Subcellular Localization of Prostaglandin Endoperoxide H Synthases-1 and -2 by Immunoelectron Microscopy*

Andrew G. Spencer, John W. WoodsDagger , Toshiya Arakawa, Irwin I. SingerDagger , and William L. Smith§

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 and the Dagger  Department of Biochemical and Molecular Pathology, Merck Research Laboratories, Rahway, New Jersey 07065

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs like aspirin and ibuprofen. These enzymes catalyze the committed step in the formation of prostanoids from arachidonic acid. Although PGHS-1 and -2 are similar biochemically, a number of studies suggest that PGHS-1 and PGHS-2 function independently to form prostanoids that subserve different cellular functions. We have hypothesized that these isozymes may reside, at least in part, in different subcellular compartments and that their compartmentation may affect their access to arachidonic acid and serve to separate the functions of the enzymes. To obtain high resolution data on the subcellular locations of PGHS-1 and -2, we employed immunoelectron microscopy with multiple antibodies specific to each isozyme. Both PGHS-1 and -2 were found on the lumenal surfaces of the endoplasmic reticulum (ER) and nuclear envelope of human monocytes, murine NIH 3T3 cells, and human umbilical vein endothelial cells. Within the nuclear envelope, PGHS-1 and -2 were present on both the inner and outer nuclear membranes and in similar proportions. Western blotting data showed a similar distribution of PGHS-1 and -2 in subcellular fractions, and product analysis using isozyme-specific inhibitors suggested that both enzymes generate the same products in NIH 3T3 cells. Thus, we are unable to attribute the independent functioning of PGHS-1 and PGHS-2 to differences in their subcellular locations. Instead, the independent operation of these isozymes may be attributable to subtle kinetic differences (e.g. negative allosteric regulation of PGHS-1 at low concentrations of arachidonate (500-1000 nM)). A further conclusion of importance from a cell biological perspective is that membrane proteins such as PGHS-1 and -2, which are located on the lumenal surface of the ER, are able to diffuse freely among the ER and the inner and outer membranes of the nuclear envelope.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Two biochemically similar enzymes with markedly different patterns of expression catalyze the committed step in the formation of prostaglandins from arachidonic acid. The prostaglandin endoperoxide H synthases (PGHS-1 and PGHS-2)1 each form PGH2 from arachidonic acid through sequential cyclooxygenase and peroxidase activities (1). Biologically active prostanoids including PGE2, PGF2, PGI2, PGD2, and thromboxane A2 are then formed by specific prostaglandin synthases. PGHS-1 is expressed constitutively in nearly all mammalian tissues and forms prostanoids central to several housekeeping functions including water reabsorption in the kidney, vascular homeostasis, and platelet aggregation. PGHS-2, although absent from most cells, can be rapidly and dramatically induced in many cell types upon treatment with inflammatory cytokines, growth factors, and tumor promoters (2). PGHS-1 and -2 share 60% primary sequence identity (3, 4), and their x-ray crystal structures are virtually superimposable (5-7). Kinetic profiles suggest similar if not identical reaction mechanisms (8, 9). There are, however, sequence differences near the active site resulting in subtle differences in substrate specificities and differential sensitivities to various nonsteroidal anti-inflammatory drugs (10, 11). These differences have been exploited in preparing a new generation of selective inhibitors of PGHS-2 that may inhibit pain, inflammation, and tumorigenesis while preserving the capacity of PGHS-1 to synthesize housekeeping prostanoids.

Despite their many biochemical similarities, results of several experiments make it difficult to argue that PGHS-2 exists primarily to augment the biosynthetic capacity of PGHS-1 (see "Discussion" in Ref. 12). PGHS expression patterns and apparently differential access to cellular pools of arachidonate (13-15) have led to the hypothesis that PGHS-1 and -2 represent two independent prostanoid biosynthetic systems. The inducible expression of PGHS-2, which is encoded by an immediate early gene, occurs in conjunction with nuclear events such as cell differentiation and replication (2). Consequently, the inducible enzyme, especially the subset of enzyme molecules localized to the nuclear envelope, has been hypothesized to be the major source of prostanoids involved in a putative peroxisome proliferator activated receptor-mediated nuclear signaling system (16-18).

PGHS-1 and PGHS-2 are both integral membrane proteins of the endoplasmic reticulum (ER) and nuclear envelope (12). Based on previous confocal immunolocalization work from our laboratory, we suggested that differential compartmentation of the two isozymes may serve, at least in part, to separate the activities of PGHS-1 and -2 within cells (12). Although both enzymes are present in the ER and nuclear envelope, the conclusion of the earlier work was that PGHS-2 was preferentially associated with the nuclear envelope (12), whereas PGHS-1 was equally distributed between the ER and nuclear envelope. To test this model, we used immunoelectron microscopy and Western blotting of PGHS-1 and -2 in subcellular fractions. In addition, to test for isozyme-specific channeling of PGH2 to downstream PG synthases, we analyzed the prostanoid products formed by PGHS-1 and -2. Immunogold labeling experiments revealed that both enzymes are on the outer nuclear membrane; this was expected due to the continuity of this membrane with the ER (19). Somewhat to our surprise, both PGHS-1 and PGHS-2 were also present on the inner nuclear membrane. Overall, our studies have indicated that the locations of the isozymes and the nature of the products are the same for both PGHS-1 and -2.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

All materials were purchased from Sigma unless otherwise noted.

