- Email: [email protected]
Coupling between cyclooxygenases and prostaglandin F2α synthase
Biochimica et Biophysica Acta 1633 (2003) 96 – 105 www.bba-direct.com
Coupling between cyclooxygenases and prostaglandin F2a synthase Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F2a-synthetic activity Karin Nakashima, Noriko Ueno, Daisuke Kamei, Toshihiro Tanioka, Yoshihito Nakatani, Makoto Murakami *, Ichiro Kudo Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan Received 13 March 2003; received in revised form 5 June 2003; accepted 11 June 2003
Abstract Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF2a only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF2a from endogenous AA, even though significant increase in PGF2a production occurred in cells transfected with COX-2 alone. This poor COX/ PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF2a-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulusinducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2. D 2003 Published by Elsevier B.V. Keywords: Prostaglandin F2a; Aldo-keto reductase; Arachidonic acid; Phospholipase A2; Cyclooxygenase; Macrophage; Lung
1. Introduction Biosynthesis of prostanoids, which exert a wide variety of biological effects, is regulated by three sequential enzymatic reactions. Arachidonic acid (AA), a precursor of prostanoids, is released from membrane glycerophospholipids by phospholipase A2 (PLA2) enzymes, such as cytosolic PLA 2 (cPLA 2 ) and secretory PLA 2 [1]. AA is Abbreviations: PG, prostaglandin; PGFS, prostaglandin F synthase; PGES, prostaglandin E synthase; mPGES-1, membrane-bound PGES-1; HPGDS, hematopoietic prostaglandin D synthase; PGIS, prostaglandin I synthase; TXS, thromboxane synthase; AA, arachidonic acid; COX, cyclooxygenase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; GSH, glutathione; GST, GSH-S-transferase; MGST, microsomal GST; MAPEG, membrane-associated protein in eicosanoid and glutathione metabolism; HEK, human embryonic kidney; AKR, aldo-keto reductase; LPS, lipopolysaccharide; IL-1h, interleukin-1h * Corresponding author. Tel.: +81-3-3784-8197; fax: +81-3-3784-8245. E-mail address: [email protected] (M. Murakami). 1388-1981/03/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/S1388-1981(03)00092-1
sequentially metabolized to prostaglandin (PG) H2, an unstable intermediate prostanoid, by either of the two cyclooxygenase (COX) enzymes, COX-1 or COX-2, and then converted to various bioactive PGs, such as PGE2, PGD2, PGI2, and thromboxane (TX) A2, by specific terminal synthases. In general, COX-1, a constitutive isozyme, regulates the immediate response, in which a burst production of prostanoids occurs in a few minutes, whereas COX-2, an inducible isozyme, is a prerequisite for the delayed response, in which PGs are produced over long periods [2]. Although molecular bases of functional segregation of the two COX enzymes have still remained obscure, it could be accounted for by subtle differences in their AA requirement and hydroperoxide sensitivity [3]. In addition, the two COX isoforms often display distinct functional coupling with upstream PLA2s [4,5] and downstream terminal PG synthases [6– 8] in distinct phases of cell activation. Importantly, spatiotemporal colocalization of COXs with upstream and downstream enzymes in proximal membrane compart-
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
ments has been proposed to be critical for their efficient functional coupling [7,8]. PGF2a is another major prostanoid that exhibits various biological activities. PGF2a is synthesized from PGH2 by PGF synthase (PGFS; PGH 9,11-endoperoxide reductase) and from PGE2 by PGE 11-ketoreductase, which belong to the aldo-keto reductase (AKR) family [9]. Two PGFS enzymes (PGFS-I and -II), purified and cloned from lung (lung-type) [10] and liver (liver-type) [11], respectively, are highly homologous, NADPH-requiring, monomeric proteins with a molecular mass of 37 kDa. These enzymes also display more potent PGD 11-ketoreductase activity to produce 9a,11h-PGF2 and are also capable of reducing various xenobiotic carbonyl compounds [11]. Besides the AKR family of PGFSs, several glutathione (GSH)-S-transferase (GST) enzymes have the capacity to produce PGF2a from PGH2 [12]. A membrane-associated, GSH-dependent PGFS with an estimated molecular mass of 16 kDa was partially purified from sheep seminal vesicles [13], although its molecular entity is unclear. We have currently analyzed the functional coupling between COX-1 or COX-2 and various terminal PG synthases, including hematopoietic PGD synthase (H-PGDS), PGI synthase (PGIS), TX synthase (TXS) and two forms of PGE synthase (PGES) [6– 8]. To extend our understanding of functional coupling between COXs and terminal PG synthases, we herein performed cotransfection analysis of COXs and PGFS-I to assess their coupling leading to PGF2a production. Our data suggest that PGFS-I can produce PGF2a from both COX isoforms only when a high concentration of exogenous AA is supplied to cells. Several lines of evidence suggests the importance of another type of PGFS, which is membrane-associated, is stimulus-inducible, and may mediate COX-2-dependent PGF2a production from endogenous AA.
2. Materials and methods 2.1. Materials Human embryonic kidney (HEK) 293 cells, HeLa cells, lung fibroblast WI-38 cells and glioblastoma U251 and GOTO cells (all from Human Science Research Resources Bank, Osaka, Japan) were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% (v/v) fetal calf serum (FCS). HEK293 cells stably expressing COX-1 or COX-2 were described previously [14]. The cDNAs for cPLA2, COX-1, COX-2 and membrane-bound PGE synthase-1 (mPGES-1) were described previously [7,14]. The cDNA for PGFS-I was kindly provided by Dr. K. Watanabe (University of East Asia, Japan). AA, PGH2 and PGE2 were purchased from Cayman Chemicals (Ann Arbor, MI). Reagents and plasmids required for transfection were obtained from Invitrogen (San Diego, CA). A23187 was purchased from Calbiochem (La Jolla, CA). Human interleu-
97
kin-1h (IL-1h) was from Genzyme (Boston, MA). Lipopolysaccharide (LPS), indomethacin, and mouse anti-FLAG antibody were from Sigma (St. Louis, MO). Goat antibodies against human COX-1 and COX-2 were purchased from Santa Cruz (Santa Cruz, CA). Horseradish peroxidase-, FITC-, and Cy3-conjugated second antibodies were obtained from Zymed (South San Francisco, CA). Animals were obtained from Nippon Bio-Supply Center (Tokyo, Japan). 2.2. Transfection studies Transfection of cDNAs into COX-expressing HEK293 cells was performed as described previously [6– 8]. Briefly, 1 Ag of plasmid (pCDNA3.1/hyg (Invitrogen) containing a specific cDNA) was mixed with 2 Al of Lipofectamine PLUS (Invitrogen) in 100 Al of Opti-MEM medium (Invitrogen) for 30 min and then added to cells that had attained 40 – 60% confluence in 12-well plates (Iwaki Glass, Tokyo, Japan) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium and culture was continued at 37 jC in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium containing 10 Ag/ml hygromycin (Invitrogen). After culture for 3 –4 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by Northern or Western blotting. The established clones were treated with AA, A23187 or IL-1h, as described previously [6 –8]. Preparation and subsequent activation of Wistar rat peritoneal macrophages with LPS were performed as described previously [7]. 2.3. Measurement of PGFS activity PGFS activity was assessed by measuring the conversion of PGH2 to PGF2a, according to the method for PGES assay [6,7]. Briefly, cells grown in 100-mm dishes were disrupted by sonication and centrifuged for 1 g at 100,000 g at 4 jC to obtain supernatant and membrane fractions. Protein concentrations were determined by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (Sigma) as a standard. An aliquot of these samples was incubated with 0.5 Ag of PGH2 for 30 s at 24 jC in 0.1 ml of 0.1 M Tris –HCl (pH 8.0) containing 1 mM GSH and 5 Ag of indomethacin. After stopping the reaction by the addition of 100 mM FeCl2, PGF2a contents in the reactions were quantified by use of the PGF2a enzyme immunoassay kit (Cayman Chemicals). 2.4. RNA blotting Extraction of total RNA from cells was performed using TRIzol reagent (Invitrogen). Approximately equal amounts ( f 5 Ag) of RNA were applied to separate lanes of 1.2% (w/
98
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore, Bedford, MA). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Bioscience, Arlington Heights, IL) by random priming (Takara Biomedicals, Ohtsu, Japan). All hybridizations were carried out as described previously [6 –8]. 2.5. Western blotting Cell lysates (105 cell equivalents) were subjected to 12.5% SDS-PAGE under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) with a semi-dry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in 10 mM Tris – HCl containing 150 mM NaCl (TBS) and 0.05% Tween-20 (TBS – Tween), the membranes were incubated with anti-COX-1 (1:20,000 dilution), anti-COX-2 (1:5,000 dilution) or anti-FLAG antibody (1:20,000 dilution) in TBS – Tween for 2 h, followed by incubation with horseradish peroxidase-conjugated antigoat (for COXs) or anti-mouse (for FLAG) antibody (1:5,000 dilution) in TBS – Tween for 2 h, and were visualized with the ECL Western blot system (NENk Life Science Products, Boston, MA) [6– 8]. 2.6. RT-PCR Synthesis of cDNA was performed using 0.5 Ag of total RNA and AMV reverse transcriptase, according to the manufacturer’s instructions supplied with the RNA PCR kit (Takara Biomedicals). Subsequent amplification of the cDNA fragments was performed using 1 Al of the reversetranscribed mixture as a template with specific primers (Greiner Japan, Tokyo, Japan). The primers sets used were as follows. For PGFS-I, 5V-ATGGATCCCAAAAGTCAGAGG-3Vand 5V-TTAATATTCTTCAGAAAATGGG-3V; for TXS, 5V-CTGCCCTATCTGGACATGGTG-3V and 5VGTCAGCGTGACACAATCTTG-3V; and for MGST-3, 5VATGGCTGTCCTCTCTAAGGAATA-3Vand 5V-TTAATGGCAGCATTTGGGTCCAC-3V. The PCR condition was 94 jC for 30 s and then 30 cycles of amplification at 94 jC for 5 s and 68 jC for 4 min, using the Advantage cDNA polymerase mix (Clontech, Palo Alto, CA). The PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide. The PCR products were ligated into the pCR3.1 vector (Invitrogen) and sequenced using a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorometric DNA sequencer 310 Genetic Analyzer (Applied Biosystems) to confirm the sequences. 2.7. Confocal laser microscopy Cells grown on collagen-coated cover glasses (Iwaki Glass) were fixed with 3% paraformaldehyde for 30 min in phosphate-buffered saline (PBS). After three washes with
PBS, the fixed cells were sequentially treated with 1% (w/v) bovine serum albumin (for blocking) and 0.2% (v/v) Triton X-100 (for permeabilization) in PBS for 1 h, with antiFLAG or anti-COX-1 antibody (1:500 dilution) for 1 h in PBS containing 1% albumin, and then with FITC-conjugated anti-mouse or Cy3-conjugated anti-goat antibody (1:200 dilution) for 1 h in PBS containing 1% albumin. After six washes with PBS, the cells were mounted on glass slides using Perma Fluor (Japan Tanner, Suita, Japan), and the signal was visualized using a laser scanning confocal microscope (IX70; Olympus, New Hyde Park, NY), as described previously [7,8]. 2.8. Other procedures Treatment of C57BL/6 mice with LPS and measurement of PGFS and PGES activities in tissue homogenates were performed as described previously [6,7]. Data were analyzed by Student’s t-test.
