Moesin-induced signaling in response to lipopolysaccharide in macrophages

Abstract

Background and 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.

Material and 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.

Conclusion: These results suggest an important role for moesin in the innate immune response and TLR4-mediated pattern recognition in periodontal disease.

Fig 1

Expression and phosphorylation of moesin after lipopolysaccharide (LPS) stimulation. (A) Moesin is increasingly expressed in LPS-stimulated macrophages as determined by Q-PCR. Changes in expression are significant at 30, 60, 90 and 120 min compared with unstimulated cells (*p < 0.05). (B) Phosphorylation of moesin and total moesin protein are compared to β-actin by western blot. Phosphorylation of moesin is rapid, appearing at 15 s followed by dephosphorylation and a second phosphorylation at 30 min. The second phosphorylation event coincides with the increase in moesin protein. (C) Total moesin protein per cell is expressed as a ratio of moesin to β-actin. There is a significant increase in total moesin protein observed at 60, 90 and 120 min (*p < 0.05). (D) Relative ratio of phospho-moesin to total moesin indicates the phosphorylation events at 15 s and 30 min (*p < 0.05).

Fig 2

Native PAGE gel shift analysis of moesin–LPS complex. Carboxyl and amine halves of recombinant moesin (rMoesin-C, rMoesin-N) and radixin (30 μg) were incubated in the absence and presence of LPS at 37°C for 30 min. A shift in the electrophoresis mobility (arrow) was only detected when rMoesin-C was incubated with LPS (500 μg/mL). No shift was observed for rMoesin-N or for either the carboxyl or the amine half of recombinant radixin (data not shown).

Fig 3

Immunoprecipitation (IP) and western blotting. Cell lysates from control and LPS-treated samples were immunoprecipitated with anti-moesin antibody, followed by western blotting using antibodies against CD14, TLR4 or MD-2. Each sample was separated by SDS-PAGE prior to immunoprecipitation, and western blotting was performed for β-actin as a control for equal loading. The results show that CD14 co-precipitated with moesin in both the control and LPS-stimulated conditions, with a stronger signal in stimulated conditions. There was no association observed between TLR4 or MD-2 and moesin in the resting cell lysates. After 5 min of stimulation, both TLR4 and MD-2 were observed to co-precipitate with moesin.

Fig 4

Inhibition of IRAK, MyD88 and IRAK activation and phosphorylation by anti-moesin antibody. Stimulated differentiated THP-1 cells, with and without prior treatment with anti-moesin antibody or an isotype-matched control antibody, were lysed (as described in the ‘Material and methods’ section) and subjected to immunoprecipitation (IP) with anti- MyD88 goat IgG followed by western blotting (WB) for IRAK (A), immunoprecipitation with IRAK rabbit IgG followed by western blotting with an antibody that recognizes threonine phosphorylation (B), or immunoprecipitation with IRAK rabbit IgG followed by western blotting with anti-TRAF6 (C). In cells pretreated with anti-moesin antibody, recruitment of IRAK to MyD88 was inhibited and subsequent IRAK phosphorylation was blocked (A and B, respectively). Furthermore, IRAK binding to TRAF6 was also inhibited (C). No effects were observed when the differentiated THP-1 cells were pre-incubated with the isotype-matched control antibody prior to LPS stimulation.

Fig 5

Effect of anti-moesin antibody on p38 MAPK activity. (A) Normal p38 activation by LPS (500 ng/mL) was initially analyzed, and maximal activation was observed at 30 min. (B) Differentiated THP-1 cells were pretreated with anti-moesin antibody (10 μg/mL), isotype- matched control antibody (10 μg/mL) or the p38 inhibitor SB202190 (25 μM). Cells pretreated with anti-moesin antibody or SB202190 prior to LPS stimulation showed significantly less p38 activation. (C) The gels were quantified by densitometry, and density was expressed as adjusted volume (OD/mm²).

Fig 6

Effect of anti-moesin antibody on p44/42 MAPK activity. (A) Normal p44/42 activation by LPS (500 ng/mL) was initially analyzed, and maximal activation was observed at 30 min. (B) For blocking experiments, macrophages were pretreated with anti-moesin antibody (10 μg/mL), isotype-matched control antibody (10 μg/mL) or the p44/42 inhibitor PD98059 (25 μM). Cells pretreated with anti-moesin antibody or PD98059 prior to LPS stimulation showed significantly less p44/42 activation. (C) The gels were quantified by densitometry, and density expressed as adjusted volume (OD/mm²).

Fig 7

Phosphorylation of IκBα, and translocation and activation NF-κB. Macrophages were stimulated with LPS (500 ng/mL). For blocking experiments, cells were treated with anti-moesin antibody (10 μg/mL) prior to LPS stimulation. Cytoplasmic and nuclear extractions were performed according to the reagent supplier’s recommendations (Imgenex). The phosphorylated form of IκBα was detected in cytoplasmic extracts by western blotting. Translocation and activation of NF-κB were measured using the active ELISA assay of nuclear extracts. (A) Phosphorylation of IκBα stimulated by LPS. No phosphorylation was observed in cells pretreated with anti-moesin antibody. (B) Activation of NF-κB and its translocation into the nucleus upon LPS stimulation. When cells were pretreated with anti-moesin antibody, no translocation was observed. No effects were observed when the differentiated THP-1 cells were pre-incubated with an isotype-matched control IgG. *p < 0.05 compared to vehicle and control.

Fig 8

Effect of moesin on TNF-α and IL-1β secretion in LPS-stimulated cells. Cells were cultured as described in the ‘Material and methods’ section and were pretreated with anti-moesin antibody or an isotype-matched control (10 μg/mL) followed by LPS stimulation (500 ng/mL) for 18 h. Control cells received no antibodies or LPS. Concentrations of TNF-α, IL-1β and IFN-β were determined by ELISA. Data are presented as means of three experiments, with standard deviations. Significance was determined by ANOVA with Bonferroni’s correction for multiple comparisons. There was a significant inhibition of the production of TNF-α and IL-1β in cells pretreated with anti-moesin antibody (*p < 0.05). However, there was no effect on anti-moesin antibody on the secretion of IFN-β.

Fig 9

Proposed model for LPS recognition and signaling. In the absence of stimulation, our model shows CD14 and moesin associated in the cell membrane in macrophages. Following LPS stimulation, moesin phosphorylation is significantly increased and the moesin binds to TLR4 and MD-2. This receptor cluster then activates the adaptor protein MyD88. The MyD88 recruits and activates IRAK through their respective death domains. The IRAK subsequently autophosphorylates, dissociates from MyD88 and interacts with TRAF6. Active TRAF6 then activates and phosphorylates MEKK-1 or MKK3/6, MKK4, MEK and NIK. Both NIK and MEKK-1 are activators of IKK, which in turn phosphorylates IκBα. The phosphorylation of IκBα results in its dissociation and degradation, freeing NF-κB. The NF-κB translocates to the nucleus, where transcription begins. TAB1, TAB2 and TAK1 are activators of p38, JNK and ERK1/2 (p44/42). The activation of these MAPKs leads to the production of proinflammatory cytokines, such as TNF-α. (LBP, LPS-binding protein; MEK, MAP kinase/ERK kinase; MKK, MAP kinase kinase; MEKK, MAP kinase kinase; NIK, B-inducing kinase).