©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Intracellular Locations for Prostaglandin Endoperoxide H Synthase-1 and -2 (*)

Ikuo Morita , Melvin Schindler , Martha K. Regier , James C. Otto , Takamitsu Hori , David L. DeWitt , William L. Smith (§)

From the (1) Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The subcellular locations of prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2) were determined by quantitative confocal fluorescence imaging microscopy in murine 3T3 cells and human and bovine endothelial cells using immunocytofluorescence with isozyme-specific antibodies. In all of the cell types examined, PGHS-1 immunoreactivity was found equally distributed in the endoplasmic reticulum (ER) and nuclear envelope (NE). PGHS-2 immunoreactivity was also present in the ER and NE. However, PGHS-2 staining was twice as concentrated in the NE as in the ER. A histofluorescence staining method was developed to localize cyclooxygenase/peroxidase activity. In quiescent 3T3 cells, which express only PGHS-1, histofluorescent staining was most concentrated in the perinuclear cytoplasmic region. In contrast, histochemical staining for PGHS-2 activity was about equally intense in the nucleus and in the cytoplasm, a pattern of activity staining distinct from that observed with PGHS-1. Our results indicate that there are significant differences in the subcellular locations of PGHS-1 and PGHS-2. It appears that PGHS-1 functions predominantly in the ER whereas PGHS-2 may function in the ER and the NE. We speculate that PGHS-1 and PGHS-2 acting in the ER and PGHS-2 functioning in the NE represent independent prostanoid biosynthetic systems.


INTRODUCTION

PGH() synthases catalyze the formation of PGH from arachidonic acid. Newly formed PGH is subsequently converted to what are considered to be the biologically active prostanoids, PGD, PGE, PGF, thromboxane A, or PGI. There are two PGH synthase isozymes called PGHS-1 (COX-1) and PGHS-2 (COX-2). Each isozyme is encoded by a different gene (1, 2) . PGHS-1 is a constitutive enzyme present in many but not all mammalian cells (3, 4, 5) . Its expression appears to be regulated developmentally (6) . PGHS-2 is undetectable in most mammalian tissues (4, 5) , but expression of this isozyme can be induced by cytokines, growth factors, and tumor promoters (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .

PGHS-1 and PGHS-2 are 60% identical at the amino acid level. The most pronounced differences occur in the signal peptides and membrane binding domains near the N termini and in the C-terminal 15-25 amino acids (1, 2, 4, 19) . The kinetic properties of the two enzymes are quite similar. Both enzymes catalyze ( a) a cyclooxygenase reaction in which arachidonic acid is converted to PGG and ( b) a peroxidase reaction in which PGG is reduced to PGH(20, 21) . Moreover, PGHS-1 and PGHS-2 have similar V and Kvalues with arachidonate (21, 22, 23) , both enzymes form a tyrosyl radical in the presence of hydroperoxides (24) , and each isozyme undergoes suicide inactivation (22, 23) . Finally, those amino acids required for catalysis by PGHS-1 are conserved in PGHS-2 (19, 25) . Despite the overall enzymic similarities between PGHS-1 and PGHS-2, it should be noted that there are subtle differences between the active sites of PGHS-1 and PGHS-2 as evidenced by their different affinities toward some fatty acid substrates() and nonsteroidal anti-inflammatory drugs (21, 22, 23, 26, 27, 28) .

Treatment of quiescent murine 3T3 cells with serum causes an increase in PGHS-2 protein levels which peak approximately 3 h post-serum treatment. During this period, there are no apparent changes in the levels of PGHS-1 protein (8, 10, 16) . However, measurements of cyclooxygenase activities in quiescent and serum-stimulated (3 h) 3T3 cells have established that platelet-derived growth factor and serum cause only a 1.5- to 2-fold increase in total activity (8, 29, 30, 31) . Similar measurements comparing the cyclooxygenase activities of control and LPS-treated macrophages have yielded similar results (16) . Thus, in those cells which already express PGHS-1, there is no dramatic increase in the total PGH biosynthetic capacity following the induction of PGHS-2. Coupled with the fact that PGHS-1 and PGHS-2 have very similar kinetic properties, it is difficult to argue that PGHS-2 is induced simply to augment the biosynthetic capacity of PGHS-1. Rather, PGHS-2 seems likely to have a function(s) independent of PGHS-1.

There is recent biochemical evidence that PGHS-1 and PGHS-2 function as separate biosynthetic systems. Reddy and Herschman (32) reported that in murine 3T3 cells and macrophages PGHS-1 and PGHS-2 utilize different pools of arachidonate for synthesizing prostanoids. In murine bone marrow-derived mast cells, PGHS-1 and PGHS-2 are coupled to different stimulus-initiated pathways (33) . These results argue that the arachidonate used by PGHS-1 and PGHS-2 for prostanoid biosynthesis is derived from different endogenous lipid pools.

