Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) is an important mediator of infection and inflammation leading to tissue damage, sepsis and septic shock. The number and sequence of binding sites/receptors and signal transduction events leading to the biologic response are not well understood. Previously our laboratory using crosslinking studies has shown that moesin plays a role as LPS receptor. Moesin is 78 kDa protein of a very diverse functional capabilities, playing major role in linking cytoskeleton to the cell membrane while controlling cell shape, and events such as adhesion, locomotion and signal transduction.
Objective: The aim of this study is to analyze the capability of moesin to work as an LPS receptor by utilizing antisense oligo experiments.
Methods: Two antisense and two respective control sense oligos were synthesized, and introduced every 4h for total of 48h in adherent macrophage-like THP-1 cells at a concentration of 2mM each. Cells were afterwards stimulated with LPS for 4h in media containing 5% human serum and the supernatants were assayed for TNF-alpha production by ELISA. Cell lysates were additionally assayed for moesin expression by Western blotting.
Results: Moesin gene expression was completely suppressed after 48h of incubation with 2 different oligos. The oligo abrogation of moesin gene expression led to a significant reduction of LPS-induced TNF-alpha in human THP-1 cell line. Restoration of moesin gene expression led to a restoration of TNF-alpha production and could be observed at least partially 4 days after the last exposure with antisense oligos.
Conclusion: The present data support the predominant role of Moesin in LPS-induced TNF-alpha production and highlight the importance of this molecule in LPS signal transduction cascade of events leading to deleterious biological responses.
NIH SUPPORT Supported by USPHS Grants DE13191