteomics revealed that cytoskeletal proteins were preferentially increased in cardiomyocytes at EAM3w, compared with preimmunized (EAM0w) hearts, and that moesin was the most remarkably upregulated among them. Immunoblot analyses demonstrated that the expression of both total and phosphorylated moesin was upregulated in isolated cardiomyocytes from EAM3w hearts. Immunofluorescence staining showed that moesin was localized at cardiomyocyte protrusions at EAM3w. Adenoviral vectors expressing wild-type, constitutively active and inactive form of moesin (wtMoesin, caMoesin, and iaMoesin, respectively) were transfected in neonatal rat cardiomyocytes. The overexpression of wtMoesin and caMoesin resulted in protrusion formation, while not iaMoesin. Finally, we found that cardiomyocyte protrusions were accompanied by cell-cell contact formation. The expression of moesin was upregulated in cardiomyocytes under inflammation, inducing protrusion formation in a phosphorylation-dependent fashion. Moesin signal could be a novel therapeutic target that stimulates myocardial repair by promoting contact formation of cardiomyocytes.

Retinoic acid induces nuclear FAK translocation and reduces breast cancer cell adhesion through Moesin, FAK, and Paxillin

Breast cancer is the most common malignancy in women, with metastases being the cause of death in 98%. In previous works we have demonstrated that retinoic acid (RA), the main retinoic acid receptor (RAR) ligand, is involved in the metastatic process by inhibiting migration through a reduced expression of the specific migration-related proteins Moesin, c-Src, and FAK. At present, our hypothesis is that RA also acts for short periods in a non-genomic action to cooperate with motility reduction and morphology of breast cancer cells. Here we identify that the administration of 10−6 M RA (10–20 min) induces the activation of the migration-related proteins Moesin, FAK, and Paxillin in T-47D breast cancer cells. The phosphorylation exerted by the selective agonists for RARα and RARβ, on Moesin, FAK, and Paxillin was comparable to the activation exerted by RA. The RARγ agonist only led to a weak activation, suggesting the involvement of RARα and RARβ in this pathway. We then treated the cells with different inhibitors that are involved in cell signaling to regulate the mechanisms of cell motility. RA failed to activate Moesin, FAK, and Paxillin in cells treated with Src inhibitor (PP2) and PI3K inhibitor (WM), suggesting the participation of Src-PI3K in this pathway. Treatment with 10−6 M RA for 20 min significantly decreased cell adhesion. However, when cells were treated with 10−6 M RA and FAK inhibitor, the RA did not significantly inhibit adhesion, suggesting a role of FAK in the adhesion inhibited by RA. By immunofluorescence and immunoblotting analysis we demonstrated that RA induced nuclear FAK translocation leading to a reduced cellular adhesion. These findings provide new information on the actions of RA for short periods. RA participates in cell adhesion and subsequent migration, modulating the relocation and activation of proteins involved in cell migration.

Proteomic analysis of fine-needle aspiration in differential diagnosis of thyroid nodules

Thyroid nodules are common in the general population and vary widely in their propensity to harbor thyroid malignancies. The category of follicular lesion of undetermined significance, for instance, carries only a 15% risk of malignancy. The overarching aim of this work was the proteomic study of thyroid cancer because more effort needs to be placed on differentiating malignant thyroid nodules to avoid unnecessary thyroidectomy. We used 2-dimensional electrophoresis coupled to nano-liquid chromatography electrospray ionization tandem mass spectrometry, to examine fine-needle aspiration (FNA), which was easily attainable from the wash of the syringe used for classical FNA biopsy. Overall, we found 25 different proteins able to discriminate benign from malignant samples. The different expression of moesin; annexin A1 (ANXA1); cornulin (CRNN); lactate dehydrogenase; enolase; protein DJ-1; and superoxide dismutase was confirmed in FNA by enzyme-linked immunosorbent assay or Western blot. Receiver operating characteristic curves were calculated to investigate the discriminative power of our marker. The best performance in diagnosis was obtained by combining ANXA1, enolase, protein DJ-1, superoxide dismutase, and CRNN. In addition, the most highly ranked proteins, from the perspective of follicular lesion of undetermined significance, were ANXA1 and CRNN. The research of these candidate biomarkers has then been widened to other biological fluids, such as serum and whole saliva. In conclusion, we believe that when a decision by a thyroid nodule biopsy cannot be distinctly made, the combination of our biomarkers may be one of the criteria to be taken into account for the final decision, together with the identification of ANXA1 in serum and saliva.

