Moesin-induced signaling in response to lipopolysaccharide in macrophages.
OBJECTIVE
Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane-organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)-mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS-induced moesin signaling pathways in macrophages.


METHODS
Macrophages were stimulated with 500 ng/mL LPS in macrophage serum-free medium. For blocking experiments, cells were pre-incubated with anti-moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real-time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD-2 and Toll-like receptor 4 (TLR4) and co-immunoprecipitation of MyD88-interleukin-1 receptor-associated kinase (IRAK) and IRAK-tumor necrosis factor receptor-activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor kappaB (NF-kappaB) and IkappaBalpha were studied. Tumor necrosis factor alpha, interleukin-1beta and interferon beta were measured by ELISA.


RESULTS
Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl-terminus. Moesin is temporally associated with TLR4 and MD-2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide-induced signaling is transferred downstream to p38, p44/42 MAPK and NF-kappaB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF-kappaB activation and translocation to the nucleus.


CONCLUSIONS
These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.
Zawawi
K H
KH
Department of Preventive Dental Science, Division of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia.
Kantarci
A
A
Schulze-Späte
U
U
Fujita
T
T
Batista
E L
EL
Amar
S
S
Van Dyke
T E
TE
D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology* / Q000378:metabolism; D000704:Analysis of Variance; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D006801:Humans; D020672:I-kappa B Proteins / Q000276:immunology / Q000378:metabolism; D007113:Immunity, Innate / Q000502:physiology*; D018836:Inflammation Mediators / Q000276:immunology / Q000378:metabolism; D016899:Interferon-beta / Q000096:biosynthesis / Q000276:immunology; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology*; D051218:Lymphocyte Antigen 96 / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000276:immunology / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051192:Receptors, Pattern Recognition / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D018709:Statistics, Nonparametric; D048029:TNF Receptor-Associated Factor 6 / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000276:immunology
D048868:Adaptor Proteins, Signal Transducing; D016207:Cytokines; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008840:Microfilament Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D051192:Receptors, Pattern Recognition; D048029:TNF Receptor-Associated Factor 6; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C071058:moesin; D016899:Interferon-beta; D053592:Interleukin-1 Receptor-Associated Kinases; D020928:Mitogen-Activated Protein Kinases