Isolation and Preparation of Human Monocytes for Electron Microscopy-- Peripheral blood mononuclear cells (PBMCs) were isolated from several healthy human volunteers. Heparinized blood was diluted 1:1 in Hanks' balanced salts solution (HBSS, Life Technologies, Inc.) and spun against a Ficol-Hypaque (LSM-Organon Teknika) gradient for 20 min at 500 × g. PBMCs were isolated as a band from the interface and washed 2 × with HBSS before resuspension in 10 ml of RPMI medium (Life Technologies, Inc.) containing 5% autologous serum and penicillin/streptomycin (100 units/ml). Cells (106/ml) were incubated in the presence or absence of 1 ng/ml lipopolysaccharide (LPS; from Jayne Chen at Merck) and incubated with gentle agitation in a water-saturated 5% CO2 atmosphere at 37 °C for 24 h. Fixation was performed after LPS- or vehicle-stimulated cells were collected by centrifugation and resuspended in appropriate media (see below). Monocytes comprise 5-8% of PBMCs. Most of the other cells in PBMC preparations are T and B lymphocytes that do not normally express PGHSs and, therefore, provide a convenient internal negative control against PGHS immunostaining. Because ultrathin sectioning makes possible the observation of more than one section from a given cell, care was taken to use several different samples in the interest of gathering data from many different cells. PBMCs from each volunteer were separated into between 5 and 10 separate pellets per experiment. Between every few grids of thin sections taken from a single pellet, several thick (1 µm) sections were cut to maximize the number of different cells observed per experiment.

Preparation of NIH 3T3 Cells and HUVEC Cells for Electron Microscopy-- Murine NIH 3T3 cells express PGHS-1 constitutively (20). Serum-starved, quiescent 3T3 cells were cultured and then stimulated by the addition of 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT) for 3 h to induce PGHS-2 as described previously (20). After serum stimulation, cells from five 100-mm culture dishes were removed from the growing surface with a rubber policeman, resuspended in DMEM (Life Technologies, Inc.), and immediately fixed (see below). HUVECs (Cell Systems, Seattle, WA) were thawed from stocks frozen at passage 1 and expanded through passage 3-4 as suggested by the manufacturer. After stimulation for 20 h with 10 ng/ml IL-1beta , cells were removed from culture dishes with a rubber policeman, immediately resuspended in CS-C media (Cell Systems, Seattle, WA), and fixed (see below). Previous PGHS immunolocalization experiments on both adherant and detached cells gave similar results, suggesting that removal of cells from the growing surface does not affect the localization of PGHS-1 or PGHS-2 (12, 40).

Fixation and Cryoprotection for Electron Microscopy-- Cells (PBMCs, NIH 3T3, and HUVEC) were obtained in a 2.5-ml suspension of an appropriate medium and immediately fixed by the addition of 2.5 ml of 2× fixative. Two different fixation methods were employed to allow for differences in the sensitivity to fixation of PGHS-1 and -2 antigens. Microwave/glutaraldehyde-fixed cells were fixed exactly as described previously (21). Cells fixed with Nakane fixative (22) alone were treated by adding 2× Nakane fixative (1× Nakane = 0.1 M NaIO4, 0.75 M lysine, 0.0375 M phosphate buffer, 2% paraformaldehyde (Fisher)) to an equal volume of cell suspension and incubated for 2 h at room temperature. These cells were then washed twice with 1× Nakane fixative and resuspended in fresh 1× Nakane before an overnight (or shorter) incubation at 4 °C. NIH 3T3 cells immunolabeled for PGHS-2 were only fixed at 4 °C for an additional 2 h after the 2-h room temperature fixation. After the overnight fixation step, both microwave/glutaraldehyde- and Nakane-fixed cells were collected by centrifugation, resuspended in 0.1 M sucrose/PBS, and pelleted in 2% low gelling temperature agarose, 0.1 M sucrose, PBS. Cryoprotection was performed for 2 h (25 °C) or overnight (4 °C) in polyvinylpyrrolidone, 2.3 M sucrose/phosphate buffer (pH 7.2) (polyvinylpyrrolidone-sucrose) (21). Cell pellets were mounted on bull's-eye specimen pins (Ted Pella, Inc.), frozen by plunging into liquid propane, and stored under liquid nitrogen until use.

Immunogold Labeling-- Immunogold labeling was performed essentially as described previously (21). Briefly, ultrathin (75-80-nm) sections of cells were cut at -106 °C on a Reichert Ultracut S ultramicrotome fitted with a Reichert FCS cryoattachment. Sections were collected on drops of 2.3 M sucrose and placed on glow discharged, Formvar-coated nickel grids (Ted Pella, Inc.). After a minimum of 1 h in blocking solution (5% milk, 1% bovine serum albumin, PBS, 0.02% sodium azide) and washing, grids were placed section-side down on 25-µl drops of primary antibody solutions of various concentrations and incubated for various times depending on the primary antibody. All antibody solutions were cleaned by filtration through 0.2-µm filters prior to use. To control for antibody specificity, primary antibodies were incubated with a 50-fold molar excess of either cognate peptide (for anti-PGHS-1 or anti-PGHS-2 peptide directed antibodies) or an approximately 10-fold molar excess of purified ovine PGHS-1 or -2 (for antibodies prepared against either whole protein). Preadsorption of whole protein antibodies with purified PGHS-1 or -2 was performed with constant agitation at 4 °C for 24 h. The samples were centrifuged at 12,000 rpm in a Beckman Microfuge for 1 h at 4 °C, the supernatants were removed, filtered through a 0.2-µm syringe filter, and used as primary antibody solutions in immunogold labeling experiments. Purified ovine PGHS-1 was from R. M. Garavito at Michigan State University; purified ovine PGHS-2 was from Cayman Chemical Co., Ann Arbor, MI. After incubation with primary antibody, each grid was washed eight times for 3 min on drops of 1% bovine serum albumin, PBS, 0.02% sodium azide before a 1-h incubation at room temperature on drops of gold-conjugated secondary antibody (Amersham Pharmacia Biotech GAR-G5 diluted 1:75 in 1% bovine serum albumin, PBS, 0.02% sodium azide). After a washing step, sections were post-fixed and stained for contrast by floating the grids sequentially on drops of 2% glutaraldehyde, 2% osmium tetroxide, and 2% uranyl acetate. Polyvinyl alcohol (2%) was used to embed the grids before observation by transmission electron microscopy (23).