If PGHS-1 and PGHS-2 do use different intracellular pools of arachidonate, there must be mechanisms for compartmentalizing PGHS-1 and PGHS-2 such that each isozyme would only have access to its own arachidonate substrate pool. One possibility is that the PGHS isozymes are located in different subcellular compartments. Previous studies, including those from our laboratory, have indicated that PGHS-1 and PGHS-2 are both found in the endoplasmic reticulum (ER) and nuclear envelope (NE) of fibroblasts and macrophages and that there is no particularly obvious difference in the subcellular distributions of the isozymes (34, 35, 36, 37) . We have now reinvestigated this issue using a variety of cell types, several antibodies for each isozyme, and a newly developed histofluorescence assay for PGHS activity. Our results confirm the previous observations that both PGHS-1 and PGHS-2 are located within the ER and NE. However, PGHS-2 protein was found to be more concentrated in the NE than PGHS-1. And when the distributions of PGHS-1 and PGHS-2 activities were monitored by histofluorescence, only PGHS-2 caused staining within the cell nucleus.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium (DMEM) was from Life Technologies, Inc. Chloroquine, horse myoglobin, saponin, digitonin, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, FITC-conjugated concanavalin A (ConA) and DEAE-dextran were from Sigma. Ellman's reagent, Sulfolink maleimide-activated gel, and maleimide-activated KLH coupling kits were from Pierce Chemical Co. Fetal calf serum and calf serum were obtained from Hyclone. Peptides were from the Howard Hughes Research Center at Harvard Medical School. Arachidonic acid and linoleic acid were from Cayman Chemical Co. 5-(and -6)-Carboxyl-2`,7`-dichlorodihydrofluorescein diacetate (CDCF-DA) and 3,3`-dihexyloxacarbocyanine iodide (DiOC(3) ) were purchased from Molecular Probes. Valerylsalicylate was synthesized as described previously (38) . Other reagents were from common commercial sources.

Cell Culture

NIH 3T3 fibroblasts were grown on 100-mm Corning culture dishes in DMEM supplemented with 8% calf serum and 2% fetal calf serum. The cells were grown to approximately 80% confluence, detached from the monolayer with 0.25% trypsin, 0.02% EDTA, diluted into 50 ml of serum-supplemented DMEM, and used to inoculate Lab-Tek chambered coverglasses. NIH 3T3 cells cultured in DMEM supplemented with 0.2% calf serum for 48 h were designated ``quiescent'' cells. To obtain ``serum-activated'' cells, the serum-starved cells were changed to a medium containing 8% calf serum and 2% fetal calf serum in DMEM and incubated for 3 h.

NIH 3T3 cells expressing PGHS-2 constitutively were prepared as follows. A 1.8-kilobase SalI restriction fragment containing the entire murine PGHS-2 coding sequence (39) was subcloned into the Lac-inducible plasmid pRSVNOT (40) . The pRSVNOT plasmid, part of the LacSwitch gene expression system (Stratagene), contains a combination Rous sarcoma virus promoter (RSV)-bacterial -galactosidase promoter (RSV/-gal) to drive transcription of the donor gene. The pRSVNOT-PGHS-2 plasmid was transfected into a murine NIH 3T3 cell line expressing a Lac repressor protein, modified so that it functions as an efficient eukaryotic transcription factor. In theory, the Lac repressor protein tonically suppresses gene expression from the RSV/-gal promoter, and suppression can be relieved by addition of isopropyl-1-thio--D-galactopyranoside, allowing regulated expression of the target gene. During characterization of pRSVNOT-PGHS-2 transformants, however, one cell line was identified that constitutively expressed PGHS-2. This line was designated as the 3T3-cPGHS-2 cell line. 3T3-cPGHS-2 cells constitutively express PGHS-1 and PGHS-2 under serum-starved culture conditions. 3T3-cPGHS-2 cells were routinely grown in DMEM supplemented with 10% fetal calf serum and 600 µg/ml geneticin and hygromycin. NIH 3T3-cPGHS-2 cells were transferred to Lab-Tek chambered coverglasses and cultured for 2 days, the medium was removed, and the cells were then cultured in DMEM supplemented with 0.2% calf serum for 24 h. These cells were used for immunocytochemistry as described below.

Human umbilical vein endothelial cells (HUVECs) (Cell Systems, passage 3-4) were cultured in Lab-Tek chambered coverglasses in Cell System CS-C venous endothelial cell growth medium for 3 days. The cells were then treated with 2 ng/ml of phorbol myristate acetate (PMA) for 3 h and used for localization experiments.