3. Results 3.1. Coupling between COXs and PGFS-I To assess functional coupling between COXs and PGFSI, we transfected these cDNAs, alone or in combination, into HEK293 cells, as we had previously done with other terminal PG synthases [6 – 8]. The expression levels of COX-1, COX-2 and PGFS-I in established transfectants and parental cells are shown in Fig. 1A. Although endogenous PGFS-I in parental HEK293 cells was below the detection limit as assessed by Northern blotting, it was detected by highly sensitive RT-PCR using two sets of primers that could amplify PGFS-I (Fig. 1B). Sequencing of an amplified fragment confirmed its identity to the sequence of the corresponding portion of PGFS-I (data not shown). The expression of endogenous PGFS-I in HEK293 cells was unaffected by IL-1 stimulation under the condition where the expression of TXS was significantly elevated (Fig. 1B). We then investigated the conversion of exogenous AA to PGF2a by these transfectants. When the cells transfected with either COX-1 or COX-2 alone were incubated for 30 min with exogenous AA, there were significant increases in the production of PGF2a in proportion to increasing concentrations of AA (Fig. 1C). In COX-1-transfected cells, PGF2a production was increased modestly at 2 – 5 AM AA and markedly at 5 – 10 AM, whereas in COX-2-transfected cells PGF2a production was increased almost linearly at 0– 5 AM and saturated at higher concentrations. This pattern was similar to PGE2 production by COX-1- or COX-2-transfected HEK293 cells, even though the amounts of PGE2 produced were 5– 10 times larger than those of PGF2a [14]. PGF2a production by cells cotransfected with either COX isozyme and PGFS-I was essentially identical to that by
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
99
Fig. 1. Cotransfection of COXs and PGFS-I in HEK293 cells. (A) Expression of each COX isoform and PGFS-I in respective transfectants as assessed by Western blotting and Northern blotting, respectively. (B) Expression of endogenous PGFS and TXS in HEK293 cells as assessed by RT-PCR. Cells were cultured with (+) or without ( ) 1 ng/ml IL-1h for 4 h. (C) Conversion of exogenous AA by COX/PGFS-I-transfected HEK293 cells. Cells were incubated for 30 min with the indicated concentrations of AA, and the resultant supernatants were taken for PGF2a enzyme immunoassay. Values are means F S.E. of four independent experiments.
cells expressing respective COX alone up to 5 AM AA, and significant increase by PGFS-I was observed only when the highest concentration of AA (10 AM) was supplied to these cells (Fig. 1C). At 10 AM AA, the amount of PGF2a produced by COX-1/PGFS-I cotransfectants was approximately 3-fold more than that produced by COX-1 single transfectants, while the increase in PGF2a production by COX-2/PGFS-I cotransfectants over COX-2 single transfectants was as much as 1.5-fold, revealing a moderate COX-1 preference. To assess the metabolism of endogenous AA released by endogenous cPLA2 to PGF2a, we stimulated the transfectants with A23187 for 30 min or with IL-1 for 4 h as models for the immediate and delayed responses, respectively [6– 8]. As shown in Fig. 2A, cells transfected with COX-2 alone produced PGF2a in response to both stimuli, whereas PGF2a production in cells transfected with COX-1 alone was minimal. Of note, transfection of PGFS-I did not increase PGF2a production in COX-1- or COX-2-expressing cells further (Fig. 2A). Since the con-
version of exogenous AA to PGF2a by PGFS-I requires high concentration of AA (Fig. 1C) and since increased supply of endogenous AA by cPLA2 overexpression allows more efficient functional coupling between COXs and various terminal synthases [6 – 8], we transfected cPLA2 into COX/PGFS-I double transfectants to increase endogenous AA levels. Although transfection of cPLA2 significantly increased PGF 2a generation in COX-1expressing cells following A23187 stimulation and in COX-2-expressing cells following both stimuli, PGFS-I was still unable to augment PGF2a generation in cPLA2/ COX/PGFS-I triple transfectants over cPLA2/COX double transfectants (Fig. 2). Since the spatiotemporal colocalization of COXs and terminal PG synthases crucially influences their efficient functional coupling [7,8], we sought to determine subcellular localization of PGFS-I in HEK293 cells. To address this, PGFS-I was N-terminally tagged with a FLAG epitope and then transfected into HEK293 cells. As shown in Fig. 3A, only a f 40 kDa protein band was detected in the
100
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
Fig. 2. COX/PGFS-I coupling from endogenous AA. (A) HEK293 cells transfected with either COX-1 isozyme, PGFS-I and cPLA2, alone or in combination, were stimulated for 30 min with 10 AM A23187 or for 4 h with 1 ng/ml IL-1h, and the resultant supernatants were taken for PGF2a enzyme immunoassay. (B) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of HEK293 lysates in the presence (+) or absence ( ) of 1 mM GSH. Values are means F S.E. of three independent experiments.
transfectants as assessed by Western blotting with antiFLAG antibody, confirming the specificity of the antibody to FLAG-tagged PGFS-I. Confocal microscopy using this antibody revealed sustained location of PGFS-I in the cytosol before and after cell activation (Fig. 3B). To investigate the spatial relationship between PGFS-I and COX enzymes, FLAG-tagged PGFS-I was transfected into COX-1- or COX-2-expressing HEK293 cells and double immunofluorescent staining with anti-FLAG and anti-COX antibodies was conducted. COX-1 (Fig. 3C) and COX-2 (data not shown) gave a perinuclear envelope signal (as reported in Refs. [7,8]), which was largely dissociated from a cytoplasmic signal for PGFS-I. That substantial amounts of PGF2a were produced by HEK293 cells transfected with COX-2, rather than COX-1, alone even without PGFS-I cotransfection (Figs. 1C and 2A) suggests the presence of a COX-2-prefering endogenous PGFS in this cell line. We therefore measured PGFS
activity in 100,000 g supernatant and pellet fractions of HEK293 lysates. As shown in Fig. 2B, significant PGFS activities were detected in both soluble and pellet fractions. Of interest, membrane-associated PGFS activity was activated markedly by GSH (Fig. 2B). Since many of the terminal PG synthases that prefer COX-2 to COX-1 are perinuclear membrane-bound [7,8], we were interested in PGFS activity in the membrane fraction. 3.2. Detection of inducible membrane-associated PGFS activity We next looked for other cell types that contained membrane-associated PGFS activity. Since activated macrophages are known to produce PGF2a in addition to PGE2 and other PGs following proinflammatory stimuli [15,16], we used rat peritoneal macrophages in primary culture. We examined the kinetic profiles of the production of PGF2a
Fig. 3. Subcellular localization of PGFS-I in HEK293 transfectants. (A) Expression of FLAG-PGFS-I in HEK293 cells as assessed by Western blotting with anti-FLAG antibody. (B) Confocal microscopic analysis of subcellular localization of FLAG-PGFS-I in HEK293 cells before and after stimulation with A23187 for 30 min or with IL-1h for 4 h. (C) Double immunofluorescent microscopy. Signals for PGFS-I and COX-1 are shown by green and red colors, respectively.