Antibodies Specific for PGHS-1 and PGHS-2-- All primary antibodies were raised in rabbits as described previously (24). For PGHS-2 staining of human monocytes and HUVECs, a polyclonal antibody raised against ovine PGHS-2 was used. This antibody (from Dr. Jilly Evans, Merck Frosst) cross-reacts with human PGHS-2 but not with ovine or human PGHS-1 (25). PGHS-1 immunostaining of NIH 3T3 mouse fibroblasts was performed with an affinity purified anti-peptide antibody directed against amino acids Leu274-Arg288 of murine PGHS-1 (26). This antibody does not cross-react with PGHS-2 in Western blotting experiments. Another antibody, an IgG fraction from a polyclonal antibody raised against PGHS-1 (27), recognizes only PGHS-1 and was used for immunolabeling of NIH 3T3 cells and HUVECs. PGHS-2 labeling of NIH 3T3 cells was performed using an affinity purified anti-peptide antibody directed against an 18-amino acid cassette near the C terminus of PGHS-2 (12, 28); this antibody does not cross-react with any known PGHS-1 but recognizes human and murine PGHS-2 on Western blots.

Distribution of PGHS-1 and PGHS-2 between Inner and Outer Membranes of the Nuclear Envelope-- We determined the distribution of PGHS-1 and PGHS-2 between the inner and outer nuclear membranes essentially as described previously (21). Gold particles lying on or within one 5-nm particle diameter of the inner nuclear membrane were designated as being on the inner nuclear membrane. Particles lying on or within one 5-nm particle diameter of the outer nuclear membrane were designated as being on the outer nuclear membrane. When a gold particle was observed in the nuclear envelope and not within one particle diameter of either membrane, it was designated as lumenal. Analysis of the distribution of PGHS-1 and PGHS-2 was performed on a total of 24 and 32 cells, respectively. Distribution analysis was limited to well-preserved sections of nuclear envelope. Only regions of cells in which the inner and outer nuclear envelope were clearly distinguishable were used for our analyses.

Statistical Analysis-- The distribution analysis described above was performed using at least three experimental groups of well preserved cells for both PGHS-1 in NIH 3T3 cells and PGHS-2 in monocytes. The analysis of PGHS-2 in NIH 3T3 cells was performed on seven cells taken from two separate experiments. Analysis of the mean distribution percentages using Student's t test showed that there was no significant difference between the inner membrane/outer membrane distribution of PGHS-1 and -2 in the cell types analyzed. It is possible for a protein of the nuclear envelope to be present in significantly different amounts on the inner and outer nuclear membranes, as evidenced by our previous work (21) on 5-lipoxygenase and FLAP.

Subcellular Fractionation and Western Analysis-- NIH 3T3 cells were prepared as whole cell lysates, microsomes, or isolated nuclei. For whole cells, harvests of three 100-mm culture dishes of serum-stimulated NIH 3T3 cells in PBS were collected by centrifugation and resuspended in Hanks' balanced salts solution (HBSS). The resuspended cells were then disrupted by sonication and Dounce homogenization to produce whole cell lysates. Microsomes were prepared as described previously (29-31) from 10 plates of cells except that the 200,000 × g pellet was resuspended in HBSS. Isolation of cell nuclei (32, 33) began with the harvest of 20 dishes of 3T3 cells in PBS followed by centrifugation at 1000 × g. Nuclei were isolated in the presence of 0.2 or 1% saponin as described below. Isolation of nuclei in the absence of detergent was begun by resuspending the nuclei in 10 ml of cold Buffer A (10 mM Tris, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg leupeptin/ml, and 3 mM MgCl2 (pH 8.0)) and incubation on ice for 5 min, followed by 5 s of gentle vortexing. After passage 10 times through a 20-gauge needle, the crude nuclei were again collected by centrifugation at 1000 × g. The pellet was resuspended in 6 ml of cold Buffer A and incubated on ice for 5 min. After an additional 10 passages through a 20-gauge needle, the suspension was subjected to gentle homogenization in a Teflon homogenizer. Half of the suspension was placed in each of two Beckman 5-ml UltraClear ultracentrifuge tubes. A nuclear spin cushion was prepared by dissolving 6.16 g of sucrose in 10 ml of Buffer B (60 mM KCl, 15 mM NaCl, 15 mM Tris, 0.15 mM spermine, 0.5 mM spermidine, and 0.5 mM dithiothreitol) to give a 1.8 M sucrose solution. After underlaying 2 ml of the sucrose solution beneath the crude nuclear suspension, the tubes were centrifuged for 19 min at 4 °C at 13,500 rpm in a Beckman SW50.1 rotor. Isolated nuclei were visible at the bottom of the tubes. The liquid was removed from the tubes by aspiration, and the nuclear pellet was resuspended in 400 µl of HBSS. Nuclei were washed 4 times with HBSS and collected by centrifugation at 1800 rpm for 3 min in a microcentrifuge. Purified nuclei and whole cell lysates were disrupted by sonication before protein concentrations were determined using a Bio-Rad Protein Assay solution. Aliquots of broken cells, microsomes, or nuclei (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting as described previously (34). Densitometric quantitation of immunoreactive PGHS-1 or -2 was performed using a Bio-Rad GS-505 Molecular Imaging System and Molecular Analyst software. For a given isozyme, densities are expressed as the ratio of nuclear to microsomal immunoreactivity. Student's t test was utilized to determine if these ratios for PGHS-1 and PGHS-2 were significantly different for each experimental condition.

