Date of Award

2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Daniel Hwang

Abstract

Genetic evidence indicating that Toll-like receptor 4 (TLR4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine TLR4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through activation of NFkappaB. Thus, we determined whether murine TLR4 confers LPS responsiveness as evaluated by activation of NFkappaB and COX-2 expression. Expression of the constitutively active form (DeltaTlr4) of TLR4 in murine macrophage cell line (RAW 264.7) is sufficient to activate NFkappaB and COX-2 expression and to stimulate prostaglandin E2 synthesis. The truncated form [DeltaTlr4 (P712H)] of the missense mutant Tlr4 (P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NFkappaB activation and COX-2 expression. Inability of DeltaTlr4 (P712H) to activate NFkappaB and to induce COX-2 expression is rescued by a constitutively active adaptor protein (MyD88) which interacts directly with the cytoplasmic domain of TLR proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild type DeltaTlr4, but not with DeltaTlr4 (P712H) mutant. Together, these results indicate that TLR4 confers LPS responsiveness in RAW 264.7 cells, and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is due to disruption of TLR4-mediated signaling pathways resulting from inability of the mutant TLR4 (P712H) to interact with MyD88. In addition, the involvement of Lyn, Src-protein tyrosine kinase family, in LPS-induced signaling pathways leading to COX-2 expression was studied. All together, the results from this study may help pave the way to understanding the signal transduction pathways for COX-2 gene expression.

ISBN

9780493443614

Pages

149

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