Immunohistochemical assessment of ezrin and moesin in colorectal carcinoma

ABSTRACTEzrin and moesin are important molecules of the ERM family of proteins, which regulate cell adhesion and migration. The aim of this study was to evaluate the intensity and pattern of ezrin and moesin expression in colorectal carcinoma (CRC) together with correlating their expression with the clinico-pathologic features of this neoplasm. This study was carried out on 48 CRC and 10 adenoma specimens. All adenoma and 95% of CRC cases showed both ezrin and moesin expression. Ezrin was predominantly cytoplasmic in adenoma cases in comparison to membranous localization in carcinoma cases. Moesin was predominantly expressed in stroma (inflammatory cells and fibroblasts) in carcinoma (89.1%) compared with adenoma (50%). High H-score of ezrin expression was associated with adenocarcinoma type (P = .024) and was inversely correlated with mitotic count (P = .005). High H-score of moesin expression was associated with early Dukes staging of CRC (P = .016), absence of lymph node involvement (P = .022), and low number of involved lymph nodes (P = .04). The association of ezrin with favorable prognostic parameters may be due to its prominent membranous localization. The stroma of CRC could stand against invasion by expression of moesin. Ezrin and moesin are independently expressed from each other.

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.

Prolactin Promotes Breast Cancer Cell Migration through Actin Cytoskeleton Remodeling.

The role of prolactin on breast cancer development and progression is debated. Breast cancer progression largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, actin-binding proteins are requested to achieve fibrillar actin de-polymerization and relocation at the cell membrane. Kinases such as focal adhesion kinase (FAK) are later required to form actin/vinculin-enriched structures called focal adhesion complexes, which mediate firm adhesion to the extracellular matrix. These controllers are regulated by c-Src, which forms multiprotein signaling complexes with membrane receptors and is regulated by a number of hormones, including ­prolactin. We here show that breast cancer cells exposed to prolactin display an elevated c-Src expression and phosphorylation. In parallel, increased moesin and FAK expression and phosphorylation are found. These molecular changes are associated to relocation to the plasma membrane of cytoskeletal actin fibers and to increased horizontal cell movement. In conclusion, prolactin regulates actin remodeling and enhances breast cancer cell movement. This finding broadens the understanding of prolactin actions on breast cancer cells, highlighting new pathways that may be relevant to on breast cancer progression.

Semaphorin 4A enhances lung fibrosis through activation of Akt via PlexinD1 receptor.

Semaphorin 4A plays a regulatory role in immune function and angiogenesis. However, its specific involvement in controlling lung fibrosis, a process that is closely related to angiogenesis and inflammation is still poorly understood. In the present study, we show that treatment of Sema4A on normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that Sema4A enhances the ability of lung fibroblasts to contract collagen gel. Sema4A treatment led to resistance to apoptosis in normal lung fibroblasts. Relative to normal lung fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease Systemic Sclerosis (SSc) showed elevated Sema4A secretion, enhanced alpha-SMA, ezrin, moesin, and paxillin expression, and high ability to induce collagen gel contraction. Using neutralizing antibody against Sema4A receptor, PlexinD1, we found that endogenous Sema4A signalling in SSc fibroblast was through PlexinD1 receptor. We then identified the signalling mechanism through which Sema4A-PlexinD1 promotes the ability of normal fibroblasts to contract a collagen gel matrix. Western blot analysis showed that Sema4A activated the Akt pathway in lung fibroblasts, and the specific inhibitor of Akt pathway, Akt inhibitor III, blocked the ability of Sema4A to promote the ability of lung fibroblasts to contract a collagen gel matrix. Thus, blocking Sema4APlexinD1- Akt cascades might be beneficial in reducing pulmonary fibrosis.