Prostanoid Product Formation by Murine NIH 3T3 Cells-- Murine NIH 3T3 cells were cultured in DMEM with 10% fetal calf serum in a water-saturated 7% CO2 incubator. At approximately 60% confluence, the cells were starved for 48 h by incubation in DMEM containing 0.2% fetal calf serum. Quiescent cells were stimulated by the addition of fetal bovine serum (final concentration of 16%) for 3 h (20). In some experiments various PGHS-1 and/or PGHS-2 inhibitors (valerylsalicylate (500 µM), NS398 (15 µM), flurbiprofen (100 µM), or aspirin (500 µM)) were added 30 min before the addition of serum (10, 35), and when used, these agents were also present in the medium during the stimulation.

NIH 3T3 cells, typically from 20 100-mm culture dishes, were scraped from the dishes with a rubber policeman and collected by centrifugation. To isolate nuclei, cell pellets were resuspended in 8 ml of phosphate-buffered saline (PBS) containing 1% (for product analysis and Western blotting) or 0.2% (for Western blotting only) saponin using a 20-gauge needle and were collected by centrifugation at 1000 × g for 10 min. The crude nuclear pellets were washed twice with 3 ml of SM buffer (250 mM sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgSO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM CaCl2, 250 µM ATP, and 1 mM dithiothreitol) and then were resuspended in 1.5 ml of SM buffer. The pellet suspensions were layered on top of 1 ml of SM buffer containing 2.1 M sucrose in a centrifuge tube and centrifuged at 100,000 × g for 1 h. The nuclear pellets were resuspended with 20 mM Tris-HCl (pH 7.4) and sonicated three times for 5 s. Protein concentrations were determined using a Bio-Rad protein assay reagent.

The Western blotting for PGHS-1 and PGHS-2 was performed as described above. Radio-thin layer chromatographic analysis of prostanoid product formation was performed essentially as described by Takahashi et al. (36) using aliquots of the same samples used for Western transfer blotting. Each assay sample contained 35 µM [1-14C]arachidonic acid in 100 mM Tris-HCl (pH 7.4), 1 µM hematin, 2 mM tryptophan, and 2 mM glutathione in a 200-µl reaction volume; assays were performed for 10 min at 37 °C. PGHS-1 and PGHS-2 inhibitors (valerylsalicylate (500 µM), NS398 (15 µM), flurbiprofen (100 µM), or aspirin (500 µM)) were included as indicated in the figure legends. Thin layer chromatography plates were developed with the organic phase of ethyl acetate/acetic acid/2,2,4-trimethylpentane/water (110:20:50:100, v/v) for 1 h, exposed to x-ray film (Kodak, Bio-MAX), and the products determined by comparison with chromatographic standards. The amounts of PGE2 and PGF2 were quantified by scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Immunogold Labeling of PGHS-2 in Human Monocytes and HUVECs-- Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human volunteers, treated for 20 h with bacterial lipopolysaccharide (LPS), and processed for electron microscopy. Western blotting of samples of these cells prior to fixation established that, as expected, PGHS-2 expression was induced by LPS but that vehicle-treated cells lacked detectable levels of this isozyme (37). Monocytes in LPS-treated samples, when incubated with an antibody raised against ovine PGHS-2, exhibited perinuclear staining for PGHS-2 (Fig. 1A). PGHS-2 labeling was completely eliminated by preadsorption of the anti-PGHS-2 antibody with purified ovine PGHS-2 (Fig. 1B). No comparable gold label was seen in vehicle-treated cells (data not shown). PGHS-2 labeling was observed on both the inner and outer membranes of the nuclear envelope in monocytes present in LPS-treated PBMC preparations (Fig. 1C). The distribution of PGHS-2 within the nuclear envelope of 26 LPS-treated monocytes was determined by counting the gold particles associated with well preserved regions of the inner and outer nuclear membranes (Table I). In performing these distribution analyses, only those segments of the nuclear envelope in which both the inner and outer nuclear membranes were clearly distinguishable were used. PGHS-2 labeling was approximately equally distributed between the inner and outer membranes of the nuclear envelope.


