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Molecular Cloning, Functional Expression, and Selective Regulation of Ovine Prostaglandin H Synthase-2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
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Molecular Cloning, Functional Expression, and Selective Regulation of Ovine Prostaglandin H Synthase-21 Vivian Zhang, Mary O’Sullivan, Hameda Hussain, William T. Roswit, and Michael J. Holtzman2 Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110 Received September 4, 1996 Structural characterization for ovine prostaglandin H synthase-1 (PGHS-1) is extensive, but the corresponding structure for the homologous ovine PGHS-2 isoform is undefined. Accordingly, we isolated a full-length (3.4 kb) ovine PGHS-2 cDNA from a primary-culture cell model (ovine tracheal epithelial cells) originally described as containing both PGHS isoforms. Analysis of ovine PGHS-2 cDNA sequence indicated conservation of critical amino acid residues, but differences in other hydrophilic regions allowed for the development of an anti-peptide antibody highly selective for PGHS-2. Enzymatic activities of the recombinant ovine PGHS isozymes indicated significant differences in response to aspirin-acetylation consistent with the characteristics of endogenous cellular PGHS activities under basal and serum-induced conditions. The results fully account for previous evidence of two distinct PGHS activities in cultured airway epithelial cells and provide for additional definition of PGHS structure–function relationships. q 1996 Academic Press, Inc.
Prostaglandin H synthase (PGHS) exists in two isoforms (PGHS-1 and PGHS-2) that each convert arachidonic acid to prostaglandin H2 (PGH2), the first committed step in the biosynthesis of biologically active prostaglandins and thromboxanes. PGHS features were defined first and remain defined most completely for ovine PGHS-1. This species was the first one cloned and sequenced (1, 2), and remains as the most extensively mutated (3-9). It is the only PGHS with three-dimensional structure defined by x-ray crystallography (10). Together, these approaches have defined critical structures governing PGHS-1 interaction with substrate and inhibitors. The subsequent identification of PGHS-2 raised the possibilities that PGHS-1 and -2 may mediate constitutive versus inducible prostanoid production, respectively, and that isozymespecific inhibitors might be designed for greater efficacy (11). Because structural features of ovine PGHS-1 are well defined, the design of inhibitors may be further facilitated by corresponding structural data for ovine PGHS-2. In fact, a PGHS isozyme (distinct from PGHS-1) exhibiting selective regulation and distinct pharmacology was also first identified using an ovine cell culture system (12, 13), but these studies did not define ovine PGHS-2 sequence or fully link the functional expression of 4.0 kb PGHS mRNA with the corresponding 72-kDa polypeptide. Accordingly, the present experiments were performed to (i) isolate the cDNA for ovine PGHS-2 from cultured ovine tracheal epithelial cells (oTECs); (ii) compare the structure for ovine PGHS-2 to PGHS-1; (iii) test the cDNA-encoded ovine PGHS-2 for enzymatic activity and for reactivity with nonsteroidal anti-inflammatory drugs (especially aspirin); and 1 The nucleotide sequence described in this paper has been submitted to the GenBank database with the Accession No. U68486 (bankit id No. 66332). 2 To whom correspondence should be addressed at Washington University School of Medicine, 660 S. Euclid Ave., Box 8052, St. Louis, MO 63110. Fax: (314) 362-8987; E-mail: [email protected]. Abbreviations: HETE, hydroxyeicosatetraenoic acid; oTEC, ovine tracheal epithelial cell; PG, prostaglandin; PGHS, prostaglandin H synthase.