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
released into the culture supernatants (Fig. 4A) and the PGF 2a-synthetic activity from PGH 2 in macrophage lysates (Fig. 4B) after stimulation with LPS for various periods. Generation of PGF2a increased gradually over 6 – 24 h (Fig. 4A) and was abolished almost completely by the COX-2 inhibitor NS-398 (data not shown). Interestingly, the PGF2a-synthetic activity in cell lysates was increased concomitantly over time (Fig. 4B). Under the same condition, enzymatic activity of mPGES-1 was increased markedly, that of H-PGDS was constant, and that of TXS was gradually declined over the culture periods, as reported previously [7,17]. Thus, as does mPGES-1 [7], the PGF2a-synthetic enzyme in macrophages appears to be stimulus-inducible. The macrophage lysates were centrifuged at 100,000 g to assess the distribution of the PGF2a-synthetic activity in soluble and membrane fractions. Without LPS stimulation, more PGF2a-synthetic activity was detected in the soluble fraction than the membrane fraction (Fig. 4C). Of note, the membrane-associated PGF2a-synthetic activity was increased several-fold, whereas the soluble PGF2a-synthetic activity was unchanged, after LPS stimulation (Fig. 4C). The membrane-associated PGF2a-synthetic activity, before and after LPS stimulation, was rather low when the enzyme assay was conducted in the absence of GSH (Fig. 4D). Thus, the inducible PGF2a-synthetic enzyme in macrophages
101
appears to be a membrane-bound, GSH-activated enzyme. To eliminate the possibility that the activity was due to PGES-mediated or aqueous conversion of PGH2 to PGE2 followed by its reduction to PGF2a by PGE 11-ketoreductase, we measured the production of PGF2a when PGE2 instead of PGH2 was added directly to the macrophage lysates. As shown in Fig. 4E, conversion of PGE2, relative to PGH2, to PGF2a was minimal in LPS-stimulated macrophages, indicating that we were measuring PGFS activity that catalyzes the isomerization of PGH2 to PGF2a. Of several other cell types tested, the human lung fibroblastic cell line WI-38 was found to produce a significant level of PGF2a in response to IL-1 (Fig. 5A and also see Fig. 7D). Increased production of PGF2a in culture medium over 12 – 24 h demonstrated a typical delayed response. The PGF2a-synthetic enzyme activity in WI-38 lysates was distributed in both soluble and membrane fractions, and the membrane-associated, but not soluble, activity was increased significantly 24 h after IL-1 treatment (Fig. 5B). As in macrophages (Fig. 4D), the membranebound PGF2a-synthetic activity in WI-38 cell lysates showed GSH dependence (data not shown). In order to assess if the increase in PGFS following proinflammatory stimulus occurs in vivo, the activity in various tissues of mice with or without LPS administration
Fig. 4. Detection of membrane-bound PGFS activity in rat peritoneal macrophages. (A) PGF2a production by macrophages stimulated for the indicated periods with 10 Ag/ml LPS. (B) PGFS activity in the lysates of macrophages stimulated for the indicated periods with LPS. (C) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of macrophage lysates with (+) or without ( ) stimulation with LPS for 12 h. (D) The membrane-bound PGFS activity with (+) or without ( ) 12-h stimulation with LPS was assayed in the presence (+) or absence ( ) of 1 mM GSH. (E) Conversion of PGH2 and PGE2 to PGF2a by 100,000 g supernatant (sup) and membrane (ppt) fractions of the lysates of LPS-stimulated macrophages. The enzyme assay condition is described in Materials and methods. In (B), (C) and (E), 1 mM GSH was added to the assay. Values are means F S.E. of three independent experiments.
102
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
detoxication [19], MGST-2 exhibits LTC4-synthetic activity in addition to canonical GST activity [20], and LTC4 synthase and FLAP play a specific role in the biosynthesis of LTs [21,22]. On the basis of these backgrounds, we were interested in MGST-3 [23], the eicosanoid-biosynthetic function of which has been poorly understood. As assessed by Northern blotting, MGST-3 was endogenously expressed in HEK293 cells and was not increased appreciably following IL-1 stimulation (Fig. 7A). When MGST-3 was overexpressed in COX-2-expressing HEK293 cells (the expression level is shown in the inset of Fig. 7B), neither exogenous AA-, A23187- (data not shown) nor IL1-elicited production of PGF2a and PGE2 was affected (Fig. 7B). Under the same condition, cells transfected with mPGES-1 produced more PGE2 and less PGF2a (shunting effect) than cells expressing COX-2 alone, as expected (Fig. 7B). Moreover, although RT-PCR revealed the expression of endogenous MGST-3 in various types of cells (Fig. 7C), their PGF2a-producing capacities did not correlate with their MGST-3 expression levels (Fig. 7D). These
Fig. 5. PGF2a production and PGFS enzymatic activity in WI-38 cells. (A) PGF2a production by WI-38 cells stimulated for the indicated periods with 1 ng/ml IL-1h. (B) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of the lysates of WI-38 cells cultured for 24 h with (+) or without ( ) IL-1h. Values are means F S.E. of three independent experiments.
was measured. As illustrated in Fig. 6A, there were significant increases in PGFS activity in the membrane fraction of several, if not all, tissues, including lung, spleen, kidney, testis and pancreas, in LPS-treated mice over control mice. In particular, the increase in PGFS activity in the lung of LPS-treated mice was remarkable (Fig. 6A). In comparison, induction of PGES activity following LPS treatment was more widespread (Fig. 6B), consistent with mPGES-1 induction in various tissues [7]. 3.3. Membrane-bound PGFS is distinct from MGST-3 As several properties (such as stimulus inducibility and GSH sensitivity) of the membrane-bound PGFS are similar to those of mPGES-1, we asked if this enzyme is attributed to another member of the MAPEG (for membrane-associated protein in eicosanoid and glutathione metabolism) family proteins. To date, six MAPEG proteins, including MGST-1,2 and-3, mPGES-1, leukotriene (LT) C4 synthase and 5lipoxygenase-activating protein (FLAP), have been identified in mammals [18]. So far known, MGST-1 is highly expressed in the liver and acts as a GST for xenobiotic
Fig. 6. PGFS and PGES enzymatic activities in the membrane fractions of LPS-treated mouse tissues. Tissues from male C57BL/6 mice before ( ) and 24 h after (+) treatment with LPS were homogenized and centrifuged at 100,000 g for 1 h to obtain membrane fractions. Then, aliquots of the membrane fractions were subjected to enzyme assays for PGFS (A) and PGES (B). A representative result of two experiments is shown.
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
103
Fig. 7. MGST-3 does not function as PGFS. (A) Expression of MGST-3 in HEK293 cells with (+) or without ( ) stimulation with 1 ng/ml IL-1h, as assessed by Northern blotting. (B) Production of PGF2a and PGE2 by HEK293 cells transfected with COX-2, mPGES-1 and MGST-3 alone or in combination (mean F S.E., n = 3). (Inset) Expression of MGST-3 in MGST-3-transfected (+) and nontransfected ( ) HEK293 cells, as assessed by Northern blotting. (C) Expression of MGST-3 in various cell lines, as assessed by RT-PCR. (D) PGF2a production by various cells stimulated for 24 h with 1 ng/ml IL-1h (mean F S.E., n = 3).
results suggest that MGST-3 does not function as PGFS in cells.
4. Discussion In this study, we aimed to investigate the modes of functional coupling between COX-1/COX-2 and PGFS-I, an AKR family enzyme [9,10], in mammalian cells by the cotransfection strategy. However, we unexpectedly found that PGFS-I can be coupled with COX enzymes only poorly in the HEK293 cell system. PGFS-I augmented both COX-1and COX-2-mediated PGF2a synthesis only from the highest concentration of exogenous AA, yet it failed to do so from endogenous AA even when cPLA2 was overexpressed to increase the level of AA (Figs. 1 and 2). This is in marked contrast to other terminal PG synthases (mPGES, cPGES, HPGDS, PGIS and TXS), each of which shows unique coupling with COX-1 and COX-2 in this setting [6 –8]. The AKR family contains PGFS-I (AKR1C3) and its closest homolog PGFS-II, PGE 9-ketoreductase (identical to 20a-hydroxysteroid dehydrogenease; AKR1C1), bile acidbinding protein (AKR1C2) and dihydrodiol dehydrogenases (AKR1C4) [9]. PGFS-I and -II catalyze the reduction of the 9-, 11-endoperoxide group of PGH2 to give rise to PGF2a as well as that of the 11-keto group of PGD2 to produce 9a-, 11h-PGF2, with the latter activity being higher than the former activity. Both enzymes also catalyze the reduction of various xenobiotic carbonyl compounds [11] and at least some of dihydrodiol dehydrogenases exhibit a significant PGFS activity [24]. Based on these facts, one can argue that the main physiological role of the AKR1C enzymes including PGFS-I and -II may be the detoxification of a class of xenobiotic substances or steroid metabolism rather than the
production of PGF2. However, our data do not rule out the possibility that the AKR1C family of PGFSs play a role in PGF2a production in particular cell types and that HEK293 cells lack certain critical factors that support the cellular PGF2a-biosynthetic function of these enzymes, as discussed below. In fact, PGFS-I is expressed in the alveolar septum and contractile interstitial cells in the lung, where PGF2a may play a role in pulmonary contraction and pulmonary capillary configuration [25]. This enzyme is also present in neuronal dendrites and somata of gray matter and vascular endothelial cells in spinal cord, which is suggestive of the neural activity of PGF2a [26]. PGFS-II is co-expressed with COX-1 in the liver sinusoidal endothelial cells, which produce PGF2a [27]. Although PGF2a plays an obligatory role in labor as evidenced by PGF-receptor knockout mice [28], the contribution of AKR1C enzymes to this system remains controversial. Some reports have shown that PGFSI and PGE 9-ketoreductase are expressed in the corpus luteum and may participate in the production of the luteolytic PGF2a [29], and that the expression of these enzyme is increased in mouse uterus during gestation [30]. On the contrary, Madore et al. [31] have recently demonstrated that bovine AKR1B5 (human AKR1B1; originally reported as aldose reductase with 20a-hydroxysteroid dehydrogenease activity), but not any known members of the AKR1C family of enzymes, is critically associated with PGF2a production in the endometrium at the time of luteolysis. AKR1B5 exhibits higher PGFS activity (i.e. direct conversion of PGH2 to PGF2a) than PGFS-I and -II, shows no PGE 9ketoreductase activity, and is induced in luminal and glandular epithelial cells of the endometrium during the luteolysis period [31]. The poor ability of PGFS-I to be coupled with COXs in HEK293 cells could be explained by following reasons.