Bovine arterial endothelial cells (BAECs) were isolated from freshly excised carotid artery obtained at a local abattoir as described previously (41) . The cells were seeded on 100-mm Corning culture dishes in DMEM supplemented with 20% fetal calf serum and subcultured in DMEM containing 10% fetal calf serum. The cells were identified as endothelial cells from their typical cobblestone appearance at confluency. The endothelial cells (passages 20-22) were cultured in Lab-Tek chambered coverglasses for 2 days, and then cells were treated with 2 ng/ml phorbol 12-myristate 13-acetate (PMA) for 3 h before use in localization experiments.

Isozyme-specific Anti-peptide Antibodies

Affinity-purified isozyme-specific antibodies to PGHS-1 and PGHS-2 were generated as described previously (36) . Antibodies reactive against both the murine and human PGHS-1 were prepared using the following KLH-coupled peptides derived from the murine protein: ( a) Leu-Met-Arg-Tyr-Pro-Pro-Gly-Val-Pro-Pro-Glu-Arg-Gln-Met-Ala-Cys (residues 274-289) or ( b) Cys-Asn-Thr-Ser-Met-Leu-Val-Asp-Tyr-Gly-Val-Glu-Ala-Leu-Val-Asp-Ala-Phe-Ser (residues 412-429). An additional anti-peptide antibody reactive with human, ovine, and bovine PGHS-1, but not murine PGHS-1, was prepared against the ovine PGHS-1 sequence, Leu-Met-His-Tyr-Pro-Arg-Gly-Ile-Pro-Pro-Gln (residues 272-283) (36) . We have previously described an anti-peptide antibody reactive with human, bovine, and murine PGHS-2 (10, 22, 35, 36, 39) ; this antibody was prepared against the peptide Ser-His-Ser-Arg-Leu-Asp-Asp-Ile-Asn-Pro-Thr-Val-Leu-Ile-Lys (residues 584-598), a sequence unique to PGHS-2. Another antibody to murine PGHS-2 was prepared against the KLH-coupled murine PGHS-2-derived peptide, Cys-Gln-Asp-Pro-Gln-Pro-Thr-Lys-Thr-Ala-Thr-Ile-Asn (residues 569-580). Peptide-specific antibodies were purified by affinity chromatography using peptide-coupled Sulfolink matrix as described previously (35, 36) and were diluted to approximately 0.5 mg/ml.

Immunocytochemistry of PGHS

Murine NIH 3T3 cells, murine NIH 3T3-cPGHS-2 cells, HUVECs, and BAECs cultured in Lab-Tek chambers were washed in phosphate buffered saline (PBS), fixed by incubation for 10 min in 2% formaldehyde in PBS, and washed in 10% calf serum in PBS (35) . Affinity-purified anti-peptide antibodies to either PGHS-1 or PGHS-2 diluted 1:20 in PBS containing 0.2% saponin and 10% calf serum were added to the chambers, and the samples were incubated for 30 min. After washing in PBS containing 10% calf serum, the samples were incubated for 30 min with a 1:40 dilution of FITC-conjugated goat anti-rabbit IgG in PBS containing 0.2% saponin and 10% calf serum. The samples were washed with PBS containing 10% calf serum and then rinsed with PBS. For negative control staining, the same procedure as described above was performed ( a) without primary antibody or ( b) with primary antibody together with a 10 µM concentration of its cognate peptide.

Cell Labeling with DiOC(3) or FITC-labeled ConA

In one experiment, after murine 3T3 cells were subjected to immunocytofluorescence with anti-peptide antibodies, the cells were then stained using either DiOC(3) or FITC-labeled ConA. The cells were treated with 1 µg/ml DiOC(3, 42) for 10 s or with 5 µg/ml FITC-conjugated ConA (43) for 1 min at room temperature. The samples were then rinsed with PBS, pH 7.4. These procedures were performed on the platform of the microscope, and the samples were visualized immediately.

Histochemical Measurements of PGHS Activity

Quiescent or serum-activated NIH 3T3 cells were washed with PBS containing 0.1 mg/ml of both calcium chloride and magnesium chloride (PBS+), incubated with 2 µM CDCF-DA in PBS+ for 5 min, and washed with PBS+. The fluorescence intensity in the cells at 0 time and 20 s after the addition of 10 µM arachidonic acid was quantified by fluorescence confocal microscopy.

Fluorescence Confocal Microscopy

An Insight Bilateral scanning confocal microscope (Meridian Instruments, Okemos, MI) was used with an argon ion laser as the excitation source (44) . A 60 objective and a laser power setting of 1 milliwatt were used for subcellular localization of enzyme activity by histofluorescence and subcellular localization of DiOC(3) and FITC-labeled ConA. A 60 objective lens and a laser power setting of 30 milliwatts were used for subcellular localization of PGHSs by immunocytochemistry.


