Date of Award

1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Environmental Sciences

First Advisor

Leslie C. Plhak

Second Advisor

Jacqueline M. Stephens

Abstract

Cyclooxygenase (COX) is the key enzyme in the biosynthesis of prostaglandins (PGs) known as pro-inflammatory mediators. COX has two isoforms: the constitutively expressed COX-1 and the mitogen-inducible COX-2. It has been demonstrated that COX-2 expression is selectively induced by lipopolysaccharide (LPS) treatment after several hours in rat alveolar macrophages (Lee et al., 1992; Chanmugam et al., 1995). The mitogen-activated protein kinases [MAPKs; extracellular signal-regulated kinases (ERKs), P38, and c-jun N-terminal kinases (JNKs)], nuclear factor kappa B (NF-kB), and Lyn kinase are known to be activated in the early stages (within minutes) of LPS signaling pathways. In the current study, the mechanism of COX-2 expression induced by LPS in RAW 264.7 macrophages was examined. To examine the involvement of MAPKs and NF-kB on COX-2 expression, RAW 264.7 cells were treated with LPS and known activators and inhibitors of MAPKs, NF-kB, or protein tyrosine kinases (PTKs). To examine the role of Lyn kinase on COX-2 expression, Phoenix ( F NX) cells, a packaging cell line, were transfected with Flag (epitope)-tagged lyn cDNAs in the retroviral vector pBPSTR1, and the infectious viral particles produced by transfected F NX cells were infected to NIH/3T3 cells, a murine fibroblast cell line. The activation of MAPKs by known activators was insufficient to induce COX2 expression. Partial inhibition of LPS-induced activation of MAPKs by PD98059 or SB203580 resulted in partial inhibition of COX-2 expression. Similarly, inhibition of LPS-induced activation of NF-kB by Z-LLF-CHO resulted in suppression of COX-2 expression. Both inhibition of LPS-induced activation of MAPKs and NF-kB by Radicicol or Herbimycin A resulted in suppression of COX-2 expression. Transfection data lead to an observation that high COX-2 expression and PGE 2 levels can be induced by overexpression of mutant Lyn in NIH/3T3 cells transfected with mutant lyn DNA. This study demonstrates that COX-2 expression induced by LPS in RAW 264.7 cells appears to be mediated via rapid activation of MAPKs and NF-kB in LPS signaling pathways.

ISBN

9780599372535

Pages

137

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