104
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
First, the intracellular AA/PGH2 levels in this cell line may not reach the threshold of PGFS-I action even if cPLA2/COX are overexpressed. The fact that the supply of a high concentration of exogenous AA allows COX/PGFS-I coupling to occur (Fig. 1C) supports this notion. In theory, PGFS-I could contribute to PGF2a production in particular cell types in which endogenous concentration of AA or PGH2 reaches a level equivalent to 10 AM exogenous AA (i.e. cells harboring very high expression or activation levels of PLA2s and COXs). Second, cytosolic localization of PGFS-I versus perinuclear localization of COXs in HEK293 cells (Fig. 3) may be disadvantageous for the transfer of the unstable substrate PGH2 from COXs to PGFS-I. Accumulating evidence suggests the importance of perinuclear compartmentalization of sequential metabolic enzymes for the biosynthesis of eicosanoids. For instance, TXS, PGIS and mPGES-1 are located in the perinuclear membranes and H-PGDS translocates from the cytosol to the perinuclear region following cell activation [7,8]. PGF2aproducing cells might contain a regulatory factor that enables PGFS-I to be relocalized into the perinuclear area, allowing efficient COX/PGFS-I coupling. Third, PGFS-I may undergo some posttranslational modifications (e.g. phosphorylation) to be fully activated, a process that might not occur in HEK293 cells. Whether PGFS-I and related AKR enzymes undergo posttranslational modifications following cell activation will await further studies using cell types in which these enzymes can exhibit the PGF2a-biosynthetic function. Despite the inability of PGFS-I to profoundly affect PGF2a production in HEK293 cells, substantial PGF2a production occurred if COX-2 was transfected into these cells (Fig. 2), suggesting the presence of an endogenous, COX-2-prererring PGFS that is distinct from PGFS-I. Indeed, a GSH-activated, membrane-associated PGF2a-synthetic activity was detected in several cells and tissues (Figs. 4 –6), and because of its stimulus inducibility, it is conceivable that this putative membrane-associated PGFS contributes to COX-2-dependent production of PGF2a in response to proinflammatory stimulus. Although several properties of the membrane-bound PGFS activity are similar to those of the MAPEG proteins, it appears to be distinct from MGST-3 (Fig. 7) and from other known MAPEG proteins [19 – 22]. Notably, the membrane-associated PGFS activity we have detected in this study appears to resemble the enzyme partially purified from sheep seminal vesicles in several terms [13], and their identity will be worthwhile to examine in the future. Nevertheless, molecular identification of the membrane-associated form of PGFS will help us to fully understand the regulation of PGF2a biosynthesis in pathophysiological states.
Acknowledgements We would like to thank Dr. Kikuko Watanabe for providing us PGFS-I cDNA. This work was supported by
Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Technology of Japan and Sankyo Foundation of Life Science.
References [1] I. Kudo, M. Murakami, Phospholipase A2 enzymes, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 3 – 58. [2] W.L. Smith, D.L. DeWitt, R.M. Garavito, Cyclooxygenases: structural, cellular, and molecular biology, Annu. Rev. Biochem. 69 (2000) 145 – 182. [3] W.L. Smith, I. Song, The enzymology of prostaglandin endoperoxide H synthases-1 and-2, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 115 – 128. [4] S.T. Reddy, H.R. Herschman, Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells, J. Biol. Chem. 272 (1997) 3231 – 3237. [5] H. Kuwata, Y. Nakatani, M. Murakami, I. Kudo, Cytosolic phospholipase A2 is required for cytokine-induced expression of type IIA secretory phospholipase A2 that mediates optimal cyclooxygenase2-dependent delayed prostaglandin E2 generation in rat 3Y1 fibroblasts, J. Biol. Chem. 273 (1998) 1733 – 1740. [6] T. Tanioka, Y. Nakatani, N. Semmyo, M. Murakami, I. Kudo, Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis, J. Biol. Chem. 275 (2000) 32775 – 32782. [7] M. Murakami, H. Naraba, T. Tanioka, N. Semmyo, Y. Nakatani, F. Kojima, T. Ikeda, M. Fueki, A. Ueno, S. Oh-Ishi, I. Kudo, Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2, J. Biol. Chem. 275 (2000) 32783 – 32792. [8] N. Ueno, M. Murakami, T. Tanioka, K. Fujimori, Y. Urade, I. Kudo, J. Biol. Chem. 276 (2001) 34918 – 34927. [9] K. Watanabe, Prostaglandin F synthase, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 401 – 407. [10] K. Watanabe, Y. Fujii, K. Nakayama, H. Ohkubo, S. Kuramitsu, H. Kagamiyama, S. Nakanishi, O. Hayaishi, Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 11 – 15. [11] T. Suzuki, Y. Fujii, M. Miyano, L.Y. Chen, T. Takahashi, K. Watanabe, DNA cloning, expression, and mutagenesis study of liver-type prostaglandin F synthase, J. Biol. Chem. 274 (1999) 241 – 248. [12] M. Chang, Y. Hong, J.R. Burgess, C.P. Tu, C.C. Reddy, Isozyme specificity of rat liver glutathione S-transferases in the formation of PGF2a and PGE2 from PGH2, Biochem. Biophys. Res. Commun. 142 (1987) 441 – 447. [13] J.R. Burgess, C.C. Reddy, Isolation and characterization of an enzyme from sheep seminal vesicles that catalyzes the glutathione-dependent reduction of prostaglandin H2 to prostaglandin F2a, Biochem. Mol. Biol. Int. 41 (1997) 217 – 226. [14] M. Murakami, T. Kambe, S. Shimbara, I. Kudo, Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways, J. Biol. Chem. 274 (1999) 3103 – 3115. [15] P. Dieter, R. Scheibe, P.J. Jakobsson, K. Watanabe, A. Kolada, S. Kamionka, Functional coupling of cyclooxygenase 1 and 2 to discrete prostanoid synthases in liver macrophages, Biochem. Biophys. Res. Commun. 276 (2000) 488 – 492. [16] U. Karck, F. Reister, W. Schafer, H.P. Zahradnik, M. Breckwoldt, PGE2 and PGF2a release by human peritoneal macrophages in endometriosis, Prostaglandins 51 (1996) 49 – 60. [17] H. Naraba, M. Murakami, H. Matsumoto, S. Shimbara, A. Ueno, I. Kudo, S. Oh-ishi, Segregated coupling of phospholipases A2, cyclo-
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
[18]
[19]
[20]
[21]
[22]
[23]
[24]
oxygenases, and terminal prostanoid synthases in different phases of prostanoid biosynthesis in rat peritoneal macrophages, J. Immunol. 160 (1998) 2974 – 2982. P.J. Jakobsson, R. Morgenstern, J. Mancini, A. Ford-Hutchinson, B. Persson, Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). A widespread protein superfamily, Am. J. Respir. Crit. Care Med. 161 (2000) S20 – S24. M.J. Kelner, R.D. Bagnell, M.A. Montoya, L.A. Estes, L. Forsberg, R. Morgenstern, Structural organization of the microsomal glutathione S-transferase gene (MGST1) on chromosome 12p13.1 – 13.2; identification of the correct promoter region and demonstration of transcriptional regulation in response to oxidative stress, J. Biol. Chem. 275 (2000) 13000 – 13006. M. Sjostrom, P.J. Jakobsson, M. Heimburger, J. Palmblad, J.Z. Haeggstrom, Human umbilical vein endothelial cells generate leukotriene C4 via microsomal glutathione S-transferase type 2 and express the CysLT1 receptor, Eur. J. Biochem. 268 (2001) 2578 – 2586. R.A. Dixon, R.E. Diehl, E. Opas, E. Rands, P.J. Vickers, J.F. Evans, J.W. Gillard, D.K. Miller, Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis, Nature 343 (1990) 282 – 284. B.K. Lam, J.F. Penrose, G.J. Freeman, K.F. Austen, Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 7663 – 7667. S.O. Fetissov, O. Schroder, P.J. Jakobsson, B. Samuelsson, J.Z. Haeggstrom, T. Hokfelt, Expression of microsomal glutathione Stransferase type 3 mRNA in the rat nervous system, Neuroscience 115 (2002) 891 – 897. A. Hara, K. Matsuura, Y. Tamada, K. Sato, Y. Miyabe, Y. Deyashiki, N. Ishida, Relationship of human liver dihydrodiol dehydrogenases to
[25]
[26]
[27]
[28]
[29]
[30]
[31]
105
hepatic bile-acid-binding protein and an oxidoreductase of human colon cells, Biochem. J. 313 (1996) 373 – 376. M. Fukui, T. Fujimoto, K. Watanabe, K. Endo, K. Kuno, Prostaglandin F synthase is localized to contractile interstitial cells in bovine lung, J. Histochem. Cytochem. 44 (1996) 251 – 257. T. Suzuki-Yamamoto, K. Toida, Y. Tsuruo, K. Watanabe, K. Ishimura, Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord, Brain Res. 877 (2000) 391 – 395. T. Suzuki-Yamamoto, H. Yokoi, Y. Tsuruo, K. Watanabe, K. Ishimura, Identification of prostaglandin F-producing cells in the liver, Histochem. Cell Biol. 112 (1999) 451 – 456. Y. Sugimoto, A. Yamasaki, E. Segi, K. Tsuboi, Y. Aze, T. Nishimura, H. Oida, N. Yoshida, T. Tanaka, M. Katsuyama, K. Hasumoto, T. Murata, M. Hirata, F. Ushikubi, M. Negishi, A. Ichikawa, S. Narumiya, Failure of parturition in mice lacking the prostaglandin F receptor, Science 277 (1997) 681 – 683. E. Asselin, M.A. Fortier, Detection and regulation of the messenger for a putative bovine endometrial 9-keto-prostaglandin E2 reductase: effect of oxytocin and interferon-tau, Biol. Reprod. 62 (2000) 125 – 131. S.K. Winchester, T. Imamura, G.A. Gross, L.M. Muglia, S.K. Vogt, J. Wright, K. Watanabe, H.H. Tai, L. Muglia, Coordinate regulation of prostaglandin metabolism for induction of parturition in mice, J. Endocrinol. 143 (2002) 2593 – 2598. E. Madore, N. Harvey, J. Parent, P. Chapdelaine, J.A. Arosh, M.A. Fortier, An aldose reductase with 20a-hydroxysteroid dehydrogenease activity is most likely the enzyme responsible for the production of prostaglandin F2a in the bovine endometrium, J. Biol. Chem. 278 (2003) 11205 – 11212.