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Fig. 1.   Immunogold labeling of PGHS-2 in human monocytes. Human peripheral blood mononuclear cells were isolated, fixed, and processed for electron microscopy as detailed in the text. Ultrathin cryosections were labeled with either a primary antibody specific for PGHS-2 (A and C) or a primary antibody specific for PGHS-2 which had been preadsorbed with purified PGHS-2 (B). This was followed by treatment with a secondary anti-rabbit IgG-gold conjugate. A and B, sections of monocytes showing a portion of the nucleus and cytoplasm with gold label denoted by small arrowheads. C, region of a nuclear envelope with small arrowheads denoting gold particles on the outer (o) nuclear membrane and large arrowheads denoting label on the inner (i) nuclear membrane. C, opposed, open arrows indicate well preserved sections of nuclear envelope representative of sections used in the analysis of PGHS-2 distribution between the inner and outer nuclear membranes. N, nucleus; er, endoplasmic reticulum. Scale bar = 0.1 µm.

                              
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Table I
Analysis of PGHS-1 and PGHS-2 distribution within the nuclear envelope
The distribution of PGHS-1 and PGHS-2 within the nuclear envelope of LPS-treated human monocytes and serum-treated murine NIH 3T3 cells were determined as detailed under "Experimental Procedures." In this analysis gold particles were counted as being associated with either the inner or outer nuclear membrane if they were touching or within one particle diameter of that membrane. Other particles, not within one particle diameter of either membrane, were designated here as lumenal. Only well preserved regions of nuclear envelopes (i.e. regions in which both the inner and outer nuclear membranes were visible) were used for this analysis. For monocytes, particles representing PGHS-2 from a total of 26 cells were counted. For NIH 3T3 cells, a total of 27 and 7 cells were analyzed for particles corresponding to PGHS-1 and PGHS-2, respectively. Analysis of the mean distribution percentages using Student's t test showed that there was no significant difference between the inner membrane/outer membrane distribution of PGHS-1 and -2 in the cell types analyzed.

To determine if the distribution pattern of PGHS-2 was the same in cell types other than monocytes, we performed additional studies with (a) serum-treated murine NIH 3T3 cells and (b) IL-1beta -treated human umbilical vein endothelial cells (HUVEC) both of which are known to express PGHS-2 (20, 38). An anti-peptide antibody raised against the C-terminal 18-amino acid cassette of PGHS-2 was used to label PGHS-2 in serum-treated NIH 3T3 cells. This antibody labeled serum-treated NIH 3T3 cells along the nuclear membrane and in the ER (not shown). PGHS-2 immunoreactivity was distributed equally between the inner and outer nuclear membranes in NIH 3T3 cells (Table I). PGHS-2 labeling of serum-treated NIH 3T3 cells was eliminated by preincubation of the antibody with its cognate peptide. Finally, PGHS-2 staining in IL-1beta -treated HUVECs was present on the inner and outer nuclear membranes and in the ER, a pattern similar to that seen in LPS-treated monocytes and serum-treated NIH 3T3 cells (data not shown).

Immunogold Labeling of PGHS-1 in Murine NIH 3T3 Cells-- Serum-starved or serum-treated NIH 3T3 cells were fixed and processed for immunoelectron microscopy. The expectation that PGHS-1 would be expressed at similar levels in both starved and treated cells (20, 39) was confirmed by Western blotting (not shown). Serum-treated (Fig. 2A) or serum-starved (not shown) NIH 3T3 cells exhibited perinuclear and ER staining for PGHS-1. PGHS-1 labeling was eliminated by preincubation of the anti-PGHS-1 antibody with its cognate peptide (Fig. 2B). The immunostaining experiments depicted in Fig. 2 were performed using an anti-PGHS-1 antibody raised against a peptide corresponding to amino acids Leu274-Arg288 of murine PGHS-1. Identical experiments performed with another anti-PGHS-1 antibody, this one raised against whole ovine PGHS-1 (27), resulted in a pattern of staining indistinguishable from that seen with the anti-peptide antibody (not shown). PGHS-1 staining by the antibody prepared against the whole protein was eliminated by preadsorption of the antibody with purified ovine PGHS-1.


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Fig. 2.   Immunogold labeling of PGHS-1 in murine NIH 3T3 cells. NIH 3T3 cells were serum-treated, fixed, and processed for electron microscopy as detailed in the text. Ultrathin cryosections were labeled with either an affinity purified rabbit anti-peptide antibody to the sequence Leu274-Arg288 of murine PGHS-1 (A and C) or the same antibody preincubated with its cognate peptide (B). This was followed by treatment with a secondary anti-rabbit IgG-gold conjugate. A and B, sections of NIH 3T3 cells showing portions of the nucleus and cytoplasm with gold label denoted by small arrowheads. C, region of a nuclear envelope with small arrowheads denoting gold particles on the outer (o) nuclear membrane and large arrowheads denoting label on the inner (i) nuclear membrane. C, opposed, open arrows indicate well-preserved sections of nuclear envelope representative of sections used in the analysis of PGHS-1 distribution between the inner and outer nuclear membranes. N, nucleus; er, endoplasmic reticulum. Scale bar = 0.1 µm.