499 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(iv) determine whether PGHS-2 is selectively regulated in (non-inflammatory) epithelial cells that express both isoforms. MATERIALS AND METHODS Materials. AMV reverse transcriptase was from Life Sciences; MMLV reverse transcriptase was from BRL; [aP]dCTP (3000 Ci/mmol) and [3H8]arachidonic acid (95.1 Ci/mmol) were from Dupont-New England Nuclear; PGHS1 purified from ovine seminal vesicle was from Cayman Chemical Co., Inc.; and purified ovine placental and recombinant chicken PGHS-2 were from Oxford Biomedical Research, Inc. Laboratory of Human Carcinogenesis (LHC) basal medium was obtained from Biofluids and was supplemented with ovine pituitary extract, epidermal growth factor, epinephrine, hydrocortisone, insulin, transferrin, triiodothyronine, L-glutamine, calcium chloride, trace elements, penicillin, and streptomycin (LHC-8e) as described previously for oTEC culture (12-14). Authentic reference compounds included (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) and PGB2 , PGD2 , PGE2 , and PGF2a obtained from Biomol Research Laboratories Inc. or Cayman Chemical Co. Antibodies. Three rabbit antisera raised against ovine seminal vesicle PGHS-1 were obtained from Oxford Biomedical Research (PG-16 and PG-20) and Cayman Chemical Co. (#160103). A rabbit antisera raised against PGHS-2 Nterminus was also obtained from Oxford Biomedical Research (PG-26). In addition, rabbit antisera were raised against deduced amino acid sequences of murine PGHS-2 using synthetic peptides conjugated to keyhole limpet hemocyanin via an N-terminal cysteine as antigens (15). IgG was purified from antisera by peptide-affinity chromatography using antigenic peptide and Sulfolink coupling gel (Pierce Chemical). Peptide sequences were selected because of their conservation across murine and chicken PGHS-2 (16-18) and their dissimilarity to ovine PGHS-1 (1, 2) as well as their predicted hydrophilicity (19). Preliminary experiments indicated that antisera (designated MH.745/6) from both rabbits immunized against one of these peptides exhibited selective reactivity with recombinant chicken and purified ovine PGHS-2 at high titer (1:50,000 dilution). Neither pre-immune sera nor antisera against other peptides exhibited PGHS reactivity. PCR-based amplification of ovine PGHS-2 mRNA. Total RNA was isolated from cultured oTECs (12) using guanidinium isothiocyanate lysis and centrifugation through cesium chloride (20). Poly(A)/ RNA was fractionated with a biotinylated oligo(dT) probe-streptavidin magnetic particle system (Promega) and was used to generate corresponding cDNA using MMLV reverse transcriptase. The cDNA product was amplified by PCR using an annealing cycle at 557C and Hot Tub DNA polymerase (Amersham). DNA primers were based on the sequences surrounding the active site Tyr385 (4) and the aspirin-acetylation site Ser532 in murine PGHS-2 (17, 18) which are conserved in ovine seminal vesicle PGHS-1 (1, 2) and chicken embryo fibroblast PGHS-2 (16) and were constructed to also contain extensions with either XhoI (CTCGAG) or SpeI (ACTAGT) restriction sites at their 5* ends. The downstream primer 5*-GATCACTCGAGATGGGATTTCCCATAAGTCCTTTCAAGGAGAA-3* contained an Xho1 site and nucleotides 1668-1698 of murine PGHS-2 which included Tyr385, and the upstream primer 5*-AATGCACTAGTGAATTCAACACACTCTATCACTGGCACCCCCT-3* contained an SpeI site and nucleotides 1219-1251 of murine PGHS-2 which included Ser532. Reverse-transcriptase PCR products were selected for further pursuit on the basis of size, Southern blotting against oligonucleotides containing internal PGHS-2 sequence, and Northern blotting against epithelial cell mRNA. PCR products were purified and ligated into XhoI/SpeI-digested pBlueScript II SK(//0) phagemid (Stratagene) with T4 DNA ligase. The resulting phagemid was used to transform E. Coli DH1 cells, and recombinant colonies were detected by white colony formation on IPTG/XGAL plates. Phagemid insert was subjected to DNA sequencing using the dideoxy chain termination method with the Sequenase