Coupling between cyclooxygenases and prostaglandin F2a synthase Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F2a-synthetic activity Karin Nakashima, Noriko Ueno, Daisuke Kamei, Toshihiro Tanioka, Yoshihito Nakatani, Makoto Murakami *, Ichiro Kudo Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan Received 13 March 2003; received in revised form 5 June 2003; accepted 11 June 2003
Abstract Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF2a only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF2a from endogenous AA, even though significant increase in PGF2a production occurred in cells transfected with COX-2 alone. This poor COX/ PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF2a-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulusinducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2. D 2003 Published by Elsevier B.V. Keywords: Prostaglandin F2a; Aldo-keto reductase; Arachidonic acid; Phospholipase A2; Cyclooxygenase; Macrophage; Lung
1. Introduction Biosynthesis of prostanoids, which exert a wide variety of biological effects, is regulated by three sequential enzymatic reactions. Arachidonic acid (AA), a precursor of prostanoids, is released from membrane glycerophospholipids by phospholipase A2 (PLA2) enzymes, such as cytosolic PLA 2 (cPLA 2 ) and secretory PLA 2 [1]. AA is Abbreviations: PG, prostaglandin; PGFS, prostaglandin F synthase; PGES, prostaglandin E synthase; mPGES-1, membrane-bound PGES-1; HPGDS, hematopoietic prostaglandin D synthase; PGIS, prostaglandin I synthase; TXS, thromboxane synthase; AA, arachidonic acid; COX, cyclooxygenase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; GSH, glutathione; GST, GSH-S-transferase; MGST, microsomal GST; MAPEG, membrane-associated protein in eicosanoid and glutathione metabolism; HEK, human embryonic kidney; AKR, aldo-keto reductase; LPS, lipopolysaccharide; IL-1h, interleukin-1h * Corresponding author. Tel.: +81-3-3784-8197; fax: +81-3-3784-8245. E-mail address: [email protected] (M. Murakami). 1388-1981/03/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/S1388-1981(03)00092-1
sequentially metabolized to prostaglandin (PG) H2, an unstable intermediate prostanoid, by either of the two cyclooxygenase (COX) enzymes, COX-1 or COX-2, and then converted to various bioactive PGs, such as PGE2, PGD2, PGI2, and thromboxane (TX) A2, by specific terminal synthases. In general, COX-1, a constitutive isozyme, regulates the immediate response, in which a burst production of prostanoids occurs in a few minutes, whereas COX-2, an inducible isozyme, is a prerequisite for the delayed response, in which PGs are produced over long periods [2]. Although molecular bases of functional segregation of the two COX enzymes have still remained obscure, it could be accounted for by subtle differences in their AA requirement and hydroperoxide sensitivity [3]. In addition, the two COX isoforms often display distinct functional coupling with upstream PLA2s [4,5] and downstream terminal PG synthases [6– 8] in distinct phases of cell activation. Importantly, spatiotemporal colocalization of COXs with upstream and downstream enzymes in proximal membrane compart-
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
ments has been proposed to be critical for their efficient functional coupling [7,8]. PGF2a is another major prostanoid that exhibits various biological activities. PGF2a is synthesized from PGH2 by PGF synthase (PGFS; PGH 9,11-endoperoxide reductase) and from PGE2 by PGE 11-ketoreductase, which belong to the aldo-keto reductase (AKR) family [9]. Two PGFS enzymes (PGFS-I and -II), purified and cloned from lung (lung-type) [10] and liver (liver-type) [11], respectively, are highly homologous, NADPH-requiring, monomeric proteins with a molecular mass of 37 kDa. These enzymes also display more potent PGD 11-ketoreductase activity to produce 9a,11h-PGF2 and are also capable of reducing various xenobiotic carbonyl compounds [11]. Besides the AKR family of PGFSs, several glutathione (GSH)-S-transferase (GST) enzymes have the capacity to produce PGF2a from PGH2 [12]. A membrane-associated, GSH-dependent PGFS with an estimated molecular mass of 16 kDa was partially purified from sheep seminal vesicles [13], although its molecular entity is unclear. We have currently analyzed the functional coupling between COX-1 or COX-2 and various terminal PG synthases, including hematopoietic PGD synthase (H-PGDS), PGI synthase (PGIS), TX synthase (TXS) and two forms of PGE synthase (PGES) [6– 8]. To extend our understanding of functional coupling between COXs and terminal PG synthases, we herein performed cotransfection analysis of COXs and PGFS-I to assess their coupling leading to PGF2a production. Our data suggest that PGFS-I can produce PGF2a from both COX isoforms only when a high concentration of exogenous AA is supplied to cells. Several lines of evidence suggests the importance of another type of PGFS, which is membrane-associated, is stimulus-inducible, and may mediate COX-2-dependent PGF2a production from endogenous AA.
2. Materials and methods 2.1. Materials Human embryonic kidney (HEK) 293 cells, HeLa cells, lung fibroblast WI-38 cells and glioblastoma U251 and GOTO cells (all from Human Science Research Resources Bank, Osaka, Japan) were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% (v/v) fetal calf serum (FCS). HEK293 cells stably expressing COX-1 or COX-2 were described previously [14]. The cDNAs for cPLA2, COX-1, COX-2 and membrane-bound PGE synthase-1 (mPGES-1) were described previously [7,14]. The cDNA for PGFS-I was kindly provided by Dr. K. Watanabe (University of East Asia, Japan). AA, PGH2 and PGE2 were purchased from Cayman Chemicals (Ann Arbor, MI). Reagents and plasmids required for transfection were obtained from Invitrogen (San Diego, CA). A23187 was purchased from Calbiochem (La Jolla, CA). Human interleu-
97
kin-1h (IL-1h) was from Genzyme (Boston, MA). Lipopolysaccharide (LPS), indomethacin, and mouse anti-FLAG antibody were from Sigma (St. Louis, MO). Goat antibodies against human COX-1 and COX-2 were purchased from Santa Cruz (Santa Cruz, CA). Horseradish peroxidase-, FITC-, and Cy3-conjugated second antibodies were obtained from Zymed (South San Francisco, CA). Animals were obtained from Nippon Bio-Supply Center (Tokyo, Japan). 2.2. Transfection studies Transfection of cDNAs into COX-expressing HEK293 cells was performed as described previously [6– 8]. Briefly, 1 Ag of plasmid (pCDNA3.1/hyg (Invitrogen) containing a specific cDNA) was mixed with 2 Al of Lipofectamine PLUS (Invitrogen) in 100 Al of Opti-MEM medium (Invitrogen) for 30 min and then added to cells that had attained 40 – 60% confluence in 12-well plates (Iwaki Glass, Tokyo, Japan) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium and culture was continued at 37 jC in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium containing 10 Ag/ml hygromycin (Invitrogen). After culture for 3 –4 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by Northern or Western blotting. The established clones were treated with AA, A23187 or IL-1h, as described previously [6 –8]. Preparation and subsequent activation of Wistar rat peritoneal macrophages with LPS were performed as described previously [7]. 2.3. Measurement of PGFS activity PGFS activity was assessed by measuring the conversion of PGH2 to PGF2a, according to the method for PGES assay [6,7]. Briefly, cells grown in 100-mm dishes were disrupted by sonication and centrifuged for 1 g at 100,000 g at 4 jC to obtain supernatant and membrane fractions. Protein concentrations were determined by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (Sigma) as a standard. An aliquot of these samples was incubated with 0.5 Ag of PGH2 for 30 s at 24 jC in 0.1 ml of 0.1 M Tris –HCl (pH 8.0) containing 1 mM GSH and 5 Ag of indomethacin. After stopping the reaction by the addition of 100 mM FeCl2, PGF2a contents in the reactions were quantified by use of the PGF2a enzyme immunoassay kit (Cayman Chemicals). 2.4. RNA blotting Extraction of total RNA from cells was performed using TRIzol reagent (Invitrogen). Approximately equal amounts ( f 5 Ag) of RNA were applied to separate lanes of 1.2% (w/
98
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore, Bedford, MA). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Bioscience, Arlington Heights, IL) by random priming (Takara Biomedicals, Ohtsu, Japan). All hybridizations were carried out as described previously [6 –8]. 2.5. Western blotting Cell lysates (105 cell equivalents) were subjected to 12.5% SDS-PAGE under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) with a semi-dry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in 10 mM Tris – HCl containing 150 mM NaCl (TBS) and 0.05% Tween-20 (TBS – Tween), the membranes were incubated with anti-COX-1 (1:20,000 dilution), anti-COX-2 (1:5,000 dilution) or anti-FLAG antibody (1:20,000 dilution) in TBS – Tween for 2 h, followed by incubation with horseradish peroxidase-conjugated antigoat (for COXs) or anti-mouse (for FLAG) antibody (1:5,000 dilution) in TBS – Tween for 2 h, and were visualized with the ECL Western blot system (NENk Life Science Products, Boston, MA) [6– 8]. 2.6. RT-PCR Synthesis of cDNA was performed using 0.5 Ag of total RNA and AMV reverse transcriptase, according to the manufacturer’s instructions supplied with the RNA PCR kit (Takara Biomedicals). Subsequent amplification of the cDNA fragments was performed using 1 Al of the reversetranscribed mixture as a template with specific primers (Greiner Japan, Tokyo, Japan). The primers sets used were as follows. For PGFS-I, 5V-ATGGATCCCAAAAGTCAGAGG-3Vand 5V-TTAATATTCTTCAGAAAATGGG-3V; for TXS, 5V-CTGCCCTATCTGGACATGGTG-3V and 5VGTCAGCGTGACACAATCTTG-3V; and for MGST-3, 5VATGGCTGTCCTCTCTAAGGAATA-3Vand 5V-TTAATGGCAGCATTTGGGTCCAC-3V. The PCR condition was 94 jC for 30 s and then 30 cycles of amplification at 94 jC for 5 s and 68 jC for 4 min, using the Advantage cDNA polymerase mix (Clontech, Palo Alto, CA). The PCR products were analyzed by 1% agarose gel electrophoresis with ethidium bromide. The PCR products were ligated into the pCR3.1 vector (Invitrogen) and sequenced using a Taq cycle sequencing kit (Takara Biomedicals) and an autofluorometric DNA sequencer 310 Genetic Analyzer (Applied Biosystems) to confirm the sequences. 2.7. Confocal laser microscopy Cells grown on collagen-coated cover glasses (Iwaki Glass) were fixed with 3% paraformaldehyde for 30 min in phosphate-buffered saline (PBS). After three washes with
PBS, the fixed cells were sequentially treated with 1% (w/v) bovine serum albumin (for blocking) and 0.2% (v/v) Triton X-100 (for permeabilization) in PBS for 1 h, with antiFLAG or anti-COX-1 antibody (1:500 dilution) for 1 h in PBS containing 1% albumin, and then with FITC-conjugated anti-mouse or Cy3-conjugated anti-goat antibody (1:200 dilution) for 1 h in PBS containing 1% albumin. After six washes with PBS, the cells were mounted on glass slides using Perma Fluor (Japan Tanner, Suita, Japan), and the signal was visualized using a laser scanning confocal microscope (IX70; Olympus, New Hyde Park, NY), as described previously [7,8]. 2.8. Other procedures Treatment of C57BL/6 mice with LPS and measurement of PGFS and PGES activities in tissue homogenates were performed as described previously [6,7]. Data were analyzed by Student’s t-test.