RESULTS

Subcellular Localization of PGHS-1 and PGHS-2 in Murine 3T3 Cells

Shown in Fig. 1are fluorescence photomicrographs of quiescent and serum-activated murine 3T3 cells stained by indirect immunofluorescence using primary antibodies specific for either PGHS-1 or PGHS-2. Staining for PGHS-1 was performed separately using two different anti-peptide antibodies directed against residues 274-289 and residues 412-429. In both quiescent and serum-activated 3T3 cells, PGHS-1 immunoreactivity was observed in the cytoplasm near the nucleus and also more peripherally. PGHS-1 is known to be present in the endoplasmic reticulum (ER) of 3T3 cells (34) , and, thus, fluorescent staining in the cytoplasm is due to staining of the ER. Anti-peptide antibodies reactive with residues 569-580 or 584-598 of murine PGHS-2 caused fluorescence staining of both the cytoplasm and the nuclear envelope (NE) of serum-activated 3T3 cells (Fig. 1); staining of the NE was clearly more prominent for PGHS-2 than for PGHS-1. No staining for PGHS-2 was observed in quiescent, serum-starved 3T3 cells which do not express this second PGHS isozyme (8, 10, 11, 35) . Moreover, preincubation of either of the anti-PGHS-1 or anti-PGHS-2 antibodies with their cognate peptides completely eliminated staining, and no staining was observed in the absence of the primary antibody.


Figure 1: Immunofluorescence staining for PGHS-1 and PGHS-2 in quiescent and serum-activated murine NIH 3T3 cells. Murine NIH 3T3 cells were subjected to indirect immunofluorescent staining using affinity-purified rabbit anti-peptide antibodies directed against PGHS-1 or PGHS-2 and then FITC-labeled goat anti-rabbit IgG as described in the text. The final concentrations of the anti-peptide antibodies used in each experiment were 25 µg/ml. Cognate peptides used to block antibodies were used at a concentration of 10 µM. A, serum-activated 3T3 cells and antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-288); B, computer enhancement shadowing of A; C, serum-activated 3T3 cells and antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-288) in the presence of cognate peptide; D, quiescent 3T3 cells and antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-288); E, serum-activated 3T3 cells and antibodies to murine PGHS-1 ((C)-NTSMLVDYGVEALVDAFS; residues 412-429); F, serum-activated 3T3 cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues 584-598); G, computer enhancement shadowing of F; H, serum-activated 3T3 cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues 584-598) in the presence of cognate peptide; I, quiescent 3T3 cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues 584-598); and J, serum-activated 3T3 cells and antibodies to murine PGHS-2 ((C)-GDPQPTKTATIN; residues 569-580). Magnification, 200.



To quantify the relative distributions of PGHS-1 and PGHS-2 in the ER and the NE of 3T3 cells, the cells were stained first with an antibody to a single PGHS isozyme, and staining intensities in the ER and on the NE were then quantified using confocal imaging microscopy. The same cells were then stained with either FITC-labeled ConA or a fluorescent dye (DiOC(3) ), both of which partition uniformly among intracellular membranes (42, 43) , and again the staining intensities were quantified in the ER and NE.() As shown in Fig. 2, the relative fluorescence intensities in the ER and NE were the same for PGHS-1, FITC-labeled ConA, and DiOC(3) . In contrast, the intensity of PGHS-2 immunofluorescence was two times higher in the NE than the ER.


Figure 2: Subcellular distribution of fluorescence staining for PGHS-1, PGHS-2, DiOC(3), and concanavalin A in serum-activated NIH 3T3 cells. Serum-activated murine NIH 3T3 cells were first subjected to indirect immunocytofluorescence staining using antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-288) or using antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues 584-598) essentially as described in the legend to Fig. 1. The same samples used to analyze PGHS immunoreactivity were subsequently treated with DiOC(3) (42) or FITC-conjugated ConA (43) as described in the text. Under the conditions used for detecting DiOC(3) or FITC-conjugated ConA fluorescence, the fluorescence attributable to PGHS staining was negligible. The intensities of fluorescence were quantified in 20 different areas (1 µm) within the cytoplasmic region neighboring the nucleus ( ER) and in 20 different areas (1 µm) over the nuclear envelope ( NE) in each cell. DiOC(3), open bars; FITC-labeled ConA, striped bars; PGHS-1, stippled bars; and PGHS-2, solid dark bars. The data represent mean ± S.E. of two cells in six individual culture chambers and are expressed as fluorescence intensities. For comparison of mean values between two sets of data, Student's t test was used, employing standard criterion of p < 0.01 ( double asterisk) to indicate statistical significance.



In a related experiment, we determined if time-dependent changes occurred in the distribution of either PGHS-1 or PGHS-2 in the NE relative to the ER of 3T3 cells after serum activation. As shown in Fig. 3 , the relative fluorescence intensities in the ER and NE were 2.0, 2.0, 1.7, and 1.7 for PGHS-2 during a 4-h treatment of initially quiescent 3T3 cells with fetal calf serum; in contrast, the relative intensities were 1.0 for PGHS-1 at all time points. When measurements were conducted at 3, 6, 9, and 12 h post-serum stimulation, the relative intensities were also the same (± 0.1) for PGHS-2 at all time points, and the relative intensities remained at 1.0 for PGHS-1. Overall, the intensities of immunostaining for PGHS-2 increased with time after serum activation, but the intensities of ER/NE staining remained relatively constant.


Figure 3: Subcellular distribution of PGHS-1 and PGHS-2 immunoreactivities during stimulation of murine NIH 3T3 cells with fetal calf serum. Murine 3T3 cells were serum-starved for 48 h and then treated with fetal calf serum for the indicated times. The cells were fixed with 2% formaldehyde and subjected to indirect immunocytofluorescent staining for either PGHS-1 ( upper panel) or PGHS-2 ( lower panel), and the intensities of fluorescence were quantified in the cytoplasm adjoining the nucleus (ER) and in the NE as described in the legend to Fig. 2; open circles, ER; closed circles, NE. The data represent mean ± S.E. of two cells in six individual culture chambers and are expressed as the percentage of the intensities in the NE relative to a cytoplasmic ( i.e. ER) fluorescence of 100%. For comparison of mean values between two sets of data, Student's t test was used, employing standard criterion of p < 0.05 ( single asterisk) and p < 0.01 ( double asterisk) to indicate statistical significance.



Subcellular Localization of PGHS-1 and PGHS-2 in Murine 3T3-cPGHS-2 Cells, Human Umbilical Vein Endothelial Cells, and Bovine Aortic Endothelial Cells

The subcellular locations of PGHS-1 and PGHS-2 in murine 3T3-cPGHS-2 cells, bovine aortic endothelial cells, and human umbilical vein endothelial cells were also determined by indirect immunofluorescence. The purpose of these studies was to determine if the distributions of PGHS-1 and PGHS-2 immunoreactivities in murine 3T3 cells were representative of other cells which express both isozymes. NIH 3T3-cPGHS-2 cells express PGHS-2 constitutively. Staining of serum-starved NIH 3T3-cPGHS-2 cells with antibodies to PGHS-2 showed intense staining of the NE with some cytoplasmic staining (Fig. 4). No staining occurred when the anti-PGHS-2 antibodies were preincubated with their respective cognate peptides (data not shown). Thus, the pattern of PGHS-2 staining observed with NIH 3T3-cPGHS-2 cells cultured under serum-starved conditions is the same as that seen under serum-activated conditions in the parent NIH 3T3 cell line. These results suggest that no secondary factor induced by serum treatment of 3T3 cells is required for PGHS-2 to become concentrated in the NE.


Figure 4: Immunofluorescence staining for PGHS-1 and PGHS-2 in quiescent murine NIH 3T3-cPGHS-2 cells which express PGHS-2 constitutively. Quiescent, serum-starved murine NIH 3T3-cPGHS-2 cells, which constitutively express both PGHS-1 and PGHS-2, were subjected to indirect immunocytofluorescence essentially as described in the legend to Fig. 1. A, quiescent 3T3-cPGHS-2 cells and antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-289); B, quiescent 3T3-cPGHS-2 cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues 584-598). Magnification, 500.



Anti-peptide antibodies to ( a) the trypsin cleavage sequence of ovine PGHS-1 (residues 272-283) (36) and ( b) the 18 amino acid cassette unique to PGHS-2 (residues 584-598 of murine PGHS-2) (35, 36) were used to stain nonstimulated and PMA-activated human umbilical vein endothelial cells (HUVECs) and bovine arterial endothelial cells (BAECs) by indirect immunofluorescence (Fig. 5). HUVECs and BAECs not treated with PMA only stained with antibodies to ovine PGHS-1; in both cases, the patterns of PGHS-1 staining were similar to that observed for PGHS-1 in NIH 3T3 cells (data not shown). Antibodies to PGHS-1 caused staining of the cytoplasm ( i.e. ER) of PMA-activated HUVECs and BAECs. This contrasted with the results observed for the PGHS-2 antigen where a very distinct nuclear ring of staining was also apparent. Again, in all cases, the immunofluorescent staining was blocked when the primary antibody was used in the presence of its cognate peptide. These data indicate that the subcellular locations of PGHS-1 and PGHS-2 observed in NIH 3T3 cells are common to endothelial cells from two different sources and species.


Figure 5: Immunofluorescence staining for PGHS-1 and PGHS-2 in PMA-treated human umbilical vein endothelial cells (HUVECs) and PMA-treated bovine arterial endothelial cells (BAECs). HUVECs and BAECs were activated by treatment with PMA as described in the text and then subjected to indirect immunofluorescent staining for PGHS-1 and PGHS-2 as described in the text and essentially as described for 3T3 cells in the legend to Fig. 1. A, PMA-treated HUVECs stained with antibodies to PGHS-1 (LMHYPRGIPPQ-(C); residues 272-282 of ovine PGHS-1); B, PMA-treated HUVECs stained with antibodies to PGHS-2 (SHSRLDDINPTVLIK; residues 584-598 of murine PGHS-2); C, PMA-treated BAECs stained using antibodies to PGHS-1 (LMHYPRGIPPQ-(C); residues 272-282 of ovine PGHS-1); and D, PMA-treated BAECs stained with antibodies to PGHS-2 (SHSRLDDINPTVLIK; residues 584-598 of murine PGHS-2). Magnification, 400.



Histochemical Staining for PGHS-1 and PGHS-2 Activities

To determine if there is a correspondence between the locations of PGHS-1 and PGHS-2 immunoreactivities and catalytic activities, we developed a histochemical procedure for visualizing PGHS activities. Briefly, NIH 3T3 cells were preloaded with CDCF-DA, and fluorescence measurements were made following the addition of arachidonic acid. CDCF is a common co-substrate for peroxidases and will serve as a reducing substrate for the peroxidase activities of PGHS-1 and PGHS-2. PGG generated by the cyclooxygenase activities of PGHSs upon the addition of arachidonate is reduced to PGH by the peroxidase activities of PGHSs with resultant oxidation of CDCF to a fluorescent compound which can be detected by confocal microscopy. As shown in Fig. 6, addition of 10 µM arachidonate to quiescent, serum-starved NIH 3T3 cells preloaded with CDCF led to an increase in fluorescence which was apparent in the cytoplasm of the cell but not in the cell nucleus. No fluorescence was observed upon addition of arachidonate to cells that had been pretreated with either 1 mM aspirin or 1 mM valerylsalicylate for 30 min prior to the addition of arachidonate; under these conditions, both aspirin and valerylsalicylate inhibit the cyclooxygenase activity (but not the peroxidase activity) of PGHS-1 (38) . When serum-activated NIH 3T3 cells were subjected to this same cytochemical assay, fluorescent staining was observed in the cytoplasm and, additionally, across the surface of the nucleus. Aspirin prevented arachidonate-induced fluorescence() in the serum-activated 3T3 cells. Valerylsalicylate, which is a relatively specific inhibitor of PGHS-1 (38) , appeared to reduce the intensity of the cytoplasmic fluorescence in serum-activated cells but did not block the nuclear staining. Thus, the nuclear staining and part of the cytoplasmic staining observed with serum-activated 3T3 cells is a result of the activity of PGHS-2, whereas only cytoplasmic fluorescence results from the activity of PGHS-1 ( cf. Fig. 6A versus 6 F). As noted earlier, quiescent 3T3-cPGHS-2 cells express PGHS-2 constitutively. When subjected to histofluorescence staining, the patterns of staining were similar to those of serum-activated 3T3 cells. These results, in which the subcellular locations of PGHS-1 and PGHS-2 activities were determined, provide support for the concept that PGHS-1 occurs mainly in association with the ER and the portion of the NE which is contiguous with the ER, whereas PGHS-2, while present in the ER, is more highly concentrated than PGHS-1 on the NE.


Figure 6: Subcellular localization of histofluorescence staining for PGHS-1 and PGHS-2 activities in quiescent and serum-activated murine NIH 3T3 cells and quiescent NIH 3T3-cPGHS-2 cells. Cells were loaded with 5-(and -6)-carboxyl-2`,7`-dichlorodihydrofluorescein (CDCF) by preincubation with the diacetate derivative (CDCF-DA) as described in the text. Arachidonate (10 µM) was added, and confocal fluorescence imaging microscopy was performed 20 s later. A, quiescent 3T3 cells incubated with 10 µM arachidonate; B, quiescent 3T3 cells pretreated for 30 min with 1 mM aspirin prior to the addition of arachidonate; C, quiescent 3T3 cells pretreated for 30 min with 1 mM valerylsalicylate prior to the addition of arachidonate; D, serum-activated 3T3 cells incubated with arachidonate; E, serum-activated 3T3 cells pretreated for 30 min with 1 mM aspirin prior to the addition of arachidonate; F, serum-activated 3T3 cells pretreated for 30 min with 1 mM valerylsalicylate prior to the addition of arachidonate; G, quiescent 3T3-cPGHS-2 cells incubated with 10 µM arachidonate; H, quiescent 3T3-cPGHS-2 cells pretreated for 30 min with 1 mM aspirin prior to the addition of arachidonate; I, quiescent 3T3-cPGHS-2 cells pretreated for 30 min with 1 mM valerylsalicylate prior to the addition of arachidonate. Magnification, 250.




DISCUSSION

The studies reported here provide evidence for differences in the subcellular compartmentation of PGHS-1 and PGHS-2. Our results provide support for an emerging view that PGHS-1 and PGHS-2 represent two independently operating prostanoid biosynthetic systems which produce prostanoids for extracellular housekeeping and intranuclear differentiative events, respectively. This concept derives from the following observations. First, PGHS-1 is expressed constitutively by many cells and is available for acute, on demand prostanoid formation to modulate processes such as platelet aggregation (45, 46) and renal water reabsorption (47, 48) ; in contrast, PGHS-2 is normally absent from most cells but can be induced in association with differentiative or replicative events (2, 4, 7, 10) . Further supporting the idea that there are two distinct prostanoid systems are findings which suggest that PGHS-1 and PGHS-2 utilize different arachidonate pools that are mobilized in response to different cellular stimuli (32, 33) . Finally, in fibroblasts and macrophages expressing PGHS-1 constitutively, induction of PGHS-2 leads to a modest 1.5- to 2-fold increase in prostanoid biosynthetic capacities (8, 16, 29, 30, 31) ; this, coupled with the fact that the kinetic properties of the isozymes are very similar, makes it hard to argue that PGHS-2, at least in fibroblasts and macrophages, exists solely to supplement PGHS-1 activity.

It had been established by immunoelectron microscopy that PGHS-1 is associated with the ER and NE but not the plasma membrane or mitochondrion (34) . When examined by either conventional or confocal microscopy, PGHS-1 and PGHS-2 exhibit quite similar patterns of cytoplasmic and perinuclear immunofluorescent staining in both murine 3T3 cells and macrophages (35, 36, 37) . These results have implied that both PGHS-1 and PGHS-2 are associated with the same intracellular membrane systems, the ER and NE. Our present results are in qualitative agreement with previous findings. However, repeated visual observations suggested that there were subtle differences between the relative levels of cytoplasmic and perinuclear immunostaining of PGHS-1 and PGHS-2 in 3T3 cells and in bovine and human endothelial cells. More precise quantitation made possible by confocal microscopy and digital imaging confirmed these observations. The intensities of staining for PGHS-1 immunoreactivity, DiOC(3) , and FITC-labeled ConA were the same in the ER and NE. In contrast, the intensities of staining for PGHS-2 immunoreactivity were 2-fold higher in the NE than the ER.

In the case of PGHS-1, we interpret the results of the immunochemical staining simply to reaffirm the concept that this isozyme is a resident ER protein which binds to the lumenal surface of the ER (36) . Membrane binding purportedly involves a series of amphipathic helices, residues 70-116 of the ovine PGHS-1 (49) .() Because the outer membrane of the NE is continuous with the ER (50) , PGHS-1 would be expected to be present in the NE and presumably at a concentration similar to that in the ER. To determine if there is a correspondence between the subcellular locations of the PGHS antigens and PGHS activity, we developed a histofluorescence assay for PGHS activity. This assay represents a modification of a histochemical method originally described by Janszen and Nugteren (51) but one which can be used to measure PGHS activity in intact cells with a fluorescent probe. When applied to the measurement of PGHS-1 activity, histofluorescent staining was observed throughout the cytoplasm, but there was little or no staining over the surface of the nucleus. Thus, there is a correspondence between the location of PGHS-1 immunoreactivity and enzyme activity, and we conclude again that PGHS-1 is present and functions primarily in the ER.

In the case of PGHS-2, our immunocytochemical localization studies raised the possibility that this isozyme might form PGH at a different subcellular location than PGHS-1. Indeed, histofluorescence measurements of enzyme activity showed PGHS-2 staining, but not PGHS-1 staining, of the nucleus. In serum-stimulated NIH 3T3 cells, PGHS-1 continues to be present at constant levels when expression of PGHS-2 is induced. Valerylsalicylate selectively blocks PGHS-1 activity (38) , and, similarly, valerylsalicylate blocked PGHS-1 histofluorescence in quiescent 3T3 cells. However, in serum-stimulated NIH 3T3 cells pretreated with valerylsalicylate, histofluorescent staining was observed, and staining was present in the nucleus as well as in the cytoplasm. This pattern is distinct from that seen with PGHS-1.

At this stage, we can only speculate about the implications of our findings that PGHS-2 is present in ER but is concentrated in the NE. First, we suggest that PGHS-2 present in the ER of serum-stimulated 3T3 cells and PMA-treated endothelial cells serves to augment PGHS-1-mediated PGH synthesis. PGHS-1 and PGHS-2 may utilize the same pool of arachidonate. Second, we speculate that at least a portion of the PGHS-2 associated with the NE is in a different subcellular location than PGHS-1. Because as discussed above, PGHS-1 is likely on the lumenal surface of the outer membrane of the NE, a subset of PGHS-2 molecules may be on the lumenal surface of the inner membrane. Differential compartmentation of PGHS-1 and PGHS-2 in the NE could easily explain why these isozymes utilize different arachidonate pools (32, 33) and raises the possibility that there are different lipases coupled to the release of arachidonate from these pools. Furthermore, in some cases there may even be different PGH metabolizing enzymes ( e.g. thromboxane A and PGI synthases).

If PGHS-2 is uniquely present on the inner membrane of the nuclear envelope, it may have the ability to produce prostanoids that function within the nucleus. This is an attractive possibility based simply on the observations that PGHS-2 is an immediate early gene (9) whose induction is consistently associated with cell differentiation or replication, events typically attributable to cell nuclear activity. The question of the whether PGHS-1 and PGHS-2 proteins have different locations in the NE can best be addressed by high resolution immunoelectron microscopy using isozyme-specific antibodies. Equally important would be the ability to visualize the sites of PGH synthesis from endogenous as opposed to exogenous arachidonate using histofluorescence staining.

Based on studies with other resident ER proteins, we presume that PGHS-1 is initially translocated during its synthesis into the ER lumen and then either retained in the ER (52) or circulated through the cis-Golgi ER retention system (53, 54, 55) . The identity of the ER targeting signal for PGHS-1 is unknown. In fact, contrary to expectations, the C-terminal -P/STEL sequence present in all PGHSs appears not to be involved in ER targeting.() We speculate that PGHS-2 also contains an ER targeting signal and that the enzyme then moves from the ER to the inner membrane of the NE directed by a secondary NE targeting signal. In this regard, it will be of particular interest to determine if the regions having the greatest sequence divergence between PGHS-1 and PGHS-2 ( e.g. the signal peptides, membrane binding domains, and the C-terminal 15-25 amino acids) are involved in either ER or NE targeting.


FOOTNOTES

*
This work was supported in part by United States Public Health Service National Institutes of Health Grants DK22042 (to W. L. S.), DK45209 (to W. L. S.), and GM40713 (to D. L. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, Rm. 513, Biochemistry Bldg., Michigan State University, East Lansing, MI 48824. Tel.: 517-353-8680; Fax: 517-353-9334; E-mail: smithww@pilot.msu.edu.

The abbreviations used are: PGHS, prostaglandin endoperoxide synthase; ER, endoplasmic reticulum; PG, prostaglandin; KLH, keyhole limpet hemocyanin; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TBS, Tris-HCl-buffered saline; PMA, phorbol 12-myristate 13-acetate; DiOC(3), 3,3`-dihexyloxacarbocyanine iodide; CDCF-DA, 5-(and -6)-carboxyl-2`,7`-dichlorodihydrofluorescein diacetate; CDCF, 5-(and -6)-carboxyl-2`,7`-dichlorodihydrofluorescein; DMEM, Dulbecco's modified Eagle's medium; ConA, concanavalin A; HUVEC, human umbilical vein endothelial cell; BAEC, bovine arterial endothelial cell.

O. Laneuville, D. K. Breuer, N. Xu, Z. H. Huang, M. Lagarde, D. L. DeWitt, and W. L. Smith, unpublished results.

The fluorescence resulting from the initial staining for PGHS-1 and PGHS-2 did not interfere with the second sets of measurements, because the staining intensities of FITC-labeled ConA and DiOC(3) were much greater than those for immunofluorescence staining for PGHS-1 and PGHS-2.

Serum-activated NIH 3T3 cells treated under these conditions do not form any detectable oxygenated products ( i.e. prostanoids or 15 R-HETE) from [1-C]arachidonic acid.

Evidence from studies of I-TID (3-trifluoromethyl-3-( m-[I]iodophenyl)diazirine) labeling of microsomal PGHS-1 indicates that the enzyme is anchored to the ER membrane via a domain located between Ala (the N terminus of the mature enzyme) and Lys (J. C. Otto and W. L. Smith, unpublished results).

M. K. Regier, J. C. Otto, D. L. DeWitt, and W. L. Smith, submitted for publication.


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