Several experiments were performed using a total of 24 different NIH 3T3 cells in which well preserved sections of the nuclear envelope were analyzed to determine the distribution of PGHS-1 between the inner and outer nuclear membranes (Fig. 2C). Gold particles representing PGHS-1 staining along the NE were present in approximately equal abundance on the inner and outer nuclear membranes (Table I). Our results confirm that PGHS-1 is a membrane protein of the ER and nuclear envelope (40) and establish that PGHS-1 is distributed equally between the membranes of the nuclear envelope. Collectively, the data demonstrate that PGHS-1 and -2 reside in the same subcellular membranes.2

Western Blotting of PGHS-1 and PGHS-2 in Subcellular Fractions-- Earlier immunofluorescence studies from our laboratory indicated that PGHS-1 and PGHS-2 are in the ER and nuclear envelope but that the concentration of PGHS-2 in the nuclear envelope is roughly twice that of the ER, whereas the concentration of PGHS-1 is the same in the nuclear envelope and the ER (12). In contrast, our quantitative immunoelectron microscopy indicated that PGHS-1 and -2 are present in the same subcellular locations in approximately equal proportions. There are significant differences between the fixation and staining protocols used for immunofluorescence and electron microscopy, any one of which could conceivably cause subtle differences in the patterns of staining. One difference involves the use of detergents to permeabilize the cells for immunofluorescence staining. Accordingly, we determined the effect of saponin, the detergent used in our earlier immunofluorescence work (12), on the distribution of PGHS-1 and PGHS-2 in nuclear and ER fractions from NIH 3T3 cells. When nuclei were isolated in the presence of 1% saponin from murine NIH 3T3 cells, immunoreactive PGHS-2 was relatively more abundant in nuclear membranes than in microsomal membranes, whereas PGHS-1 immunoreactivity was equally distributed between nuclear and microsomal membranes in the same experiments (Fig. 3A).3 Qualitatively similar results were obtained when nuclei were isolated in the presence of 0.2% saponin, although the difference between PGHS-1 and PGHS-2 distribution were not statistically significant (Fig. 3A). However, when Western blotting experiments were conducted on subcellular fractions isolated from serum-stimulated murine NIH 3T3 cells in the absence of detergent (Fig. 3A), PGHS-1 and PGHS-2 were similarly distributed between nuclear and microsomal membranes. We measured the ratios of immunoreactive PGHS-1 and -2 in nuclei versus microsomes using densitometry (Fig. 3B). In three experiments in the presence of 1% saponin, the ratios of nuclear to microsomal immunoreactive PGHS-1 and -2 were 1.1 ± 0.1 and 1.9 ± 0.2, respectively. In the presence of 0.2% saponin, the same ratios for PGHS-1 and -2 were 1.14 ± 0.1 and 1.26 ± 0.1. Exclusion of detergent resulted in nuclear to microsomal immunoreactivity ratios for PGHS-1 and -2 of 1.08 ± 0.1 and 1.10 ± 0.2, respectively. These data suggest that PGHS-1 and PGHS-2 can be differentially solubilized from nuclear membranes by detergents such as saponin. This may account for the differential patterns of PGHS-1 and PGHS-2 staining that have been observed in immunofluorescence studies (12).


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Fig. 3.   Western blot analysis of PGHS-1 and PGHS-2 in subcellular fractions of murine NIH 3T3 cells. A, whole cells, microsomes, or nuclei were prepared from murine NIH 3T3 cells in the presence of 0.2% saponin, 1% saponin, or no detergent as described in the text. After separation of the protein fractions by SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose, isozyme-specific antibodies were used as indicated to visualize PGHS-1 and PGHS-2 immunoreactivity in each membrane fraction. B, densitometric analysis was used to determine the ratio of immunoreactive PGHS-1 or PGHS-2 in nuclear versus microsomal membranes. *, significantly different from each other.

Products Formed via PGHS-1 and -2 in NIH 3T3 Cells-- As shown in Fig. 4A, quiescent murine NIH 3T3 cells express PGHS-1 but not PGHS-2, whereas after serum stimulation, PGHS-2 is detectable, and there is no significant change in the level of immunoreactive PGHS-1. To determine if a different set of products are produced via PGHS-1 versus PGHS-2, serum stimulated NIH 3T3 cells were treated with various PGHS-1 and PGHS-2 inhibitors, and the products formed from [1-14C]arachidonic acid were separated and quantified. The main prostanoid product derived from arachidonic acid by PGHS-1 and -2 in NIH 3T3 cells was PGE2 in all cases (Fig. 4B). Following serum stimulation, the amount of product increased by about 20%. We presume that this results from the increase in PGHS-2 (Fig. 4B). In support of this presumption are the following findings. Following treatment of serum-starved 3T3 cells with valerylsalicylate (500 µM), an inhibitor which is relatively specific for PGHS-1 (35), prostanoid production was almost completely inhibited, whereas valerylsalicylate caused incomplete (~80%) inhibition of prostanoid formation by serum-stimulated 3T3 cells. NS398 is a PGHS-2-specific inhibitor (41), and NS398 (15 µM) caused a 15-20% inhibition of prostanoid formation by serum-stimulated cells but had little effect on synthesis by serum-starved cells. Flurbiprofen inhibits both PGHS-1 and PGHS-2 (10), and flurbiprofen (100 µM) completely inhibited synthesis by both serum-starved and serum-stimulated 3T3 cells.


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Fig. 4.   Prostanoid production by serum-starved and serum-stimulated murine NIH 3T3 cells treated with various inhibitors of PGHS-1 and and PGHS-2. Serum-starved or serum-stimulated 3T3 cells were treated with the indicated inhibitors and incubated with 35 µM [1-14C]arachidonic acid, and products were analyzed by radio-thin layer chromatography as described in the text. A, Western blotting analyses of PGHS-1 and -2 in NIH 3T3 cells with or without NSAIDs; w/o, no drug; w/VAL, with valerylsalicylate (500 µM); w/NS, with NS398 (15 µM); w/FBP, with flurbiprofen (100 µM); w/ASP, with aspirin (500 µM). Minus indicates serum-starved cells and plus denotes serum-stimulated cells. B, audioradiogram of prostanoid products. The products are indicated on the left side. The ratios of relative amount of PGE2 formation are indicated below each lane. AA, arachidonate acid; HETEs, 11- and 15-hydroxyeicosatetraenoic acids; HHT, 10-hydroxyheptadecatrienoic acid.