Kit (U. S. Biochemicals) and primers based on flanking sequences of the T3 and T7 promoters in pBluescript. cDNA library construction and screening. Poly(A)/RNA from oTECs was fractionated by oligo(dT) affinity chromatography and converted to double-stranded blunt-ended cDNA (21). EcoRI(NotI) linker-adapters were added using the reagents contained in the Copy Kit (Invitrogen Corp.). After size selection by agarose gel electrophoresis, the cDNA was ligated into a phage cloning vector (lambda ZAP II, Stratagene) and then packaged in vitro (Gigapack II Gold, Stratagene). The library was amplified in E. Coli (host strain XL1-Blue, Stratagene) and was screened with 32 P-labeled cDNA generated from reverse transcriptase-PCR as noted above and then radiolabeled with [a-32P]dCTP by random primer synthesis using the Klenow fragment of DNA polymerase I (Multiprime, Amersham Corp.) to a specific activity of 109 cpm/mg. Phage from positive plaques was subjected to secondary screening and then rescued into pBluescript using f1 helper phage (R408, Stratagene). Insert size was determined by agarose gel eletrophoresis after digestion with NotI. Initial sequence was obtained using primers based on flanking sequences contained in pBluescript. Both strands of a full-length cDNA for ovine PGHS-2 (designated oPGHS-2) were sequenced using a linear sequencing strategy. Preparation of expression vectors and transfection of Cos-7 cells. For ovine PGHS-1, contiguous 0.5 kb and 1.5 kb cDNAs (2) were released from M13 phage by digestion with XbaI and EcoRI and then were gel purified and successively ligated into the EcoRI/XbaI-digested expression vector pECE (22) to form a 2.0 kb cDNA (oPGHS-1coding) containing the complete open-reading frame. In addition, this insert was released with KpnI/XbaI, blunt-ended with T4 DNA polymerase, and ligated into a new SmaI site in the expression vector pOSMLv. The parent vector 32
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(pOSML) lacking this SmaI site was obtained from W. Smith (Michigan State Univ.) and Genetics Institute, Inc (23). For the newly cloned PGHS-2, oPGHS-2 was released from pBluescript by digestion with NotI, blunt ended with the Klenow fragment of DNA polymerase I, and ligated into the SmaI site of pECE or pOSMLv. In addition, the coding region of ovine PGHS-2 (designated oPGHS-2-coding) was expressed in pECE and pOSMLv. To generate pECEoPGHS-2-coding, pECE was cut with BalI/XbaI (removing the 3*-UTR and a portion of the coding region of oPGHS2) followed by PCR to restore the full coding region. To generate pOSMLv-oPGHS-2-coding, the coding region was released from pECE with XbaI/HindIII, filled in with Klenow, and blunt ligated into the SmaI site in pOSMLv. Expression vector constructs (pECE- and pOSMLv-oPGHS-1-coding and pECE- and pOSMLv-oPGHS-2 and -oPGHS2-coding) were verified by agarose gel electrophoresis after digestion with PstI or PstI/XbaI, and all inserts were sequenced to insure proper orientation and sequence reproduction. The plasmids were grown in ampicillin-containing T broth, then purified through cesium chloride, and used to transfect Cos-7 cells (ATCC CRL-1651) by lipofectinmediated gene transfer (24). Northern analysis of recombinant and epithelial cell PGHS. Total cellular RNA was prepared from transfected Cos-7 cells and cultured oTECs and was subjected to electrophoresis in a 1.0% agarose gel containing 1 M formaldehyde. Equal amounts of RNA were loaded/lane on the basis of absorbance at 260 nm, and equivalency of sample amounts was initially verified by the intensity of ethidium bromide staining of the 28S and 18S rRNA bands. RNA was electroblotted to nylon membranes which were prehybridized for 15 min and then hybridized for 1 h at 687C in 10 ml of QuikHyb solution (Stratagene) with 32P-labeled cDNAs for ovine PGHS-1 or -2 prepared as described above. 