Mechanisms of Stromal Fibrosis

PurposeThis study was designed to uncover new drug targets to deal with the problems of fibrosis of the corneal stroma which can occur following an injury or infection, preventing the normal recovery of vision. Although, TGF‐β is clearly involved the detailed intracellular mechanisms necessary for a practical drug target are not available. The cellular target is the transition of the sessile keratocyte to a myofibroblast.MethodsCorneas of 8 week old C57BL6 mice underwent either an anterior keratectomy (AK) wound or infection with Pseudomonas aeruginosa (PA), and animals were sacrificed at 2 and 7 days, as well as 2 and 4 weeks after the procedures. The isolated stroma was used to monitor the expression and location of moesin, phospho‐moesin, TGF‐β1 and α‐SMA.ResultsTGF‐β1 and phospho‐moesin were not detected in normal corneal stromas. However, after either treatment, TGF‐β1 expression increased, along with phospho‐moesin and moesin in the wounded corneal stroma from day 2 to 7, and decreased after 2 weeks. No expression of TGF‐β1 and phospho‐moesin was found at PO week 4. Myofibroblasts positive for α‐SMA associated with either treatment were detected from day 2 to week 4 and peaked at week 2.ConclusionThese studies show that moesin interactions with actin fiber growth may be a focus drug development in fibrosis decreases; however, it remains to determine the exact details of that interaction.

Abstract 338: Immunohistochemical expression of CD44/ERM complex proteins in penile carcinoma

Abstract Background: The loco-regional involvement is considered the main complication and cause of death in the natural history of penile carcinoma. Understanding the molecular processes that may be involved in metastatic dissemination would improve the prognostic tools to better evaluate the patients, avoiding the morbidity of unnecessary surgery. Some studies have emphasized the influence of cellular adhesion in the development of tumorigenesis and metastasis as the proteins of complex CD44/ERM (Ezrin/Radixin/Moesin). Therefore, this study aimed to analyze the expression of proteins CD44 and its variant CD44v6, Ezrin and Moesin in squamous cell penile carcinoma, correlating their expression with the clinical-pathological criteria. Material and Methods: Eighty four patients were selected, retrospectively, undergoing surgery for squamous cell penile carcinoma at AC Camargo Cancer Center. The expression evaluation for CD44, CD44v6 Ezrin and Moesin was performed by immunohistochemistry using Ventana® equipment, subsequently analyzed by digital microscopy in Aperio® system. The immunoreactivity of each protein was categorized as positive and negative using a cut-off criteria established by the average percentage obtained. Results: CD44 expression was present in 19 (22.6%) positive cases, CD44v6 in 50 (59.5%), Ezrin in 30 (35.7%) and Moesin in 53 (63.1%). It was observed that the loss of CD44 expression was significantly correlated with recurrence (p = 0.002) and with the glans invasion (p = 0.035); the positive expression of CD44v6 was significantly correlated with the tumor size (p = 0.009) and with recurrence (p = 0.018); the loss of Ezrin expression was significantly correlated with macroscopic invasion (p = 0.028) and with foreskin of invasion (p = 0.015); the positive expression of Moesin was significantly correlated with macroscopic invasion (p = 0.010) and the loss of its expression was significantly correlated with recurrence (p = 0.035). Disease-free survival was significantly influenced by CD44, CD44v6 and Moesin. Conclusion: The expression of CD44, CD44v6, Ezrin and Moesin seems to significantly influence the prognostic factors of penile carcinoma and could be an important molecular marker in the identification of recurrence risk. Citation Format: Juliana B. de Andrade, Fernando A. Soares. Immunohistochemical expression of CD44/ERM complex proteins in penile carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 338. doi:10.1158/1538-7445.AM2015-338

Abstract 3320: Identification of potential molecular targets related to radioresistance in human oral cancers

