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CD14-mediated translocation of nuclear factor-kappa B induced by lipopolysaccharide does not require tyrosine kinase
R L Delude1, M J Fenton, R Savedra
1Maxwell Finland Laboratories for Infectious Diseases, Boston City Hospital, Massachusetts.
Abstract:
During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. CD14, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which CD14, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that CD14-mediated signaling is effected through a receptor-associated tyrosine kinase (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human CD14 (CHO/CD14). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/CD14 cells and to elucidate the relationship of NF-kappa B activation to the CD14 receptor system. LPS-stimulated translocation of NF-kappa B in CHO/CD14 cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, demonstrating that NF-kappa B translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for CD14-mediated signal transduction to occur in response to LPS.
Insights
Lipopolysaccharide (LPS) signaling via CD14 in immune cells does not require tyrosine kinase activity. NF-kappa B translocation occurs early, independent of protein synthesis inhibitors or corticosteroids, and is not blocked by tyrosine kinase inhibitors.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Serious bacterial infections involve lipopolysaccharide (LPS) interacting with macrophage receptors, leading to inflammation and shock.
- CD14 acts as an LPS signaling receptor but lacks a transmembrane domain, raising questions about signal transduction mechanisms.
- A hypothesis suggests CD14 signaling involves a receptor-associated tyrosine kinase (TK), proposing a multicomponent receptor model for LPS response.
Purpose of the Study:
- To investigate the role of NF-kappa B translocation in CD14-mediated LPS signaling.
- To determine if tyrosine kinase (TK) activity is essential for CD14 signal transduction in response to LPS.
Main Methods:
- Transfected Chinese hamster ovary (CHO)-K1 cells with human CD14 (CHO/CD14) to study LPS-induced responses.
- Compared NF-kappa B translocation in CHO/CD14 cells and RAW 264.7 macrophage cells.
- Utilized protein synthesis inhibitors, corticosteroids, and TK inhibitors (herbimycin A, genistein) to assess their effects on NF-kappa B translocation and TK activity.
Main Results:
- LPS stimulation induced NF-kappa B translocation in CHO/CD14 cells, similar to RAW 264.7 cells.
- NF-kappa B translocation was an early event, unaffected by protein synthesis inhibitors or corticosteroids.
- LPS-induced TK activity was observed in RAW 264.7 cells but not in CHO/CD14 cells; TK inhibitors blocked TK activity but not NF-kappa B translocation in either cell type.
Conclusions:
- Tyrosine kinase (TK) activity is not obligatory for CD14-mediated signal transduction in response to lipopolysaccharide (LPS).
- NF-kappa B translocation represents an early event in the LPS-CD14 signaling pathway.
- The findings challenge the proposed multicomponent receptor model involving TKs for CD14-LPS signaling.