32 P-labeled cDNAs were added to the hybridization mixture in 300 ml of 10 mg/ml salmon sperm DNA to achieve a final probe concentration of 1-2 1 106 cpm/ml. Blots were washed twice with 21 SSC plus 0.1% SDS for 15 min at 257C and then once in 0.11 SSC (SSC Å 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) plus 0.1% SDS for 30 min at 607C and then subjected to autoradiography. Immunoblotting of recombinant and epithelial cell PGHS. For recombinant PGHS, transfected Cos-7 cells were sonicated at 105 watts for 60 s in 50 mM Tris-HCl (pH 7.4) containing 5 mM EGTA, 1 mM EDTA, 1 mM phenol, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mM diethyldithiocarbamate (3, 12). The sonicated cell mixtures were subjected to differential centrifugation at 47C, and the 100,000 x g pellets (containing microsomes) were resuspended in sonication buffer (12). For immunoblotting, the mixture was subjected to SDS-PAGE using an 8% resolving gel followed by electrophoretic transfer to PVDF membranes (Immobilon-P, Millipore Corp.) and incubation with anti-PGHS Ab and anti-rabbit IgG conjugated to horseradish peroxidase. Binding of primary Ab was detected using enhanced chemiluminescence (Amersham). For epithelial PGHS, cultured oTECs were sonicated in 50 mM Tris-HCl (pH 7.4) containing 10 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mM diethyldithiocarbamate and the sonicate centrifuged at 15,000 1 g for 30 min at 47C. The resulting supernatant was diluted in extraction buffer and then treated with anti-PGHS Ab for 2 h followed by Protein A sepharose (Pharmacia) for 1 h at 47C. The immune complex was washed with 50 mM Tris-HCl (pH 7.4) in 0.9% NaCl containing 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 0.5% Triton X-100, and 0.1% SDS and then with the same buffer lacking detergents. Next, the immune-complex pellet was heated at 1007C for 10 min in reducing SDS-PAGE sample buffer. The mixture was centrifuged, and the resulting supernatant was subjected to SDS-PAGE and immunoblotting as described above. Assay of recombinant and epithelial cell PGHS activities. For recombinant PGHS activity, transfected Cos-7 cells (5 1 106 cells/condition) were incubated with or without 100 mM aspirin for 30 min at 377C and then with 20 mM [3H]arachidonic acid in Hepes-buffered HBSS (pH 7.4) for 5-15 min at 377C. Cell supernatants were extracted with acidified chloroform/propanol using PGB2 as an internal standard for product recovery, and the lipid extracts were analyzed by reverse-phase HPLC as described previously (12). For epithelial cell PGHS activity, cultured oTECs were switched to basal culture conditions (24 h culture in LHC-basal medium) and then treated with or without serum (20% fetal bovine serum for 1-24 h) in the presence or absence of actinomycin D (5 mg/ml) or cycloheximide (5 mg/ ml). Untreated and treated oTECs (1 1 106 cells/condition) were then incubated with 20 mM [3H]arachidonic acid, and cell supernatants were extracted and analyzed for arachidonate metabolites as described above.
RESULTS AND DISCUSSION
Molecular cloning and sequencing of ovine PGHS-2 cDNA. Reverse-transcriptase PCR amplification of poly(A)/RNA from cultured oTECs resulted in the generation of a 0.5 kb cDNA that selectively recognized a 4.0 kb (PGHS-2) mRNA in Northern blots of oTEC RNA. The 32P-labeled 0.5 kb cDNA identified three positive clones in a lambda ZAP II cDNA library prepared from cultured oTECs. Phage DNA from three plaques that strongly hybridized with the probe were rescreened at lower plating density, then released with NotI, and the products were analyzed by agarose gel electrophoresis. Two of these pPGHSov-2-S10 and pPGHSov-2S24 contained overlapping sequences and a third clone PGHSov-2-S21 encoded for the entire 501
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FIG. 1. Nucleotide and deduced amino acid sequence for ovine PGHS-2. Nucleic acid and amino acid positions are indicated on the far right. Critical residues for catalysis (Arg105, Glu509, Tyr340, Tyr370, Phe514, Ser515, Leu516, Lys517, Gly518), aspirin-acetylation (Ser515), N-glycosylation (Asn 52, 129, 395, and 579), heme coordination (His 192, 294, and 373), and inhibitor interaction (Arg120 and Val508) are each underlined and italicized. Each 3*-ATTTA motif is also underlined.