3. Results 3.1. Coupling between COXs and PGFS-I To assess functional coupling between COXs and PGFSI, we transfected these cDNAs, alone or in combination, into HEK293 cells, as we had previously done with other terminal PG synthases [6 – 8]. The expression levels of COX-1, COX-2 and PGFS-I in established transfectants and parental cells are shown in Fig. 1A. Although endogenous PGFS-I in parental HEK293 cells was below the detection limit as assessed by Northern blotting, it was detected by highly sensitive RT-PCR using two sets of primers that could amplify PGFS-I (Fig. 1B). Sequencing of an amplified fragment confirmed its identity to the sequence of the corresponding portion of PGFS-I (data not shown). The expression of endogenous PGFS-I in HEK293 cells was unaffected by IL-1 stimulation under the condition where the expression of TXS was significantly elevated (Fig. 1B). We then investigated the conversion of exogenous AA to PGF2a by these transfectants. When the cells transfected with either COX-1 or COX-2 alone were incubated for 30 min with exogenous AA, there were significant increases in the production of PGF2a in proportion to increasing concentrations of AA (Fig. 1C). In COX-1-transfected cells, PGF2a production was increased modestly at 2 – 5 AM AA and markedly at 5 – 10 AM, whereas in COX-2-transfected cells PGF2a production was increased almost linearly at 0– 5 AM and saturated at higher concentrations. This pattern was similar to PGE2 production by COX-1- or COX-2-transfected HEK293 cells, even though the amounts of PGE2 produced were 5– 10 times larger than those of PGF2a [14]. PGF2a production by cells cotransfected with either COX isozyme and PGFS-I was essentially identical to that by
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
99
Fig. 1. Cotransfection of COXs and PGFS-I in HEK293 cells. (A) Expression of each COX isoform and PGFS-I in respective transfectants as assessed by Western blotting and Northern blotting, respectively. (B) Expression of endogenous PGFS and TXS in HEK293 cells as assessed by RT-PCR. Cells were cultured with (+) or without ( ) 1 ng/ml IL-1h for 4 h. (C) Conversion of exogenous AA by COX/PGFS-I-transfected HEK293 cells. Cells were incubated for 30 min with the indicated concentrations of AA, and the resultant supernatants were taken for PGF2a enzyme immunoassay. Values are means F S.E. of four independent experiments.
cells expressing respective COX alone up to 5 AM AA, and significant increase by PGFS-I was observed only when the highest concentration of AA (10 AM) was supplied to these cells (Fig. 1C). At 10 AM AA, the amount of PGF2a produced by COX-1/PGFS-I cotransfectants was approximately 3-fold more than that produced by COX-1 single transfectants, while the increase in PGF2a production by COX-2/PGFS-I cotransfectants over COX-2 single transfectants was as much as 1.5-fold, revealing a moderate COX-1 preference. To assess the metabolism of endogenous AA released by endogenous cPLA2 to PGF2a, we stimulated the transfectants with A23187 for 30 min or with IL-1 for 4 h as models for the immediate and delayed responses, respectively [6– 8]. As shown in Fig. 2A, cells transfected with COX-2 alone produced PGF2a in response to both stimuli, whereas PGF2a production in cells transfected with COX-1 alone was minimal. Of note, transfection of PGFS-I did not increase PGF2a production in COX-1- or COX-2-expressing cells further (Fig. 2A). Since the con-
version of exogenous AA to PGF2a by PGFS-I requires high concentration of AA (Fig. 1C) and since increased supply of endogenous AA by cPLA2 overexpression allows more efficient functional coupling between COXs and various terminal synthases [6 – 8], we transfected cPLA2 into COX/PGFS-I double transfectants to increase endogenous AA levels. Although transfection of cPLA2 significantly increased PGF 2a generation in COX-1expressing cells following A23187 stimulation and in COX-2-expressing cells following both stimuli, PGFS-I was still unable to augment PGF2a generation in cPLA2/ COX/PGFS-I triple transfectants over cPLA2/COX double transfectants (Fig. 2). Since the spatiotemporal colocalization of COXs and terminal PG synthases crucially influences their efficient functional coupling [7,8], we sought to determine subcellular localization of PGFS-I in HEK293 cells. To address this, PGFS-I was N-terminally tagged with a FLAG epitope and then transfected into HEK293 cells. As shown in Fig. 3A, only a f 40 kDa protein band was detected in the
100
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
Fig. 2. COX/PGFS-I coupling from endogenous AA. (A) HEK293 cells transfected with either COX-1 isozyme, PGFS-I and cPLA2, alone or in combination, were stimulated for 30 min with 10 AM A23187 or for 4 h with 1 ng/ml IL-1h, and the resultant supernatants were taken for PGF2a enzyme immunoassay. (B) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of HEK293 lysates in the presence (+) or absence ( ) of 1 mM GSH. Values are means F S.E. of three independent experiments.
transfectants as assessed by Western blotting with antiFLAG antibody, confirming the specificity of the antibody to FLAG-tagged PGFS-I. Confocal microscopy using this antibody revealed sustained location of PGFS-I in the cytosol before and after cell activation (Fig. 3B). To investigate the spatial relationship between PGFS-I and COX enzymes, FLAG-tagged PGFS-I was transfected into COX-1- or COX-2-expressing HEK293 cells and double immunofluorescent staining with anti-FLAG and anti-COX antibodies was conducted. COX-1 (Fig. 3C) and COX-2 (data not shown) gave a perinuclear envelope signal (as reported in Refs. [7,8]), which was largely dissociated from a cytoplasmic signal for PGFS-I. That substantial amounts of PGF2a were produced by HEK293 cells transfected with COX-2, rather than COX-1, alone even without PGFS-I cotransfection (Figs. 1C and 2A) suggests the presence of a COX-2-prefering endogenous PGFS in this cell line. We therefore measured PGFS
activity in 100,000 g supernatant and pellet fractions of HEK293 lysates. As shown in Fig. 2B, significant PGFS activities were detected in both soluble and pellet fractions. Of interest, membrane-associated PGFS activity was activated markedly by GSH (Fig. 2B). Since many of the terminal PG synthases that prefer COX-2 to COX-1 are perinuclear membrane-bound [7,8], we were interested in PGFS activity in the membrane fraction. 3.2. Detection of inducible membrane-associated PGFS activity We next looked for other cell types that contained membrane-associated PGFS activity. Since activated macrophages are known to produce PGF2a in addition to PGE2 and other PGs following proinflammatory stimuli [15,16], we used rat peritoneal macrophages in primary culture. We examined the kinetic profiles of the production of PGF2a
Fig. 3. Subcellular localization of PGFS-I in HEK293 transfectants. (A) Expression of FLAG-PGFS-I in HEK293 cells as assessed by Western blotting with anti-FLAG antibody. (B) Confocal microscopic analysis of subcellular localization of FLAG-PGFS-I in HEK293 cells before and after stimulation with A23187 for 30 min or with IL-1h for 4 h. (C) Double immunofluorescent microscopy. Signals for PGFS-I and COX-1 are shown by green and red colors, respectively.
