Role for Moesin in Lipopolysaccharide-Stimulated Signal Transduction

Abstract

Moesin is a 78-kDa protein with diverse functions in linking the cytoskeleton to the membrane while controlling cell shape, adhesion, locomotion, and signaling. The aim of this study was to characterize the expression and localization of moesin in mononuclear phagocytes by using confocal microscopy, flow cytometry, immunoprecipitation, and Western blotting and to analyze the function of moesin as a lipopolysaccharide receptor, utilizing an antisense oligonucleotide approach to knock down the moesin gene. Results revealed that moesin is expressed on the surface of monocytes/macrophages and surface expression is increased after lipopolysaccharide stimulation. The total protein mass of moesin is increased in monocytes after lipopolysaccharide stimulation. Immunoprecipitation showed that moesin coprecipitates with TLR4, a well-known lipopolysaccharide receptor, suggesting an early role of moesin in the formation of the initiation complex for lipopolysaccharide signaling. Two antisense and two control sense oligonucleotides were synthesized and introduced every 4 h for 48 h in adherent macrophage-like cells. Cells were then stimulated with lipopolysaccharide for 4 h, and the supernatants were assayed for tumor necrosis factor alpha (TNF-␣) production. Cell lysates were assayed for moesin expression by Western blotting immediately after the 48-h treatment period and also after 116 h of recovery to assess the return of moesin expression and function. Moesin gene expression was completely suppressed after 48 h of incubation with antisense oligonucleotides. The antisense elimination of moesin gene expression led to a significant reduction of lipopolysaccharide-induced TNF-␣ secretion. Restoration of moesin gene expression led to restoration of TNF-␣ production. These data suggest an important role for moesin in lipopolysaccharide-induced TNF-␣ production, highlighting its importance in lipopolysaccharide-mediated signal transduction.The pathology associated with gram-negative bacterial infections is tightly linked to the direct and indirect biological impact of bacterial endotoxin (lipopolysaccharide [LPS]). Once released into the host circulation, LPS has been shown to cause a wide variety of pathophysiological events, including cell injury, septic shock, and death (27). Microbial endotoxin is a potent inducer of cytokine synthesis, and although proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣), interleukin (IL)-1␤, IL-6, and IL-12 are in general part of the innate immune response to infection, they can play crucial roles in the adverse development of systemic toxicity and septic shock (36). Peripheral mononuclear phagocytes are the host cells mainly responsible for binding and eliminating bacterial toxins. Activation with LPS leads to a significant increase in TNF-␣ protein synthesis in mononuclear phagocytes and macrophages (8). In addition, purified TNF-␣ has been shown to induce many of the deleterious effects of LPS in vivo (45). Despite intense research and numerous advances, the outcome of gra...

Inhibitory Effect of Alopecurus aequalis Sobol Ethanol Extracts on LPS-induced Inflammatory Response in RAW 264.7 Cells

“…5). (Iontcheva et al, 2004). 이 중 NO 형성은 박테리아를 죽이 거나 종양을 제거시키는 중요한 역할을 하지만, 병리적인 원 인에 의한 과도한 NO 형성은 염증을 유발시키게 되며 조직의 손상, 유전자 변이 및 신경 손상 등을 유발한다 (Stuehr et al, 1991;McCartney-Francis et al, 1993;Weisz et al, 1996).…”

MaxiK Blockade Selectively Inhibits the Lipopolysaccharide-Induced IκB-α/NF-κB Signaling Pathway in Macrophages

“…However, for most of these proteins, a functional participation in signal transduction is not clear, yet. For other molecules, such as MOESIN (24,25) and mLBP (26), a functional participation in cell activation by LPS has been shown. Several publications including our own have reported that potassium channels are involved in the activation of monocytes, macrophages, and endothelial cells by LPS (27)(28)(29)(30)(31)(32)(33).…”

The Anti-inflammatory Effects of Methylsulfonylmethane on Lipopolysaccharide-Induced Inflammatory Responses in Murine Macrophages

“…Following activation, macrophages modulate the expression of accessory molecules such as CD14 and toll-like receptor (TLR) 4. 4,5) Stimulation of TLR4 by lipopolysaccharide (LPS) triggers the recruitment of the cytoplasmic adaptor protein MyD88 and subsequently culminates in the activation of downstream signaling pathways: the transcription factor nuclear factor-kB (NF-kB) pathway. These pathways induce the expression of various inflammatory mediators, including nitric oxide (NO), prostaglandins (PGs), and inflammatory cytokines.…”