Role ofvav1in the Lipopolysaccharide-Mediated Upregulation of Inducible Nitric Oxide Synthase Production and Nuclear Factor for Interleukin-6 Expression Activity in Murine Macrophages

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Abstract
vav1has been shown to play a key role in lymphocyte development and activation, but its potential importance in macrophage activation has received little attention. We have previously reported that exposure of macrophages to bacterial lipopolysaccharide (LPS) leads to increased activity ofhckand othersrc-related tyrosine kinases and to the prompt phosphorylation ofvav1on tyrosine. In this study, we tested the role ofvav1in macrophage responses to LPS, focusing on the upregulation of nuclear factor for interleukin-6 expression (NF-IL-6) activity and inducible nitric oxide synthase (iNOS) protein accumulation in RAW-TT10 murine macrophages. We established a series of stable cell lines expressing three mutant forms ofvav1in a tetracycline-regulatable fashion: (i) a form producing a truncated protein,vavC; (ii) a form containing a point mutation in the regulatory tyrosine residue,vavYF174; and (iii) a form with an in-frame deletion of 6 amino acids required for the guanidine nucleotide exchange factor (GEF) activity ofvav1for rac family GTPases,vavGEFmt. Expression of the truncated mutant (but not the other two mutants) has been reported to interfere with T-cell activation. In contrast, we now demonstrate that expression of any of the three mutant forms ofvav1in RAW-TT10 cells consistently inhibited LPS-mediated increases in iNOS protein accumulation and NF-IL-6 activity. These data provide direct evidence for a role forvav1in LPS-mediated macrophage activation and iNOS production and suggest thatvav1functions in part via activation of NF-IL-6. Furthermore, these findings indicate that the GEF activity ofvav1is required for its ability to mediate macrophage activation by LPS.

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