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
released into the culture supernatants (Fig. 4A) and the PGF 2a-synthetic activity from PGH 2 in macrophage lysates (Fig. 4B) after stimulation with LPS for various periods. Generation of PGF2a increased gradually over 6 – 24 h (Fig. 4A) and was abolished almost completely by the COX-2 inhibitor NS-398 (data not shown). Interestingly, the PGF2a-synthetic activity in cell lysates was increased concomitantly over time (Fig. 4B). Under the same condition, enzymatic activity of mPGES-1 was increased markedly, that of H-PGDS was constant, and that of TXS was gradually declined over the culture periods, as reported previously [7,17]. Thus, as does mPGES-1 [7], the PGF2a-synthetic enzyme in macrophages appears to be stimulus-inducible. The macrophage lysates were centrifuged at 100,000 g to assess the distribution of the PGF2a-synthetic activity in soluble and membrane fractions. Without LPS stimulation, more PGF2a-synthetic activity was detected in the soluble fraction than the membrane fraction (Fig. 4C). Of note, the membrane-associated PGF2a-synthetic activity was increased several-fold, whereas the soluble PGF2a-synthetic activity was unchanged, after LPS stimulation (Fig. 4C). The membrane-associated PGF2a-synthetic activity, before and after LPS stimulation, was rather low when the enzyme assay was conducted in the absence of GSH (Fig. 4D). Thus, the inducible PGF2a-synthetic enzyme in macrophages
101
appears to be a membrane-bound, GSH-activated enzyme. To eliminate the possibility that the activity was due to PGES-mediated or aqueous conversion of PGH2 to PGE2 followed by its reduction to PGF2a by PGE 11-ketoreductase, we measured the production of PGF2a when PGE2 instead of PGH2 was added directly to the macrophage lysates. As shown in Fig. 4E, conversion of PGE2, relative to PGH2, to PGF2a was minimal in LPS-stimulated macrophages, indicating that we were measuring PGFS activity that catalyzes the isomerization of PGH2 to PGF2a. Of several other cell types tested, the human lung fibroblastic cell line WI-38 was found to produce a significant level of PGF2a in response to IL-1 (Fig. 5A and also see Fig. 7D). Increased production of PGF2a in culture medium over 12 – 24 h demonstrated a typical delayed response. The PGF2a-synthetic enzyme activity in WI-38 lysates was distributed in both soluble and membrane fractions, and the membrane-associated, but not soluble, activity was increased significantly 24 h after IL-1 treatment (Fig. 5B). As in macrophages (Fig. 4D), the membranebound PGF2a-synthetic activity in WI-38 cell lysates showed GSH dependence (data not shown). In order to assess if the increase in PGFS following proinflammatory stimulus occurs in vivo, the activity in various tissues of mice with or without LPS administration
Fig. 4. Detection of membrane-bound PGFS activity in rat peritoneal macrophages. (A) PGF2a production by macrophages stimulated for the indicated periods with 10 Ag/ml LPS. (B) PGFS activity in the lysates of macrophages stimulated for the indicated periods with LPS. (C) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of macrophage lysates with (+) or without ( ) stimulation with LPS for 12 h. (D) The membrane-bound PGFS activity with (+) or without ( ) 12-h stimulation with LPS was assayed in the presence (+) or absence ( ) of 1 mM GSH. (E) Conversion of PGH2 and PGE2 to PGF2a by 100,000 g supernatant (sup) and membrane (ppt) fractions of the lysates of LPS-stimulated macrophages. The enzyme assay condition is described in Materials and methods. In (B), (C) and (E), 1 mM GSH was added to the assay. Values are means F S.E. of three independent experiments.
102
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
detoxication [19], MGST-2 exhibits LTC4-synthetic activity in addition to canonical GST activity [20], and LTC4 synthase and FLAP play a specific role in the biosynthesis of LTs [21,22]. On the basis of these backgrounds, we were interested in MGST-3 [23], the eicosanoid-biosynthetic function of which has been poorly understood. As assessed by Northern blotting, MGST-3 was endogenously expressed in HEK293 cells and was not increased appreciably following IL-1 stimulation (Fig. 7A). When MGST-3 was overexpressed in COX-2-expressing HEK293 cells (the expression level is shown in the inset of Fig. 7B), neither exogenous AA-, A23187- (data not shown) nor IL1-elicited production of PGF2a and PGE2 was affected (Fig. 7B). Under the same condition, cells transfected with mPGES-1 produced more PGE2 and less PGF2a (shunting effect) than cells expressing COX-2 alone, as expected (Fig. 7B). Moreover, although RT-PCR revealed the expression of endogenous MGST-3 in various types of cells (Fig. 7C), their PGF2a-producing capacities did not correlate with their MGST-3 expression levels (Fig. 7D). These
Fig. 5. PGF2a production and PGFS enzymatic activity in WI-38 cells. (A) PGF2a production by WI-38 cells stimulated for the indicated periods with 1 ng/ml IL-1h. (B) PGFS activity in 100,000 g supernatant (sup) and membrane (ppt) fractions of the lysates of WI-38 cells cultured for 24 h with (+) or without ( ) IL-1h. Values are means F S.E. of three independent experiments.
was measured. As illustrated in Fig. 6A, there were significant increases in PGFS activity in the membrane fraction of several, if not all, tissues, including lung, spleen, kidney, testis and pancreas, in LPS-treated mice over control mice. In particular, the increase in PGFS activity in the lung of LPS-treated mice was remarkable (Fig. 6A). In comparison, induction of PGES activity following LPS treatment was more widespread (Fig. 6B), consistent with mPGES-1 induction in various tissues [7]. 3.3. Membrane-bound PGFS is distinct from MGST-3 As several properties (such as stimulus inducibility and GSH sensitivity) of the membrane-bound PGFS are similar to those of mPGES-1, we asked if this enzyme is attributed to another member of the MAPEG (for membrane-associated protein in eicosanoid and glutathione metabolism) family proteins. To date, six MAPEG proteins, including MGST-1,2 and-3, mPGES-1, leukotriene (LT) C4 synthase and 5lipoxygenase-activating protein (FLAP), have been identified in mammals [18]. So far known, MGST-1 is highly expressed in the liver and acts as a GST for xenobiotic
Fig. 6. PGFS and PGES enzymatic activities in the membrane fractions of LPS-treated mouse tissues. Tissues from male C57BL/6 mice before ( ) and 24 h after (+) treatment with LPS were homogenized and centrifuged at 100,000 g for 1 h to obtain membrane fractions. Then, aliquots of the membrane fractions were subjected to enzyme assays for PGFS (A) and PGES (B). A representative result of two experiments is shown.
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
103
Fig. 7. MGST-3 does not function as PGFS. (A) Expression of MGST-3 in HEK293 cells with (+) or without ( ) stimulation with 1 ng/ml IL-1h, as assessed by Northern blotting. (B) Production of PGF2a and PGE2 by HEK293 cells transfected with COX-2, mPGES-1 and MGST-3 alone or in combination (mean F S.E., n = 3). (Inset) Expression of MGST-3 in MGST-3-transfected (+) and nontransfected ( ) HEK293 cells, as assessed by Northern blotting. (C) Expression of MGST-3 in various cell lines, as assessed by RT-PCR. (D) PGF2a production by various cells stimulated for 24 h with 1 ng/ml IL-1h (mean F S.E., n = 3).
results suggest that MGST-3 does not function as PGFS in cells.
4. Discussion In this study, we aimed to investigate the modes of functional coupling between COX-1/COX-2 and PGFS-I, an AKR family enzyme [9,10], in mammalian cells by the cotransfection strategy. However, we unexpectedly found that PGFS-I can be coupled with COX enzymes only poorly in the HEK293 cell system. PGFS-I augmented both COX-1and COX-2-mediated PGF2a synthesis only from the highest concentration of exogenous AA, yet it failed to do so from endogenous AA even when cPLA2 was overexpressed to increase the level of AA (Figs. 1 and 2). This is in marked contrast to other terminal PG synthases (mPGES, cPGES, HPGDS, PGIS and TXS), each of which shows unique coupling with COX-1 and COX-2 in this setting [6 –8]. The AKR family contains PGFS-I (AKR1C3) and its closest homolog PGFS-II, PGE 9-ketoreductase (identical to 20a-hydroxysteroid dehydrogenease; AKR1C1), bile acidbinding protein (AKR1C2) and dihydrodiol dehydrogenases (AKR1C4) [9]. PGFS-I and -II catalyze the reduction of the 9-, 11-endoperoxide group of PGH2 to give rise to PGF2a as well as that of the 11-keto group of PGD2 to produce 9a-, 11h-PGF2, with the latter activity being higher than the former activity. Both enzymes also catalyze the reduction of various xenobiotic carbonyl compounds [11] and at least some of dihydrodiol dehydrogenases exhibit a significant PGFS activity [24]. Based on these facts, one can argue that the main physiological role of the AKR1C enzymes including PGFS-I and -II may be the detoxification of a class of xenobiotic substances or steroid metabolism rather than the
production of PGF2. However, our data do not rule out the possibility that the AKR1C family of PGFSs play a role in PGF2a production in particular cell types and that HEK293 cells lack certain critical factors that support the cellular PGF2a-biosynthetic function of these enzymes, as discussed below. In fact, PGFS-I is expressed in the alveolar septum and contractile interstitial cells in the lung, where PGF2a may play a role in pulmonary contraction and pulmonary capillary configuration [25]. This enzyme is also present in neuronal dendrites and somata of gray matter and vascular endothelial cells in spinal cord, which is suggestive of the neural activity of PGF2a [26]. PGFS-II is co-expressed with COX-1 in the liver sinusoidal endothelial cells, which produce PGF2a [27]. Although PGF2a plays an obligatory role in labor as evidenced by PGF-receptor knockout mice [28], the contribution of AKR1C enzymes to this system remains controversial. Some reports have shown that PGFSI and PGE 9-ketoreductase are expressed in the corpus luteum and may participate in the production of the luteolytic PGF2a [29], and that the expression of these enzyme is increased in mouse uterus during gestation [30]. On the contrary, Madore et al. [31] have recently demonstrated that bovine AKR1B5 (human AKR1B1; originally reported as aldose reductase with 20a-hydroxysteroid dehydrogenease activity), but not any known members of the AKR1C family of enzymes, is critically associated with PGF2a production in the endometrium at the time of luteolysis. AKR1B5 exhibits higher PGFS activity (i.e. direct conversion of PGH2 to PGF2a) than PGFS-I and -II, shows no PGE 9ketoreductase activity, and is induced in luminal and glandular epithelial cells of the endometrium during the luteolysis period [31]. The poor ability of PGFS-I to be coupled with COXs in HEK293 cells could be explained by following reasons.
