Nucleobindin co-localizes and associates with cyclooxygenase (COX)-2 in human neutrophils.
The inducible cyclooxygenase isoform (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, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca(2+)-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 on PGE(2) biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, 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 PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.
Leclerc
Patrick
P
Centre de Recherche en Rhumatologie et Immunologie and Department of Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada.
Biarc
Jordane
J
St-Onge
Mireille
M
Gilbert
Caroline
C
Dussault
Andrée-Anne
AA
Laflamme
Cynthia
C
Pouliot
Marc
M
D001483:Base Sequence; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000097:blood*; D051546:Cyclooxygenase 2 / Q000097:blood*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000097:blood*; D015232:Dinoprostone / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism*; D000080866:Nucleobindins; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D051546:Cyclooxygenase 2; D015232:Dinoprostone