3.4 kb mRNA (Fig. 1). This insert (designated oPGHS-2) also selectively hybridized with a 4.0 kb (PGHS-2) mRNA whereas the cDNA for the coding region of PGHS-1 (oPGHS-1coding) selectively recognized a 2.8 kb (PGHS-1) mRNA in Northern analysis of oTEC RNA (see below). 502
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FIG. 2. Expression of ovine PGHS-1 and -2 assessed by Northern (A) and Western (B) blot analysis. In A, Cos7 cells were transfected with pOSMLv (Vector, lane 1), pOSMLv-oPGHS-1-coding (PGHS-1, lane 2) or pOSMLvoPGHS-2 (PGHS-2, lane 3), and then poly(A)/ RNA (2 mg/lane) from each condition was subjected to Northern blotting using 32P-labeled ovine PGHS-1 or -2 cDNA. Arrows indicate the positions of 3.0 kb PGHS-1 (left blot) and 4.4 kb PGHS-2 mRNA (right blot), each fused to the 1.0 kb DHFR mRNA. PGHS-2 mRNA also runs as an unfused 3.4 kb species (lower arrow in right blot). Control analyses for 0.8 kb GAPDH mRNA showed identical levels in each lane (lower arrow in left blot). Controls for transfection efficiency (using a pBH-RSV-luc luciferase-reporter plasmid) also showed identical levels in each condition (data not shown). In B, the same transfection conditions were used to prepare microsomal protein (25 mg/lane) for Western blot analysis using anti-PGHS-1 Ab (Oxford PG-16) or anti-PGHS-2 Ab (MH.746) and detection by enhanced chemiluminescence.
The predicted amino acid sequence of ovine PGHS-2 conserves each of the amino acid residues identified in ovine PGHS-1 as critical for catalysis (4, 6, 8-10), heme ligation (5), aspirin-acetylation (3, 6), interaction with carboxylic acid-containing inhibitors (25), and Nlinked glycosylation (7) (Fig. 1). In addition, ovine PGHS-2 contains an N-linked glycosylation site at Asn579 that may account for variable glycosylation and doublet formation during electrophoresis (see below) as has been suggested for murine PGHS-2 (7). Ovine PGHS-2 also differs from PGHS-1 at an active site residue (Val508) that confers selective inhibition by diarylheterocycle PGHS inhibitors (11). Primary sequence alignment indicates that ovine PGHS-2 is 80-88% homologous with PGHS-2 from mitogen-stimulated human vascular endothelial cells or transformed chicken and murine fibroblasts and 61% homologous with ovine seminal vesicle PGHS-1. 503
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FIG. 3. Effect of aspirin on the profile of arachidonate products generated by recombinant ovine PGHS-1 (A and B) and PGHS-2 (C and D). Cos-7 cells transfected with pOSMLv-oPGHS-1-coding or pOSMLv-oPGHS-2 were treated with vehicle (A, C) or aspirin (B, D) and then incubated with 20 mM [3H]arachidonic acid (2.2 1 105 dpm/ ml) in Hepes-buffered HBSS for 15 min at 377C. Products were extracted from cell supernatants (5 1 106 cells) and analyzed by reverse-phase HPLC. Major peaks of radioactivity coeluted with reference PGF2a (F), PGE2 (E), PGD2 (D), 15-HETE (15H), and arachidonic acid (AA). Results were identical for full-length (oPGHS-2) and coding-region (oPGHS-2-coding) constructs; control transfections with pOSMLv gave no detectable PG-forming activity (data not shown).
