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

Leclerc P, Biarc J, St-Onge M, Gilbert C, Dussault AA, Laflamme C, Pouliot M - PLoS ONE (2008)

Bottom Line: 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.Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie and Department of Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada.

ABSTRACT
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.

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Expression of Nuc in human neutrophils.A) Real-time PCR determination of Nuc, COX-1 and COX-2 messenger RNA expression in neutrophils. Cells were stimulated for 60 min with lipopolysaccharide (LPS ; 100 ng/ml), a mixture of granulocyte/monocyte colony stimulation factor and tumor necrosis factor-α (GM/TNF; 1.4 nM and 100 ng/ml respectively), formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) or with PMA (10 nM). Samples were processed for the determination of GAPDH, COX-1, COX-2 and Nuc mRNA expression by real-time-PCR. Shown are integrated results from n = 4 (±SEM) separate experiments performed in identical conditions with different donors. B) Nuc protein expression in neutrophils, as determined by western immunoblotting. Cells were incubated for 2 h with diluent (saline), or with GM/TNF. Samples were processed for the determination of Nuc expression by western immunblotting. Nuc is constitutively present in unstimulated neutrophils. Note that hrNuc migrated slightly slower than neutrophil native Nuc, due to the presence of the signal peptide and of the additional His-Tag sequence. Shown is one immunoblot, representative of four identical experiments performed with different donors.
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pone-0002229-g001: Expression of Nuc in human neutrophils.A) Real-time PCR determination of Nuc, COX-1 and COX-2 messenger RNA expression in neutrophils. Cells were stimulated for 60 min with lipopolysaccharide (LPS ; 100 ng/ml), a mixture of granulocyte/monocyte colony stimulation factor and tumor necrosis factor-α (GM/TNF; 1.4 nM and 100 ng/ml respectively), formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) or with PMA (10 nM). Samples were processed for the determination of GAPDH, COX-1, COX-2 and Nuc mRNA expression by real-time-PCR. Shown are integrated results from n = 4 (±SEM) separate experiments performed in identical conditions with different donors. B) Nuc protein expression in neutrophils, as determined by western immunoblotting. Cells were incubated for 2 h with diluent (saline), or with GM/TNF. Samples were processed for the determination of Nuc expression by western immunblotting. Nuc is constitutively present in unstimulated neutrophils. Note that hrNuc migrated slightly slower than neutrophil native Nuc, due to the presence of the signal peptide and of the additional His-Tag sequence. Shown is one immunoblot, representative of four identical experiments performed with different donors.

Mentions: Expression of Nuc in neutrophils was first assessed. To this end, cells were incubated with agonists known to stimulate inflammatory gene expression in these cells: lipopolysaccharide (LPS), the formylated synthetic peptide fMLP, the phorbol ester PMA, or a mixture of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α (GM/TNF). Following stimulations, mRNA levels of COX-1, COX-2 and Nuc were determined by real-time PCR. While each of the agonists elicited an increase in expression of COX-2 mRNA, as previously reported [3], [43], that of Nuc only varied in a modest fashion and, in most conditions, variations were comparable to that of COX-1, a constitutively-expressed gene in neutrophils (Fig. 1A). Similar results were obtained in human monocytes stimulated with LPS (data not shown). These results are consistent with the reported structure for the promoter region of the Nuc gene, featuring typical elements of house-keeping genes [32]. Data obtained at the protein level also indicate that resting neutrophils constitutively express Nuc (Fig. 1B).


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

Leclerc P, Biarc J, St-Onge M, Gilbert C, Dussault AA, Laflamme C, Pouliot M - PLoS ONE (2008)

Expression of Nuc in human neutrophils.A) Real-time PCR determination of Nuc, COX-1 and COX-2 messenger RNA expression in neutrophils. Cells were stimulated for 60 min with lipopolysaccharide (LPS ; 100 ng/ml), a mixture of granulocyte/monocyte colony stimulation factor and tumor necrosis factor-α (GM/TNF; 1.4 nM and 100 ng/ml respectively), formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) or with PMA (10 nM). Samples were processed for the determination of GAPDH, COX-1, COX-2 and Nuc mRNA expression by real-time-PCR. Shown are integrated results from n = 4 (±SEM) separate experiments performed in identical conditions with different donors. B) Nuc protein expression in neutrophils, as determined by western immunoblotting. Cells were incubated for 2 h with diluent (saline), or with GM/TNF. Samples were processed for the determination of Nuc expression by western immunblotting. Nuc is constitutively present in unstimulated neutrophils. Note that hrNuc migrated slightly slower than neutrophil native Nuc, due to the presence of the signal peptide and of the additional His-Tag sequence. Shown is one immunoblot, representative of four identical experiments performed with different donors.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2373884&req=5

pone-0002229-g001: Expression of Nuc in human neutrophils.A) Real-time PCR determination of Nuc, COX-1 and COX-2 messenger RNA expression in neutrophils. Cells were stimulated for 60 min with lipopolysaccharide (LPS ; 100 ng/ml), a mixture of granulocyte/monocyte colony stimulation factor and tumor necrosis factor-α (GM/TNF; 1.4 nM and 100 ng/ml respectively), formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) or with PMA (10 nM). Samples were processed for the determination of GAPDH, COX-1, COX-2 and Nuc mRNA expression by real-time-PCR. Shown are integrated results from n = 4 (±SEM) separate experiments performed in identical conditions with different donors. B) Nuc protein expression in neutrophils, as determined by western immunoblotting. Cells were incubated for 2 h with diluent (saline), or with GM/TNF. Samples were processed for the determination of Nuc expression by western immunblotting. Nuc is constitutively present in unstimulated neutrophils. Note that hrNuc migrated slightly slower than neutrophil native Nuc, due to the presence of the signal peptide and of the additional His-Tag sequence. Shown is one immunoblot, representative of four identical experiments performed with different donors.
Mentions: Expression of Nuc in neutrophils was first assessed. To this end, cells were incubated with agonists known to stimulate inflammatory gene expression in these cells: lipopolysaccharide (LPS), the formylated synthetic peptide fMLP, the phorbol ester PMA, or a mixture of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α (GM/TNF). Following stimulations, mRNA levels of COX-1, COX-2 and Nuc were determined by real-time PCR. While each of the agonists elicited an increase in expression of COX-2 mRNA, as previously reported [3], [43], that of Nuc only varied in a modest fashion and, in most conditions, variations were comparable to that of COX-1, a constitutively-expressed gene in neutrophils (Fig. 1A). Similar results were obtained in human monocytes stimulated with LPS (data not shown). These results are consistent with the reported structure for the promoter region of the Nuc gene, featuring typical elements of house-keeping genes [32]. Data obtained at the protein level also indicate that resting neutrophils constitutively express Nuc (Fig. 1B).

Bottom Line: 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.Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie and Department of Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec City, Quebec, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus