(Received for publication, May 5, 1995; and in revised form, June 30, 1995)
From the
Two isoforms of prostaglandin H synthase have been described: isoform-1 (PGHS-1), which is ascribed a role in basal or housekeeping prostaglandin synthesis; and isoform-2 (PGHS-2), which has been found to be strongly inducible in many tissues and has been associated with inflammatory processes. Recent observations have indicated that cyclooxygenase catalysis by the two isoforms can be differentially regulated when both are present simultaneously (Reddy, S. T., and Herschman, H. R.(1994) J. Biol. Chem. 269, 15473-15480). The requirement of the cyclooxygenase for hydroperoxide initiator has been proposed as an important limit on cellular prostaglandin synthesis (Marshall, P. J., Kulmacz, R. J., and Lands, W. E. M.(1987) J. Biol. Chem. 262, 3510-3517). To compare the levels of hydroperoxide required for cyclooxygenase initiation in the two PGHS isoforms, we have examined the ability of a hydroperoxide scavenger, glutathione peroxidase, to suppress the cyclooxygenase activity of purified preparations of human PGHS-2, ovine PGHS-2, and ovine PGHS-1. Half-maximal prostaglandin synthetic activity was found to require a much lower hydroperoxide level with human PGHS-2 (2.3 nM) and ovine PGHS-2 (2.2 nM) than with ovine PGHS-1 (21 nM). Similar results were obtained when cyclooxygenase activity was monitored by chromatographic analyses of radiolabeled arachidonate metabolites or with oxygen electrode measurements. Mixing four parts of ovine PGHS-1 with one part of human PGHS-2 did not markedly change the sensitivity of the overall cyclooxygenase activity to inhibition by glutathione peroxidase, indicating that the PGHS-1 activity was not easily initiated by PGHS-2 activity in the same vessel. Effective catalysis by PGHS-2 can thus proceed at hydroperoxide levels too low to sustain appreciable catalysis by PGHS-1. This difference in catalytic characteristics provides a biochemical mechanism for differential control of prostaglandin synthesis by the two PGHS isoforms, even when both are present in the same intracellular compartment.
Prostaglandin H synthase (PGHS) ()catalyzes a key
irreversible step in the biosynthesis of prostaglandins, the
oxygenation and rearrangement of arachidonic acid to form prostaglandin
G
(Samuelsson et al., 1978). Two isoforms of PGHS
have been described. The first, PGHS-1, was purified from ovine and
bovine seminal vesicles (Hemler et al., 1976; Miyamoto et
al., 1976; van der Ouderaa et al., 1977). The second
isoform, PGHS-2, was discovered more recently (Xie et al.,
1991; Kujubu et al., 1991; O'Banion et al.,
1991; Sirois and Richards, 1992) and has been purified from
heterogenous expression systems (Barnett et al., 1994;
Percival et al., 1994). The two human isoforms have about 60%
sequence identity overall, with much higher sequence conservation in
catalytically important regions (Hla and Nielson, 1992). Currently, it
is thought that PGHS-2, which is strongly induced by various mitogens,
has a role in inflammation, whereas PGHS-1, whose cellular levels vary
over a smaller range, is a housekeeping enzyme (Mitchell et
al., 1993; Masferrer et al., 1994).
It has been found recently that the two PGHS isoforms can be regulated separately even when both enzymes are present in the same cell (Reddy and Herschman, 1994; Murakami et al., 1994). Differential control of the isoforms was particularly striking in fibroblast and macrophage systems, where prostaglandin synthesis from endogenous substrate was due almost entirely to PGHS-2 catalysis, even though high levels of latent PGHS-1 were present (Reddy and Herschman, 1994).
The cyclooxygenase activity of PGHS-1 has long been known to require initiation by hydroperoxide (Smith and Lands, 1972), and it has been proposed that cellular prostaglandin synthesis is controlled in part by intracellular hydroperoxide levels (Marshall et al., 1987). Hydroperoxide initiation represents a potential route for differential control of cyclooxygenase catalysis by the two PGHS isoforms. To investigate this, the hydroperoxide requirements of cyclooxygenase activity in purified preparations of human PGHS-2 and ovine PGHS-2 were compared with that of ovine PGHS-1. The results indicate that cyclooxygenase initiation in PGHS-2 requires considerably lower levels of hydroperoxide than required by PGHS-1. This would allow prostaglandin synthesis by PGHS-2 to proceed at cellular hydroperoxide levels too low to support cyclooxygenase catalysis by PGHS-1 in the same cell.
Hematin, Tween 20, reduced glutathione, erythrocyte
glutathione peroxidase, glutathione reductase, and NADPH were obtained
from Sigma. Glutathione peroxidase activity was assayed
spectrophotometrically (Lawrence et al., 1974). One unit of
glutathione peroxidase represents an initial velocity of 1 nmol of GSH
oxidized/min. Unlabeled arachidonate was obtained from NuChek Preps
(Elysian, MN) and [1-C]arachidonate from
Amersham Corp. Working stocks of arachidonate (0.50 mM in 50
mM Tris, pH 8.5) were pretreated with a small amount of GSP to
remove residual hydroperoxides as described previously (Kulmacz et
al., 1994b).
oPGHS-1 was purified to homogeneity from ovine seminal vesicles (Kulmacz and Lands, 1987) and reconstituted with a 3-fold molar excess of hematin. In some cases the partially purified enzyme from the first gel-filtration chromatography step was used, after addition of excess hematin. oPGHS-2 (purified to about 70% electrophoretic homogeneity from placenta) was purchased from Cayman Chemical and reconstituted with excess hematin. Immunoblotting of the oPGHS-2 with rabbit polyclonal antibody raised against the C-terminal insert found in murine PGHS-2, but not the PGHS-1 isoform, revealed a single band with apparent molecular weight of 72 kDa (data not shown), confirming that the ovine placental enzyme is isoform 2.
hPGHS-2 was
expressed in Sf9 (American Type Culture Collection) or High-5
(Invitrogen) cells using a Baculovirus vector containing the cDNA for
human PGHS-2 under control of the polyhedrin promoter. Insect cell
culture and Baculovirus manipulations followed procedures recommended
by the suppliers. The coding region of the hPGHS-2 cDNA was released by NotI digestion from a pcDNAI/neo expression vector (generously
provided by Dr. Timothy Hla, American Red Cross) and ligated into the NotI site of the pVL1393 transfer vector (Pharmingen). The
1.9-kilobase pair insert includes about 30 base pairs of 5`- and 60
base pairs of 3`-untranslated sequence. A clone with the hPGHS-2 insert
in the correct orientation was confirmed by restriction enzyme digest.
This clone was mixed with linearized DNA from Baculovirus containing a
lethal deletion (Baculogold, Pharmingen) and used to transfect Sf9
insect cells. The resulting recombinant virus was amplified in fresh
Sf9 cells; membranes from these cells had considerable cyclooxygenase
activity and immunoreactive protein of the expected molecular weight,
confirming expression of hPGHS-2. Large scale expression of recombinant
hPGHS-2 was done in either Sf9 or High-5 cells in suspension culture.
Insect cells were harvested 3-4 days after infection, homogenized
by brief sonication, and a membrane fraction isolated by centrifugation
at 100,000 g for 1 h. The recombinant hPGHS-2 was
solubilized with 1% Tween 20 and chromatographed on an AcA34
gel-filtration column essentially as described for oPGHS-1 (Kulmacz and
Lands, 1987). The specific activity (measured in 0.1 M potassium phosphate, pH 7.2, with 1 mM phenol and 100
µM arachidonate) of the purified hPGHS-2 used in these
studies averaged 9 units/mg of protein.
Protein levels were assayed as described by Peterson(1983). Proteins were analyzed by electrophoresis under denaturing conditions following the procedure of Laemmli(1970). Immunoblotting was done as described earlier (Kulmacz and Wu, 1989) with rabbit polyclonal antibody raised against residues 579-598 of hPGHS-2 (kindly provided by Dr. Paul Marshall, Ciba-Geigy); this antibody did not stain oPGHS-1 under the same conditions.
Cyclooxygenase activity was measured at 30 °C with an oxygen electrode (Kulmacz and Lands, 1987). The standard reaction mixture contained 3 ml of 0.1 M potassium phosphate, pH 7.2, 1 mM phenol, 0.5 mM GSH, and 50 µM arachidonate. Reaction was started by addition of enzyme. One unit of cyclooxygenase activity represents a peak velocity of 1 nmol of oxygen/min.
The hydroperoxide activator requirements of oPGHS-1, oPGHS-2, and hPGHS-2 were assessed by titration with a hydroperoxide scavenger, GSP (Kulmacz et al., 1994b). Briefly, a fixed amount of PGHS was injected into a reaction mixture containing a variable amount of GSP and the fraction of activated cyclooxygenase determined from the oxygen consumption kinetics.
Theoretical
predictions of the sensitivities of the cyclooxygenase activities of
oPGHS-1, hPGHS-2, and mixtures of the two enzymes to inhibition by GSP
were generated with an expanded version of an earlier kinetic model
(Kulmacz and Lands, 1983). In the expanded model, written in Microsoft
QuickBasic, hydroperoxide (PGG) generation and
decomposition by PGHS-1, PGHS-2, and GSP was governed by the following
equations.
The parameter values were: K1, 21
nM; K
2, 2.3 nM; K
1, 2.5 µM; K
2, 2.5 µM; K
GSP, 1.5 µM. The total
cyclooxygenase activity was set at 0.38 µM O
/s
and divided between PGHS-1 and PGHS-2 activity as desired. The total
PGHS peroxidase activity (assuming saturating substrate levels) was
1.37 µM ROOH/s and divided between PGHS-1 and PGHS-2 in
the same proportion as the cyclooxygenase activity. The GSP activity
was set to the level desired. The initial hydroperoxide concentration
was 1 nM and the hydroperoxide produced and consumed by the
five activities was calculated for a 0.001-s increment using . The
reiterative program then adjusted the hydroperoxide level to reflect
the net change before repeating the calculations for the next time
increment. Self-inactivation was accounted for by decreasing PGHS-1 (or
PGHS-2) cyclooxygenase and peroxidase activities in proportion to
cyclooxygenase catalysis by that isoform in the previous interval, so
that each PGHS-1 (or PGHS-2) molecule synthesized 1200 PGG
molecules before self-inactivation. The highest incremental
cyclooxygenase velocity during a simulated reaction was taken as the
predicted peak cyclooxygenase velocity for that particular set of
PGHS-1, PGHS-2, and GSP concentrations.
The current kinetic model
was found to predict a slightly different relationship between the
GSP/Cox ratio needed for complete suppression of cyclooxygenase
activity and the K value. For example, with the
current model a GSP/Cox ratio of 75 corresponded to a K
value of 21 nM; with the old model (Kulmacz and Lands,
1985) the same GSP/Cox ratio implied a K
value of
16 nM. This small difference was due to the smoother reaction
course predicted with the shorter time increments used in the current
model. A standard curve was generated with the current model for 10
separate K
values between 1.5 and 30 nM using the procedure described previously (Kulmacz and Lands,
1985). The points on this standard curve were fitted to an equation of
the following form.
The value of the constant, C, was found to be 1556
± 4 nM. This standard curve was used in the present
study to calculate K values from the observed
GSP/Cox ratios.
Reactions with
[C]arachidonate contained 0.6 ml of the standard
reaction mixture in a glass tube thermostatted at 30 °C. The
oxygenase was added and the mixture vortexed briefly to start the
reaction. After 1 min of further incubation, the reaction was stopped,
and lipid metabolites were extracted by addition of 3 volumes of
ice-cold diethyl ether/methanol/1 M citric acid (30:4:1)
(Miyamoto et al., 1976). To minimize decomposition of
prostaglandin endoperoxide, the lipid extracts were dried over
anhydrous sodium sulfate and stored at -20 °C until analysis
by thin layer chromatography at 0 °C (Tsai et al., 1992).
Radioactive bands were located by autoradiography and quantitated by
liquid scintillation counting. The concentration of metabolite in each
band was calculated by multiplying the fraction of total radioactivity
in that band by the initial arachidonate concentration. PGG
and PGH
standards were prepared by reacting oPGHS-1
with arachidonate in the absence of cosubstrate or with 1 mM phenol present (Hecker et al., 1987). Other standard
eicosanoids were obtained from Cayman Chemical Co. (Ann Arbor, MI).
Figure 1: Electrophoretic analysis of recombinant hPGHS-2. The chromatographically purified preparation was separated by polyacrylamide gel electrophoresis and either stained with Coomassie Blue (lane 1; 7 µg of total protein) or transferred to a nitrocellulose membrane and probed with antibody specific for the hPGHS-2 C-terminal insert peptide (lane 2; 9 µg of total protein). The positions of molecular mass markers are indicated at left. Details are described under ``Materials and Methods.''
Figure 2:
Sensitivity of the cyclooxygenase (Cox) activities of oPGHS-1, oPGHS-2, and hPGHS-2 to
inhibition by GSP, as measured by oxygen electrode. A fixed amount of
either pure oPGHS-1 (filled triangles), partially purified
oPGHS-1 (s), oPGHS-2 (filled diamonds), or hPGHS-2 (filled circles) was injected into cuvettes containing 3 ml of
0.1 M potassium phosphate, pH 7.2, 1 mM phenol, 0.5
mM GSH, 50 µM arachidonate and variable amounts
of GSP. For controls, oPGHS-1 (open triangles) or hPGHS-2 (open circles) was added to reaction mixtures without GSH. The
peak oxygenase velocities were normalized to the corresponding control
values without GSP (pure oPGHS-1, 41 units; purified oPGHS-1, 44 units;
oPGHS-2, 42 units; and hPGHS-2, 44 units) and plotted as a function of
the ratio of added GSP units to added cyclooxygenase units. Details are
described under ``Materials and Methods.'' Quantitatively
similar results were obtained in duplicate experiments with different
batches of hPGHS-2 and oPGHS-2.
The GSP sensitivity of the cyclooxygenase activity of partially purified oPGHS-1, at the same gel-filtration stage as the hPGHS-2 used in this study, was essentially the same as that of fully purified oPGHS-1 (Fig. 2). This indicates that the sensitivity to inhibition by hydroperoxide scavenger is an intrinsic property of the synthase, not a function of the extent of purification. The present observation that both oPGHS-2 and hPGHS-2 share a relatively low sensitivity to inhibition by added hydroperoxide scavenger, which indicates that a low requirement for activator hydroperoxide is a general characteristic of PGHS-2 isoforms.
The sensitivity of the cyclooxygenase to inhibition
by GSP can be used to quantitate the hydroperoxide (PGG)
level required for effective cyclooxygenase initiation (see
``Materials and Methods''; Kulmacz and Lands, 1983, 1985). In
this process, the observed GSP/Cox ratio needed for complete
suppression of cyclooxygenase activity is translated into a
corresponding value for K
, the level of
hydroperoxide required to sustain half-maximal cyclooxygenase
initiation (and activity). The data from the experiment in Fig. 2lead to K
values of 2.3 nM for purified hPGHS-2, 21 nM for pure oPGHS-1, and 16
nM for partially purified oPGHS-1. The response of oPGHS-2
activity to GSP was nonlinear (Fig. 2), but extrapolation of the
middle part of the curve to the x axis gives a suppression
ratio of about 700 and a K
estimate of 2.2
nM. Effective cyclooxygenase initiation in hPGHS-2 and oPGHS-2
thus requires about 9-fold lower hydroperoxide levels than in oPGHS-1.
Figure 3:
Thin layer chromatographic analysis of
[C]arachidonate metabolites from reactions of
oPGHS-1 and hPGHS-2 in the presence of GSP. oPGHS-1 (41 units) or
hPGHS-2 (44 units) was added to standard reaction mixtures containing
enough GSP to give the indicated GSP/Cox ratios. The lipid metabolites
were extracted and analyzed by thin layer chromatography as described
under ``Materials and Methods.'' The mobilities of standard
compounds are indicated.
The amount of total eicosanoid metabolites produced by oPGHS-1 declined sharply as the GSP/Cox ratio increased, with an extrapolated suppression ratio of about 80 (Fig. 4). Total metabolite production by hPGHS-2 was less sensitive to inhibition by the hydroperoxide scavenger, with an extrapolated suppression ratio of about 700. The GSP/Cox ratios producing suppression of arachidonate metabolism by oPGHS-1 and hPGHS-2 (Fig. 4) are in good agreement with those obtained from oxygen consumption measurements (Fig. 2), providing an important confirmation of the reliability of the more detailed and convenient kinetic measurements made with the oxygen electrode. While this manuscript was in preparation, Capdevila et al.(1995) reported GSP inhibition results with oPGHS-2 similar to those found in the present study with hPGHS-2 (Fig. 4).
Figure 4: Effect of added GSP on total arachidonate metabolism by oPGHS-1 (filled triangles) and hPGHS-2 (open triangles). The radioactivity in bands on the TLC plate shown in Fig. 3was used to calculate the overall amount of oxygenated products formed for each incubation. The total product levels were normalized to the control values (without GSP) and plotted as a function of the GSP/Cox ratio.
Figure 5:
Sensitivity of the cyclooxygenase (Cox) activity of a mixture of oPGHS-1 and hPGHS-2 to
inhibition by GSP. oPGHS-1 (42 units) (open squares) or a
mixture (filled squares) of oPGHS-1 (34 units) and hPGHS-2 (8
units) was injected into cuvettes containing 3 ml of 0.1 M potassium phosphate, pH 7.2, 1 mM phenol, 0.5 mM GSH, 50 µM arachidonate and variable amounts of GSP.
The peak oxygenase velocities were normalized to the corresponding
control values without GSP and plotted as a function of the ratio of
added GSP units to added cyclooxygenase units. The predicted behaviors
of oPGHS-1 alone (K = 21 nM) (dotted line), hPGHS-2 alone (K
=
2.3 nM) (solid line), and of the oPGHS-1/hPGHS-2
mixture (dashed line) are also shown. Details are described
under ``Materials and Methods.''
The sensitivities of the cyclooxygenase activities of the PGHS isoforms to suppression by GSP in vitro are likely to reflect the responses of the activities to hydroperoxide scavengers in vivo. GSP itself is a major peroxidase activity in many cells (Flohe, 1978), and other hydroperoxide scavengers have been shown to suppress cyclooxygenase activity, including the peroxidase activity of aspirin-treated oPGHS-1 (Kulmacz et al., 1985). It is worth noting that the GSP/Cox ratios found to suppress the cyclooxygenase activities of the purified PGHS isoforms fall in the range of those observed in several cell and tissue homogenates (Marshall et al., 1987). The general cellular hydroperoxide scavenging ``potential'' furnished by GSP is thus roughly consistent with a role for GSP in modulating cyclooxygenase activity.
The very different hydroperoxide initiator requirements found here for the isolated PGHS isoforms have important implications for the regulation of cellular prostanoid catalysis. Because of its lower requirement for initiator hydroperoxide, PGHS-2 could be fully activated at hydroperoxide levels activating only a small fraction of any PGHS-1 in the same cellular compartment. This can account for the observations with mitogen-treated 3T3 fibroblasts, where the bulk of endogenous prostaglandin synthesis was catalyzed by PGHS-2, even though large amounts of latent PGHS-1 were present (Reddy and Herschman, 1994). A limiting cellular supply of unesterified fatty acid would tend to decrease the rate of cyclooxygenase propagation and thus dampen the feedback activation of both cyclooxygenase activities. This may explain the observation that addition of excess exogenous arachidonate leads to cyclooxygenase catalysis by the otherwise latent PGHS-1 in 3T3 fibroblasts (Lin et al., 1989; Reddy and Herschman, 1994).
An initial immunofluorescence study in 3T3 fibroblasts concluded that the subcellular localization of PGHS-2 was the same as that of PGHS-1 (Regier et al., 1993). Results supporting a differential subcellular localization of the two isoforms were reported very recently for the same 3T3 fibroblasts (Morita et al., 1995). However, the discrimination between the two isoforms was not large, with less than a 2-fold difference in the ratio of nuclear envelope/endoplasmic reticulum staining. It remains to be seen if a partial difference in subcellular localization of the two isoforms in the fibroblasts contributes to the apparently large difference in their catalytic control (Reddy and Herschman, 1994).
Recent studies have indicated that phospholipid bilayers and biological membranes are rapidly traversed by fatty acids and even more polar lipids (Kamp and Hamilton, 1993; Hamilton et al., 1994). This implies that intracellular membranes do little to retard the movement of the moderately polar lipid hydroperoxides, so that intracellular compartments will have similar hydroperoxide levels. Under these circumstances, modulation of the hydroperoxide level could provide differential catalytic control of the isoforms no matter what their intracellular location(s). In any case, the large intrinsic difference between the isoforms in cyclooxygenase initiation efficiency provides a biochemical mechanism which may help explain the differential regulation of PGHS-1 and PGHS-2 catalysis in cells where the two enzymes serve very different physiological roles.