Shown is one immunoblot, representative of four identical experiments performed with different donors. Nuc and COX-2 are localized in the Golgi and ER of neutrophils The subcellular localization of Nuc, and of the enzymatic machinery responsible for prostaglandin biosynthesis was investigated in human neutrophils. 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 comprehended. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain name. 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 PGE2 biosynthesis. Complementary subcellular localization methods 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 PGE2 biosynthesis in response to arachidonic acid. 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. Introduction Cyclooxygenase (COX) mediates a critical metabolic step in prostanoid biosynthesis. The inducible isoform, COX-2, largely predominates over the constitutive isoform, COX-1, in the production of prostaglandin (PG)E2 and thromboxane (TX)A2 in inflammatory cells including neutrophils [1]C[4]. PGE2 and TXA2 biosynthesis in neutrophils is initiated by the release of esterified arachidonic Rabbit Polyclonal to Fyn acid (AA) by type IV cytosolic (c)PLA2 [5]C[8]. COX-2 then catalyzes two reactions by which AA is usually transformed into PGH2, the common precursor to all prostanoids. PGH2 can EGFR-IN-7 be isomerized in PGE2, either non-enzymatically [9], or EGFR-IN-7 by the microsomal PGE2 synthase (mPGES)-1 isoform [8], while formation of TXA2 chiefly results from TXA2 synthase activity. It is well-established that PGE2 is usually implicated in pain, vasodilation and eodema. On the other hand, PGE2 inhibits major inflammatory responses of phagocytes. In neutrophils, PGE2 can prevent chemotaxis, aggregation, superoxide production, lysosomal enzyme release and generation of leukotriene B4 [4], [10]C[14]. The COX-2 enzyme has generated particular interest for its implication in inflammation, cellular proliferation, differentiation and tumorigenesis, [15], [16], and has recently emerged as a therapeutic target in the treatment and prevention of human cancers [17]C[20]. Also, COX-2 mediates physiological events such as kidney functions, post-natal development and female reproductive processes [21]C[23]. In spite of the pivotal functions of COX-2 in many aspects of biology, much remains to be discovered round the regulation of its activity in inflammatory cells. In particular, proteins that associate with COX-2 have yet to be recognized. Nucleobindin (Nuc) is usually a EGFR-IN-7 ubiquitous protein featuring multiple putative functional domains, indicating its potential implication in a number of cellular processes [24]C[28]. As such, Nuc has been the focus of reports originating from diverse fields including autoimmunity [29], intracellular signaling [30], osteogenesis [26], malignancy [31] and inflammation [27]. At the protein level, Nuc is usually constituted of 460 amino acids, including an N-terminal 25 amino acid signal peptide responsible for its initial localization to the endoplasmic reticulum (ER) [32]. In addition, Nuc contains several classical conversation domains: a DNA binding site, a heterodimerization domain name, two EF-hand Ca2+-binding sites, a nuclear EGFR-IN-7 localization transmission [24]C[26] as well as non-classical protein-protein conversation domains including a G-protein-binding region and an high affinity COX-binding domain name, as evidenced by a yeast two-hybrid assay [27], [28]. Depending on the model at hand, Nuc has been detected in various subcellular structures such as the nucleus [31], [33], mitochondria [34] the cytoplasm [34]C[37], the endoplasmic reticulum (ER) [33], [34], [36] and the Golgi apparatus [35]. The Golgi, like the ER, plays.