Abstract In India, oral cancer is the most common cancer in males and ranks third among females, attributed predominantly to the use of smokeless tobacco. Radiotherapy is an integral part of oral cancer treatment either alone or in combination with surgery and chemotherapy. However the development of radioresistance creates a hurdle in the efficacy of radiotherapy. Therefore, exploring the differential molecular profile of established radioresistant versus parental oral cancer cells may help in predicting the clinical effectiveness of radiotherapy. The present study aims to profile the radioresistant cell lines established by low dose radiation from their parental oral cancer cell lines, to identify the molecules and the possible mechanisms associated with radioresistance in oral cancer. We have established three radioresistant oral cancer cell lines i.e. 70Gy-AW13516, 70Gy-AW8507 and 70Gy-SCC029B derived from their parental AW13516, AW8507 and SCC029B cell lines respectively using in-vitro fractionated ionizing radiation (FIR). A clinically admissible 2Gy radiation dose was given using a 60Co-γ linear accelerator (Bhabhatron-2, ACTREC) upto 70Gy totally for each of the cell lines. The radioresistant character was determined by clonogenic cell survival assay and proteomic profiling of parental versus radioresistant cells was done by two-dimensional gel electrophoresis. Gels were analysed by PD-Quest software (Bio-Rad) and differential spot identities were revealed by MALDI-TOF/TOF (Bruker Ultra flex-II). To explore changes at transcript level; cDNA microarray was performed by Affymetirx Gene Chip array and analysed by Gene Spring GX-12.5 software. Mass spectrometry (MS) identified a total of 106 differentially expressed proteins among the three replicate sets of each parental and radioresistant 2-D gels with significant MS/MS score. Some of the differentially expressed proteins like Prohibitin, Moesin, PCNA, Keratin-8, Vimentin, HSP-70 and 14-3-3 sigma were validated by western blotting. We further, transiently knocked down Moesin expression by siRNA strategies and demonstrated a significant decrease in the radioresistant character of 70Gy-AW13516 cells as compared to control siRNA treated cells, indicating a role for Moesin in acquired radioresistance of oral cancer cells. Also differentially expressed genes (&amp;gt;3 fold) of radioresistant cells, were mapped by David (NIH) &amp; Panther (USC) functional tools. The pathway analysis revealed genes related to cell survival (PI3K, P38), Wnt and Apoptosis signalling. The proteomic and transcriptomic profiling of radioresistant oral cancer cell lines have revealed several radioresistance associated molecules that may be utilised in predicting cellular response to radiotherapy and the pathways identified may provide new insights in understanding the mechanism underlying clinical radioresistance in oral cancers. Citation Format: Tanuja R. Teni, Mohd Yasser, Sagar Pawar. Identification of potential molecular targets related to radioresistance in human oral cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3320. doi:10.1158/1538-7445.AM2015-3320

Moesin regulates the motility of oral cancer cells via MT1-MMP and E-cadherin/p120-catenin adhesion complex

The present study aimed to clarify the role of Moesin in oral squamous cell carcinoma (OSCC) progression, especially in regulation of cell motility. Immunohistochemistry and western blotting were used to investigate the expression of Moesin, E-cadherin, p120-catenin and MT1-MMP in normal epithelia, dysplasia and OSCCs. Then, Moesin was knockdown by siRNA in OSCC cell lines, WSU-HN6 and CAL27, and the biological role of Moesin in cell adhesion and motility was evaluated by transwell system, cell spreading and aggregation assays. The interactions between Moesin, MT1-MMP and E-cadherin/p120-catenin complex were determined by co-immunoprecipitation and immunofluorescence. Moesin expression was found decreased in the membrane and increased in cytoplasm during the malignant transformation of oral epithelia, and cytoplasmic overexpression of Moesin correlated with nodal metastasis and poor prognosis of OSCCs. Furthermore, Moesin-silencing induced an increased cell-cell adhesion but decreased invasiveness, which was subsequently demonstrated might due to Moesin-mediated E-cadherin and p120-catenin interaction. Meantime, Moesin-silencing significantly down-regulated MT1-MMP expression, accompanied by reduced cell motility and impaired filopodia formation, which was also observed when MT1-MMP knockdown by RNAi or tissue inhibitor (TIMP2), indicating the involvement of MT1-MMP in Moesin-mediated cell motility. Finally, the relationship between Moesin, E-cadherin and MT1-MMP was confirmed in OSCC tissue samples. Taken together, our results indicate Moesin may regulate cell motility through its interactions with MT1-MMP and E-cadherin/p120-catenin adhesion complex and cytoplasmic expression of Moesin correlates with nodal metastasis and poor prognosis of OSCCs, indicating Moesin may be a potential candidate for targeted gene therapy for OSCCs.

The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility.

Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.

Role of moesin in HMGB1-stimulated severe inflammatory responses.