Expression of recombinant ovine PGH Synthases in Cos-7 cells. Expression of recombinant ovine PGHS-2 (and PGHS-1) in Cos-7 cells was accomplished using pECE and pOSMLv expression vectors (Fig. 2). Higher levels of expression attained with pOSMLv may depend on including coding sequence for murine DHFR, since this sequence appears to enhance the stability of the hybrid mRNA (26). In that context, expression of PGHS-1 and several PGHS2 constructs (with and without the 3*-UTR) all gave similar levels of PGHS mRNA and protein (Fig. 2) and corresponding enzymatic activity (1.3-2.2 nmol PG/mg protein/15 min). Comparisons of activity in untreated to aspirin-treated Cos-7 cells expressing ovine PGHS-1 or -2 indicated that recombinant ovine PGHS-1 was completely inactivated by aspirin-acetylation, but PGHS-2 still converted arachidonate to 15-HETE (Fig. 3). This profile precisely matches PG- and 15-HETE-forming activities for endogenous PGHS isozymes in cultured ovine (and human) TECs (12, 27). Regulation of PGHS activity in cultured oTECs. Among a series of cytokines and growth 504
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FIG. 4. Selective serum-stimulation of PGHS-2 in cultured oTECs assessed at the level of mRNA by Northern blot (A), protein by Western blot (B), and enzyme activity by HPLC-based assay (C). In each case, cells were untreated (0) or treated (/) with 20% fetal bovine serum for 1 h (to detect mRNA) or 4 h (to detect protein and activity). In A, oTEC RNA (15 mg/lane) was hybridized with 32P-labeled ovine PGHS-1 or PGHS-2 cDNA under high stringency conditions (50% formamide, 687C). Control analysis for GAPDH mRNA showed identical levels in each lane (lower arrow). In B, oTEC protein (15 mg/lane) was immunoblotted against anti-PGHS-2 Ab (MH.746) and detected by enhanced chemiluminescence. In C, oTEC supernatants (1 1 106 cells) were extracted and analyzed by HPLC as described in the Fig. 3 legend.
factors (including PMA, IL-1, TNF, and LPS), serum was the most potent stimulus of PGHS2 mRNA levels in cultured oTECs (data not shown). Serum treatment caused little change in the level of a 2.8 kb PGHS-1 mRNA, but a 2-4x increase in the 4.0 kb PGHS-2 mRNA (Fig. 4) confirming that PGHS-2 is selectively regulated in this cell system (13). Serum treatment caused corresponding increases in 72-kDa PGHS-2 and a two-fold increase in PGE2-forming activity to a final level of 1.5 nmol/mg protein/15 min (Fig. 4). Serum stimulation of PGHS2 mRNA was completely inhibited by actinomycin D but was unchanged by cycloheximide treatment (data not shown), indicating dependence on mRNA transcription but not protein translation for the rapid serum effect. Thus, PGHS-2 expression may develop more rapidly at sites of epithelial inflammation (and apoptosis) than at other sites that depend on monocyte/ macrophage influx and activation (28). In summary, we have characterized a full-length cDNA for ovine epithelial PGHS-2 that conserves critical amino-acid residues used by ovine PGHS-1 for interaction with substrate, inhibitors, heme prosthetic groups, and carbohydrate moieties. The data provide direct evidence for ovine epithelial PGHS-2 that differs from PGHS-1 in its response to aspirin505
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acetylation and serum-stimulation, and the results form the basis for directed analysis of PGHS structure-function relationships in the context of mutagenesis and x-ray crystallographic data for ovine PGHS-1. ACKNOWLEDGMENTS This research was supported by Grants HL-40078, HL-07317, DK-38111, and HL/AI-51071 from the National Institutes of Health and the Alan A. and Edith L. Wolff Charitable Trust.
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Molecular Cloning, Functional Expression, and Selective Regulation of Ovine Prostaglandin H Synthase-21 Vivian Zhang, Mary O’Sullivan, Hameda Hussain, William T. Roswit, and Michael J. Holtzman2 Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110 Received September 4, 1996 Structural characterization for ovine prostaglandin H synthase-1 (PGHS-1) is extensive, but the corresponding structure for the homologous ovine PGHS-2 isoform is undefined. Accordingly, we isolated a full-length (3.4 kb) ovine PGHS-2 cDNA from a primary-culture cell model (ovine tracheal epithelial cells) originally described as containing both PGHS isoforms. Analysis of ovine PGHS-2 cDNA sequence indicated conservation of critical amino acid residues, but differences in other hydrophilic regions allowed for the development of an anti-peptide antibody highly selective for PGHS-2. Enzymatic activities of the recombinant ovine PGHS isozymes indicated significant differences in response to aspirin-acetylation consistent with the characteristics of endogenous cellular PGHS activities under basal and serum-induced conditions. The results fully account for previous evidence of two distinct PGHS activities in cultured airway epithelial cells and provide for additional definition of PGHS structure–function relationships. q 1996 Academic Press, Inc.