To determine if different prostanoid products were formed via PGHS-1 or PGHS-2 in either serum-starved or serum-stimulated cells by different subcellular fractions, we assayed whole cell, nuclear, and microsomal preparations from serum-starved and serum-stimulated cells in the presence of PGHS-1- and PGHS-2-specific inhibitors. The main product formed in the presence of NS398 was PGE2 in all three preparations from both starved and stimulated cells (Fig. 5). Similarly, PGE2 was the major product observed when the experiments were performed in the presence of valerylsalicylate (data not shown).


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Fig. 5.   Prostanoid products formed by subcellular fractions of serum-starved and serum-stimulated murine NIH 3T3 cells in the presence of NS398. Cell, whole cell; Microsome, microsomal fraction; Nuclear, nuclear fraction from serum-starved (-) and serum-stimulated (+) cells were incubated with 35 µM [1-14C]arachidonic acid in the presence of NS398 (15 µM). Prostanoid products were analyzed by radio-thin layer chromatography as described in the text. A, Western blotting of PGHS-2 in NIH 3T3 cell fractions with (+) or without (-) serum stimulation. B, autoradiogram of prostanoid products. Abbreviations are as indicated in the legend to Fig. 4.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

PGHS-1 and PGHS-2 have structurally homologous membrane binding domains containing four amphipathic helices which anchor the proteins to one leaflet of the lipid bilayer (5-7, 40, 42, 43). Previous work has established that both isozymes are located on the lumenal surfaces of the ER and nuclear envelope (24, 40). However, immunocytofluorescence studies suggested that PGHS-2 was more concentrated in the nuclear envelope than the ER, whereas PGHS-1 was equally distributed in both compartments (12). One possibility to account for these latter findings was that PGHS-2 was uniquely localized on the inner membrane of the nuclear envelope. The major aim of the present studies was to examine this possibility using immunoelectron microscopy. The issue of PGHS-1 versus PGHS-2 localization is of particular interest because of the potential relationship between PGHS-2-derived products generated at the nuclear envelope and nuclear signaling associated with cell replication or differentiation.

The major finding of our EM study is that in NIH 3T3 cells both PGHS-1 and PGHS-2 are present in equal proportions on both the inner and outer membranes of the nuclear envelope. Human monocytes and umbilical vein endothelial cells stained for PGHS-1 or PGHS-2 also exhibited identical distribution patterns. Our results make it clear that both isozymes are present in the same subcellular compartments and at comparable concentrations. Of course, we cannot rule out the possibility that PGHS-1 and PGHS-2 are associated with different microdomains within these compartments.

Earlier results from immunofluorescence studies which employed low concentrations of saponin to permeabilize cell membranes and had suggested that PGHS-2 is preferentially localized to the nuclear envelope (12) can be explained on the basis of differential solubilization of the two proteins from the nuclear envelope. When ER and nuclear membranes were prepared in the presence of 1% saponin, PGHS-2 was more concentrated in the nuclear fraction, whereas PGHS-1 was found in equal abundance in the ER and nuclear membranes; in the absence of saponin, PGHS-2 was present at the same concentration in the ER and nuclear envelope. Presumably these differences in affinities of the two proteins for the nuclear envelope are a result of the significant differences in the amino acid composition of the membrane binding domains of the two isozymes (1, 44).

Studies of prostaglandin product formation by whole cell, ER, and nuclear fractions from murine 3T3 cells indicated that all fractions produced the same set of products in the presence and absence of inhibitors specific for PGHS-1 or PGHS-2. These results indicate that at least in 3T3 cells, there is no isozyme-specific channeling of the endoperoxide intermediate to specific PGH2 metabolizing synthases (e.g. PGE synthase).

A model has emerged suggesting that PGHS-1 and PGHS-2 act independently and that, at least in part, the inducible isozyme, PGHS-2, provides prostaglandins for a nuclear eicosanoid signaling system (1, 17, 45). Although this model may still be correct, our results imply that any specific connection between PGHS-2 and the generation of products that function in the nucleus would have to result from differences in the expression of the activities of PGHS-1 and PGHS-2 and not from gross differences in the subcellular distributions of PGHS-2 versus PGHS-1. That is because both PGHS-1 and PGHS-2 appear to be present in the same membranes, factors other than compartmentation account for the separation of their activities into two independent systems. The most likely factors are differences in interactions with different phospholipases and/or differences in enzyme kinetics. Arm, Austen, Herschmann, and co-workers (13, 15, 46-48) have demonstrated that two separate phases of PGD2 synthesis in mast cells are independently coupled to PGHS-1 (early phase) and PGHS-2 (late phase) by different phospholipases A2. Kinetic mechanisms for separating the actions of PGHS-1 and PGHS-2 have also been described. For example, PGHS-2 has a significantly lower threshold for hydroperoxide activation than PGHS-1 thereby enabling PGHS-2 to oxygenate arachidonic acid in the presence of lower peroxide concentrations (9, 49). In addition, negative allosteric regulation of PGHS-1 by arachidonic acid, at concentrations between 0.5 nM and 1 µM, has the overall effect of causing a 2-4-fold greater rate of PGHS-2-mediated prostanoid formation (50). These kinetic differences between PGHS-1 and PGHS-2 have been identified with purified or partially purified enzyme preparations. However, it may be possible to test for kinetic differences between the two isozymes in intact cells using histochemical assays of enzyme activity (12).

