Figure 1.
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.
Figure 2.
Neutrophil subcellular fractionation and localization of Nuc.
A) Resting (left panel) or GM/TNF-stimulated (right panel) neutrophils were processed for cavitation and subcellular fractionation, as described in Experimental procedures. Nuc and COX-2 co-localized in ER/Golgi-containing fractions, as determined by western immunoblotting. B) In GM/TNF-stimulated neutrophils, samples were processed as in A) for the determination of the indicated proteins. C) In GM/TNF, and GM/TNF+fMLP (100 nM) stimulated neutrophils, samples were processed as in A) for the determination of the indicated proteins. In each panel, all immunoblots originate from the same membrane. Shown is one immunoblot, representative of four identical experiments performed with different donors. GRP-78: ER/Golgi marker; lactoferrin: marker of specific granules; albumin; marker of secretory vesicles; 58k: 58k Golgi protein (Golgi marker); mPGES-1: microsomal prostaglandin E2 synthase-1; TXA2-Synthase: thromboxane A2 synthase; cPLA2: Type IV cytosolic phospholipase A2. D) Schematized protein structure of human Nuc and putative functional domains. The main characterized domains found in Nuc are: a signal peptide directing the protein to the ER; a COX-binding site; a putative nuclear localization signal embedded in to a DNA-binding site; two EF-hand Ca2+-bindins sites; a leucine zipper region.
Figure 3.
Subcellular localization of Nuc in neutrophils, as assessed by indirect immunofluorescence and confocal microscopy.
Resting neutrophils were processed for the detection of Nuc by immunofluorescence, as described in Experimental Procedures. A) Nuclei were stained in blue. Immunoreactive Nuc, in green, was mainly observed in the center of cells, typically as two to three main spots between nuclear lobes and, to a lesser extent, within the cytoplasm. Left panel: with anti-Nuc antibody. Right panel: anti-Nuc antibody was omitted. B) For confocal microscopy experiments, the nucleus was stained with propidium iodine and appeared red-orange; darker regions are distinctive of euchromatin; immunoreactive Nuc, in green, appeared at the center of the cell. Left panel: composite confocal image; most of the immunoreactive Nuc was at the center of the cell, with a limited number of smaller spots also in the vicinity of the nucleus. Right panel, (from left to right, top to bottom): represented are 9 equidistant slices from the composite image shown in left panel.
Figure 4.
Demonstration of co-localization for Nuc and COX-2 in neutrophils, by electron microscopy.
Neutrophils stimulated with GM/TNF were processed for the detection of Nuc (A), COX-2 (B) and Golgi (C), by indirect immunostaining and electron microscopy. For each protein, labeling was mainly found in a single cluster situated between nuclear lobes, in the center of the cell. D) Samples were processed for the double detection of Nuc and of COX-2 by electron microscopy. To this end, a polyclonal chicken anti-Nuc antibody and a polyclonal rabbit anti-COX-2 antibody, were used in sequence, as described in the Experimental procedures. Co-localization of Nuc (18 nm beads, indicated by thick arrows) and COX-2 (6 nm beads, thin arrows) is clearly seen in a cluster between nuclear lobes. In each panel, a single neutrophil is shown in the upper-left corner; the magnified region of interest is represented by the respective white square.
Figure 5.
Direct interaction between neutrophil Nuc and COX-2.
A) Nuclear-free extracts from unstimulated or PMA-stimulated neutrophils were incubated with sepharose beads linked to hrNuc for a pull-down assay, then processed for western immunoblotting for the detection of COX-2. Lane 1: whole cells, lane 2: nucleus-free extracts, lane 3: nucleus-free extracts incubated with sepharose beads linked to hrNuc (Seph-Nuc). B) Nuclear-free extracts from PMA-stimulated cells were processed as in A), in presence of 1.5 mM Ca2+ (left panel) or in presence of 5 mM EGTA (right panel; without Ca2+). Lane 1: whole cells, lane 2: nucleus-free extracts, lane 3: nucleus-free extracts incubated with inactivated sepharose beads, lane 4: nucleus-free extracts incubated with sepharose beads linked to hrNuc (Seph-Nuc). For each panel, results shown are from one experiment, typical of two separate experiments performed in identical conditions with different donors. C) Immunomagnetic beads coated with anti-Nuc IgGs or with irrelevant IgGs were incubated with an aliquot from the positive fractions (10 to 12) showed in Fig. 1. Anti-Nuc-coated beads immunoprecipitated a structure that was positive for COX-2 and GRP-78. Results are from one experiment, representative of n = 3 distinct experiments performed in identical conditions. D) Left panel: Nuc was immunoprecipitated from COX-2-expressing neutrophils, using anti-Nuc or irrelevant anti-IgG antibodies, as described in the Experimental procedures; samples were processed for the detection of COX-2 by western immunoblot. Right panel: COX-2 was immunoprecipitated using anti-COX-2 or irrelevant anti-IgG antibodies and samples were processed for the detection of Nuc by western immunoblot (IP: Immunoprecipitation; WB: western immunoblot). Results are from one experiment, representative of n = 2 distinct experiments performed in identical conditions.
Figure 6.
Nuc increases COX-2-dependent PGE2 biosynthesis.
A) Recombinant human (hr)COX-2 (1 U/sample) alone or in presence of indicated amounts of hrNuc, were incubated with AA. PGE2 production was measured by ELISA. Results are expressed as percentages of maximum production and are the mean±s.e.m. of four separate experiments performed in identical conditions. B) hrNuc (10 µg) was treated with a polyclonal anti-Nuc antibody prior to incubation with hrCOX-2. The COX-2 specific inhibitor NS-398 (50 µM) was used to confirm a COX-2-mediated event. Results are expressed as percentage of inhibition of PGE2 production, when compared to the production obtained in the absence of antibody or inhibitor (mean±s.e.m., n = 4. *: significantly higher than samples incubated without hrNuc; **: significantly higher than samples incubated with 2.0 µg hrNuc or less).
Figure 7.
Nuc increases PGE2 biosynthesis in a COX-2-dependent manner in human neutrophils.
A) COX-2-expressing neutrophil extracts were incubated with AA (10 µM) for 30 min, alone or in the presence of indicated quantities of hrNuc. PGE2 production was measured by ELISA. Results are expressed as percentages of maximum PGE2 production (mean±s.e.m., n = 3). B) Left bar; hrNuc (1 µg) was treated with a polyclonal anti-Nuc antibody prior to incubation with the cellular extracts. Right bar; the COX-2 specific inhibitor NS-398 (50 µM) was used to confirm a COX-2-mediated PGE2 biosynthesis. Results are expressed as inhibition of PGE2 production, when compared to the production obtained in the absence of antibody or inhibitor (mean %±s.e.m., n = 4). C) Bar graph; GM/TNF-treated neutrophils were transfected with full-length hrNuc, or lacking a COX-binding domain (NucΔCBD) using the Pro-Ject procedure as described in Experimental procedures, then stimulated with AA (10 µM). PGE2 production was measured by ELISA. (mean±s.e.m., n = 3. *: significantly higher than samples incubated without Nuc). Western immunoblots; Cells treated as described above were processed for the determination of cellular COX-2, Nuc, hrNuc and NucΔCBD protein levels. Note that hrNuc migrates higher than endogenous Nuc (or NucΔCBD), because of the signal peptide sequence still being present. Immunoblots are from one experiment, typical of three independent experiments performed in identical conditions.