Sepsis is a life-threatening condition that arises when the body's response to infection causes systemic inflammation. High-mobility group box 1 (HMGB1), as a late mediator of sepsis, enhances hyperpermeability, and it is therefore a therapeutic target. Despite extensive research into the underlying mechanisms of sepsis, the target molecules controlling vascular leakage remain largely unknown. Moesin is a cytoskeletal protein involved in cytoskeletal changes and paracellular gap formation. The objectives of this study were to determine the roles of moesin in HMGB1-mediated vascular hyperpermeability and inflammatory responses and to investigate the mechanisms of action underlying these responses. Using siRNA knockdown of moesin expression in primary human umbilical vein endothelial cells (HUVECs), moesin was found to be required in HMGB1-induced F-actin rearrangement, hyperpermeability, and inflammatory responses. The mechanisms involved in moesin phosphorylation were analysed by blocking the binding of the HMGB1 receptor (RAGE) and inhibiting the Rho and MAPK pathways. HMGB1-treated HUVECs exhibited an increase in Thr558 phosphorylation of moesin. Circulating levels of moesin were measured in patients admitted to the intensive care unit with sepsis, severe sepsis, and septic shock; these patients showed significantly higher levels of moesin than healthy controls, which was strongly correlated with disease severity. High blood moesin levels were also observed in cecal ligation and puncture (CLP)-induced sepsis in mice. Administration of blocking moesin antibodies attenuated CLP-induced septic death. Collectively, our findings demonstrate that the HMGB1-RAGE-moesin axis can elicit severe inflammatory responses, suggesting it to be a potential target for the development of diagnostics and therapeutics for sepsis.

Role of moesin in renal fibrosis.

BackgroundRenal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.MethodHuman proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.ResultHK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.ConclusionWe find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin.

Moesin expression in fibrosis in the mouse cornea after sterile mechanical trauma or infection.

The aim of this study was to compare the expression patterns of 3 important biochemical characteristics of fibrosis-moesin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) in the mouse cornea with fibrosis induced by common etiologies-sterile mechanical injury and infection. Corneas of 8-week-old C57BL6 mice were either wounded after an anterior keratectomy or were infected by Pseudomonas aeruginosa, and the animals were killed on days 2 and 7, and on weeks 2 and 4 after the procedure. Western blot and immunofluorescence were used to analyze the expression of moesin and phospho-moesin, and the presence of myofibroblasts identified by the expression of α-SMA in the corneal stroma. The expression of TGF-β1 was analyzed by immunofluorescence. By immunofluorescent analysis, TGF-β1, α-SMA, and phospho-moesin were not detected in the normal corneal stroma. However, after either treatment, TGF-β1 expression increased, along with phospho-moesin in the wounded corneal stroma until day 7, and decreased after week 2. No expression of TGF-β1 and phospho-moesin was found at postoperative week 4. Moesin expression increased until week 2. Myofibroblasts positive for α-SMA were detected on day 2 until week 4 and peaked at week 2. Western blot analysis confirmed the immunofluorescent data for moesin, phospho-moesin, and α-SMA. The similar expression pattern of moesin, phospho-moesin, TGF-β1, and α-SMA in the mouse cornea with fibrosis caused by sterile mechanical injury or infection indicated a role for moesin signaling in corneal fibrosis. Interference with the action of moesin may be a potential approach for intervention strategies to avert fibrosis after infection or mechanical injury.

Cytoskeletal regulators are implicated in fibrosis in mechanical injury and infection in the mouse cornea

AbstractPurpose: Mechanical injury as well as infection can result in corneal fibrosis and loss of transparency. We have used a mouse model of cornea insult using sterile mechanical injury and infection. Methods: Corneas of 8 week old C57BL6 mice underwent either an anterior keratectomy (AK) wound or infection with Pseudomonas aeruginosa (PA), and animals were sacrificed at 2 and 7 days, as well as 2 and 4 weeks after the procedures. The isolated stroma was used to monitor the expression and location of moesin, phospho‐moesin, TGF‐β1 and α‐SMA. Results: TGF‐β1 and phospho‐moesin were not detected in normal corneal stromas. However, after either treatment, TGF‐β1 expression increased, along with phospho‐moesin and moesin in the wounded corneal stroma from day 2 to 7, and decreased after 2 weeks. No expression of TGF‐β1 and phospho‐moesin was found at PO week 4. Myofibroblasts positive for α‐SMA associated with either treatment were detected from day 2 to week 4 and peaked at week 2. Conclusion: The results suggest a role for moesin signaling in corneal fibrosis. Interfering with the action of moesin is a potential target for interventive strategies to avert corneal fibrosis.

G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.