Nucleobindin co-localizes and associates with cyclooxygenase (COX)-2 in human neutrophils

Abstract

The inducible cyclooxygenase isoforms (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, nuch remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact om PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescense,confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acis. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis. © 2008 Leclerc et al.