Another more speculative possibility to account for the independent operation of PGHS-1 and PGHS-2 in cells where both isozymes are expressed is the existence of accessory proteins which differentially affect the rate of prostaglandin endoperoxide formation by PGHS-1 versus PGHS-2. Although no such protein(s) has been identified in the prostanoid biosynthetic system, there is a precedent for an accessory protein in the leukotriene pathway. Leukotrienes synthesized through 5-lipoxygenase arise from arachidonic acid apparently delivered to the 5-lipoxygenase by an activating protein, FLAP (51-55).

The observation that both PGHS-1 and PGHS-2 are located on the inner nuclear membrane is of interest from a cell biology perspective. To our knowledge, no other endogenous integral membrane proteins of the ER have been demonstrated to be present on the inner nuclear membrane. FLAP, another integral membrane protein involved in eicosanoid biosynthesis, has been localized to the inner and outer membranes of the NE (21, 56). However, in contrast to PGHS-1 and -2, FLAP is predominantly localized to the nuclear envelope; the orientation of FLAP in the membrane is not well characterized. One model of how integral membrane proteins synthesized in the ER reach the inner nuclear membrane involves lateral diffusion through the membrane bilayers of the NE (57-59). According to this model, membrane proteins are subject to a size constraint imposed by the lateral channel diameter of the nuclear pore complex, which serves to separate the inner and outer nuclear membranes. Both PGHS-1 and PGHS-2 are targeted initially to the ER by a KDEL-like C-terminal targeting signal (60). We propose that both isozymes are then able to bypass the nuclear pore complex and reach the inner nuclear membrane by lateral diffusion. Our reasoning is based on the nature of their interaction of PGHS-1 and PGHS-2 with membranes (i.e. via a monotopic membrane binding domain) and the fact that the proteins are on the lumenal surface of the membrane. Consistent with this concept are studies with the lamin B receptor, an integral membrane protein that is targeted exclusively to the inner nuclear membrane via specific targeting signals (58). When the nucleoplasmic/cytoplasmic oriented extramembrane domain of lamin B receptor is artificially enlarged, the protein is retained in the ER (and outer nuclear membrane). Other ER membrane proteins with large extramembrane domains oriented toward the cytoplasm are also thought to be restricted to the outer membrane of the nuclear envelope and the ER (58, 61). P450s such as thromboxane synthase and prostacyclin synthase are integral membrane proteins of the ER and have relatively large cytoplasmic domains. Thus, these proteins are not likely to be present on the nucleoplasmic face of the inner nuclear membrane and would be unable to metabolize efficiently PGH2 generated by PGHS-1 or PGHS-2 present on this membrane.

We conclude that PGHS-1 and PGHS-2 are present in similar proportions on the endoplasmic reticulum, outer nuclear membrane, and inner nuclear membrane of NIH 3T3 cells, human monocytes, and HUVECs. Fatty acid substrates for PGHSs appear to be supplied via both an sPLA2 that functions on the phospholipids on the cell surface and a cPLA2 that undergoes a Ca2+-dependent translocation to the cytosolic face of the ER and outer membrane of the nuclear envelope (62-66) and perhaps the inner membrane of the nuclear envelope (67). However, the issue of whether there is preferential coupling of different phospholipases A2 to PGHS-1 versus PGHS-2 is currently unresolved. PGH2 formed through both PGHS-1 and PGHS-2 can apparently diffuse readily through membranes (68). Any PGH2 that diffuses from the ER lumen into the cytoplasm is likely to be metabolized by enzymes such as thromboxane A2 synthase or PGI2 synthase located on the cytoplasmic surface of the ER and nuclear envelope (69). The fate of the PGH2 that diffuses into the nucleoplasm is presently unknown.

    ACKNOWLEDGEMENTS

We thank Jeanne Barker, Sol Scott, Jamie Flanagan, Doug Kawka, and Jayne Chen at Merck for helpful suggestions and Dr. John Heckman at the Michigan State University Center for Electron Optics for discussions and the use of equipment. We also thank Dr. Howard Worman for advice relating to integral membrane protein targeting.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK22042.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824. Tel.: 517-353-8680; Fax: 517-353-9334; E-mail: smithww{at}pilot.msu.edu.

1 The abbreviations used are: PGHS-1 and PGHS-2, prostaglandin endoperoxide H synthases-1 and -2; LPS, bacterial lipopolysaccharide; ER, endoplasmic reticulum; NE, nuclear envelope; IL-1beta , the beta -isoform of interleukin-1; PBMC, peripheral blood mononuclear cells; HUVEC, human umbilical vein endothelial cells; PG, prostaglandin; DMEM, Dulbecco's modified Eagle's medium; FLAP, 5-lipoxygenase activating protein; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline.

2 No immunogold labeling of PGHS-1 in vehicle- or LPS-treated monocytes in PBMC preparations was observed using several different anti-PGHS-1 antibodies, presumably due to the low level of PGHS-1 expression in these cells (37).

3 Similar results were observed in the mouse macrophage line RAW 264.7. Although PGHS-1 was always approximately equally distributed between microsomal and nuclear membranes, PGHS-2 was more concentrated in nuclear membranes 70% of the time in more than 20 experiments.

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Abstract
Introduction
Procedures
Results
Discussion
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