104
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
First, the intracellular AA/PGH2 levels in this cell line may not reach the threshold of PGFS-I action even if cPLA2/COX are overexpressed. The fact that the supply of a high concentration of exogenous AA allows COX/PGFS-I coupling to occur (Fig. 1C) supports this notion. In theory, PGFS-I could contribute to PGF2a production in particular cell types in which endogenous concentration of AA or PGH2 reaches a level equivalent to 10 AM exogenous AA (i.e. cells harboring very high expression or activation levels of PLA2s and COXs). Second, cytosolic localization of PGFS-I versus perinuclear localization of COXs in HEK293 cells (Fig. 3) may be disadvantageous for the transfer of the unstable substrate PGH2 from COXs to PGFS-I. Accumulating evidence suggests the importance of perinuclear compartmentalization of sequential metabolic enzymes for the biosynthesis of eicosanoids. For instance, TXS, PGIS and mPGES-1 are located in the perinuclear membranes and H-PGDS translocates from the cytosol to the perinuclear region following cell activation [7,8]. PGF2aproducing cells might contain a regulatory factor that enables PGFS-I to be relocalized into the perinuclear area, allowing efficient COX/PGFS-I coupling. Third, PGFS-I may undergo some posttranslational modifications (e.g. phosphorylation) to be fully activated, a process that might not occur in HEK293 cells. Whether PGFS-I and related AKR enzymes undergo posttranslational modifications following cell activation will await further studies using cell types in which these enzymes can exhibit the PGF2a-biosynthetic function. Despite the inability of PGFS-I to profoundly affect PGF2a production in HEK293 cells, substantial PGF2a production occurred if COX-2 was transfected into these cells (Fig. 2), suggesting the presence of an endogenous, COX-2-prererring PGFS that is distinct from PGFS-I. Indeed, a GSH-activated, membrane-associated PGF2a-synthetic activity was detected in several cells and tissues (Figs. 4 –6), and because of its stimulus inducibility, it is conceivable that this putative membrane-associated PGFS contributes to COX-2-dependent production of PGF2a in response to proinflammatory stimulus. Although several properties of the membrane-bound PGFS activity are similar to those of the MAPEG proteins, it appears to be distinct from MGST-3 (Fig. 7) and from other known MAPEG proteins [19 – 22]. Notably, the membrane-associated PGFS activity we have detected in this study appears to resemble the enzyme partially purified from sheep seminal vesicles in several terms [13], and their identity will be worthwhile to examine in the future. Nevertheless, molecular identification of the membrane-associated form of PGFS will help us to fully understand the regulation of PGF2a biosynthesis in pathophysiological states.
Acknowledgements We would like to thank Dr. Kikuko Watanabe for providing us PGFS-I cDNA. This work was supported by
Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Technology of Japan and Sankyo Foundation of Life Science.
References [1] I. Kudo, M. Murakami, Phospholipase A2 enzymes, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 3 – 58. [2] W.L. Smith, D.L. DeWitt, R.M. Garavito, Cyclooxygenases: structural, cellular, and molecular biology, Annu. Rev. Biochem. 69 (2000) 145 – 182. [3] W.L. Smith, I. Song, The enzymology of prostaglandin endoperoxide H synthases-1 and-2, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 115 – 128. [4] S.T. Reddy, H.R. Herschman, Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells, J. Biol. Chem. 272 (1997) 3231 – 3237. [5] H. Kuwata, Y. Nakatani, M. Murakami, I. Kudo, Cytosolic phospholipase A2 is required for cytokine-induced expression of type IIA secretory phospholipase A2 that mediates optimal cyclooxygenase2-dependent delayed prostaglandin E2 generation in rat 3Y1 fibroblasts, J. Biol. Chem. 273 (1998) 1733 – 1740. [6] T. Tanioka, Y. Nakatani, N. Semmyo, M. Murakami, I. Kudo, Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis, J. Biol. Chem. 275 (2000) 32775 – 32782. [7] M. Murakami, H. Naraba, T. Tanioka, N. Semmyo, Y. Nakatani, F. Kojima, T. Ikeda, M. Fueki, A. Ueno, S. Oh-Ishi, I. Kudo, Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2, J. Biol. Chem. 275 (2000) 32783 – 32792. [8] N. Ueno, M. Murakami, T. Tanioka, K. Fujimori, Y. Urade, I. Kudo, J. Biol. Chem. 276 (2001) 34918 – 34927. [9] K. Watanabe, Prostaglandin F synthase, Prostaglandins Other Lipid Mediat. 68 – 69 (2002) 401 – 407. [10] K. Watanabe, Y. Fujii, K. Nakayama, H. Ohkubo, S. Kuramitsu, H. Kagamiyama, S. Nakanishi, O. Hayaishi, Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 11 – 15. [11] T. Suzuki, Y. Fujii, M. Miyano, L.Y. Chen, T. Takahashi, K. Watanabe, DNA cloning, expression, and mutagenesis study of liver-type prostaglandin F synthase, J. Biol. Chem. 274 (1999) 241 – 248. [12] M. Chang, Y. Hong, J.R. Burgess, C.P. Tu, C.C. Reddy, Isozyme specificity of rat liver glutathione S-transferases in the formation of PGF2a and PGE2 from PGH2, Biochem. Biophys. Res. Commun. 142 (1987) 441 – 447. [13] J.R. Burgess, C.C. Reddy, Isolation and characterization of an enzyme from sheep seminal vesicles that catalyzes the glutathione-dependent reduction of prostaglandin H2 to prostaglandin F2a, Biochem. Mol. Biol. Int. 41 (1997) 217 – 226. [14] M. Murakami, T. Kambe, S. Shimbara, I. Kudo, Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways, J. Biol. Chem. 274 (1999) 3103 – 3115. [15] P. Dieter, R. Scheibe, P.J. Jakobsson, K. Watanabe, A. Kolada, S. Kamionka, Functional coupling of cyclooxygenase 1 and 2 to discrete prostanoid synthases in liver macrophages, Biochem. Biophys. Res. Commun. 276 (2000) 488 – 492. [16] U. Karck, F. Reister, W. Schafer, H.P. Zahradnik, M. Breckwoldt, PGE2 and PGF2a release by human peritoneal macrophages in endometriosis, Prostaglandins 51 (1996) 49 – 60. [17] H. Naraba, M. Murakami, H. Matsumoto, S. Shimbara, A. Ueno, I. Kudo, S. Oh-ishi, Segregated coupling of phospholipases A2, cyclo-
K. Nakashima et al. / Biochimica et Biophysica Acta 1633 (2003) 96–105
[18]
[19]
[20]
[21]
[22]
[23]
[24]
oxygenases, and terminal prostanoid synthases in different phases of prostanoid biosynthesis in rat peritoneal macrophages, J. Immunol. 160 (1998) 2974 – 2982. P.J. Jakobsson, R. Morgenstern, J. Mancini, A. Ford-Hutchinson, B. Persson, Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). A widespread protein superfamily, Am. J. Respir. Crit. Care Med. 161 (2000) S20 – S24. M.J. Kelner, R.D. Bagnell, M.A. Montoya, L.A. Estes, L. Forsberg, R. Morgenstern, Structural organization of the microsomal glutathione S-transferase gene (MGST1) on chromosome 12p13.1 – 13.2; identification of the correct promoter region and demonstration of transcriptional regulation in response to oxidative stress, J. Biol. Chem. 275 (2000) 13000 – 13006. M. Sjostrom, P.J. Jakobsson, M. Heimburger, J. Palmblad, J.Z. Haeggstrom, Human umbilical vein endothelial cells generate leukotriene C4 via microsomal glutathione S-transferase type 2 and express the CysLT1 receptor, Eur. J. Biochem. 268 (2001) 2578 – 2586. R.A. Dixon, R.E. Diehl, E. Opas, E. Rands, P.J. Vickers, J.F. Evans, J.W. Gillard, D.K. Miller, Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis, Nature 343 (1990) 282 – 284. B.K. Lam, J.F. Penrose, G.J. Freeman, K.F. Austen, Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 7663 – 7667. S.O. Fetissov, O. Schroder, P.J. Jakobsson, B. Samuelsson, J.Z. Haeggstrom, T. Hokfelt, Expression of microsomal glutathione Stransferase type 3 mRNA in the rat nervous system, Neuroscience 115 (2002) 891 – 897. A. Hara, K. Matsuura, Y. Tamada, K. Sato, Y. Miyabe, Y. Deyashiki, N. Ishida, Relationship of human liver dihydrodiol dehydrogenases to
[25]
[26]
[27]
[28]
[29]
[30]
[31]
105
hepatic bile-acid-binding protein and an oxidoreductase of human colon cells, Biochem. J. 313 (1996) 373 – 376. M. Fukui, T. Fujimoto, K. Watanabe, K. Endo, K. Kuno, Prostaglandin F synthase is localized to contractile interstitial cells in bovine lung, J. Histochem. Cytochem. 44 (1996) 251 – 257. T. Suzuki-Yamamoto, K. Toida, Y. Tsuruo, K. Watanabe, K. Ishimura, Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord, Brain Res. 877 (2000) 391 – 395. T. Suzuki-Yamamoto, H. Yokoi, Y. Tsuruo, K. Watanabe, K. Ishimura, Identification of prostaglandin F-producing cells in the liver, Histochem. Cell Biol. 112 (1999) 451 – 456. Y. Sugimoto, A. Yamasaki, E. Segi, K. Tsuboi, Y. Aze, T. Nishimura, H. Oida, N. Yoshida, T. Tanaka, M. Katsuyama, K. Hasumoto, T. Murata, M. Hirata, F. Ushikubi, M. Negishi, A. Ichikawa, S. Narumiya, Failure of parturition in mice lacking the prostaglandin F receptor, Science 277 (1997) 681 – 683. E. Asselin, M.A. Fortier, Detection and regulation of the messenger for a putative bovine endometrial 9-keto-prostaglandin E2 reductase: effect of oxytocin and interferon-tau, Biol. Reprod. 62 (2000) 125 – 131. S.K. Winchester, T. Imamura, G.A. Gross, L.M. Muglia, S.K. Vogt, J. Wright, K. Watanabe, H.H. Tai, L. Muglia, Coordinate regulation of prostaglandin metabolism for induction of parturition in mice, J. Endocrinol. 143 (2002) 2593 – 2598. E. Madore, N. Harvey, J. Parent, P. Chapdelaine, J.A. Arosh, M.A. Fortier, An aldose reductase with 20a-hydroxysteroid dehydrogenease activity is most likely the enzyme responsible for the production of prostaglandin F2a in the bovine endometrium, J. Biol. Chem. 278 (2003) 11205 – 11212.