Prostaglandin H synthase (PGHS) exists in two isoforms (PGHS-1 and PGHS-2) that each convert arachidonic acid to prostaglandin H2 (PGH2), the first committed step in the biosynthesis of biologically active prostaglandins and thromboxanes. PGHS features were defined first and remain defined most completely for ovine PGHS-1. This species was the first one cloned and sequenced (1, 2), and remains as the most extensively mutated (3-9). It is the only PGHS with three-dimensional structure defined by x-ray crystallography (10). Together, these approaches have defined critical structures governing PGHS-1 interaction with substrate and inhibitors. The subsequent identification of PGHS-2 raised the possibilities that PGHS-1 and -2 may mediate constitutive versus inducible prostanoid production, respectively, and that isozymespecific inhibitors might be designed for greater efficacy (11). Because structural features of ovine PGHS-1 are well defined, the design of inhibitors may be further facilitated by corresponding structural data for ovine PGHS-2. In fact, a PGHS isozyme (distinct from PGHS-1) exhibiting selective regulation and distinct pharmacology was also first identified using an ovine cell culture system (12, 13), but these studies did not define ovine PGHS-2 sequence or fully link the functional expression of 4.0 kb PGHS mRNA with the corresponding 72-kDa polypeptide. Accordingly, the present experiments were performed to (i) isolate the cDNA for ovine PGHS-2 from cultured ovine tracheal epithelial cells (oTECs); (ii) compare the structure for ovine PGHS-2 to PGHS-1; (iii) test the cDNA-encoded ovine PGHS-2 for enzymatic activity and for reactivity with nonsteroidal anti-inflammatory drugs (especially aspirin); and 1 The nucleotide sequence described in this paper has been submitted to the GenBank database with the Accession No. U68486 (bankit id No. 66332). 2 To whom correspondence should be addressed at Washington University School of Medicine, 660 S. Euclid Ave., Box 8052, St. Louis, MO 63110. Fax: (314) 362-8987; E-mail: [email protected]. Abbreviations: HETE, hydroxyeicosatetraenoic acid; oTEC, ovine tracheal epithelial cell; PG, prostaglandin; PGHS, prostaglandin H synthase.
499 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(iv) determine whether PGHS-2 is selectively regulated in (non-inflammatory) epithelial cells that express both isoforms. MATERIALS AND METHODS Materials. AMV reverse transcriptase was from Life Sciences; MMLV reverse transcriptase was from BRL; [aP]dCTP (3000 Ci/mmol) and [3H8]arachidonic acid (95.1 Ci/mmol) were from Dupont-New England Nuclear; PGHS1 purified from ovine seminal vesicle was from Cayman Chemical Co., Inc.; and purified ovine placental and recombinant chicken PGHS-2 were from Oxford Biomedical Research, Inc. Laboratory of Human Carcinogenesis (LHC) basal medium was obtained from Biofluids and was supplemented with ovine pituitary extract, epidermal growth factor, epinephrine, hydrocortisone, insulin, transferrin, triiodothyronine, L-glutamine, calcium chloride, trace elements, penicillin, and streptomycin (LHC-8e) as described previously for oTEC culture (12-14). Authentic reference compounds included (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) and PGB2 , PGD2 , PGE2 , and PGF2a obtained from Biomol Research Laboratories Inc. or Cayman Chemical Co. Antibodies. Three rabbit antisera raised against ovine seminal vesicle PGHS-1 were obtained from Oxford Biomedical Research (PG-16 and PG-20) and Cayman Chemical Co. (#160103). A rabbit antisera raised against PGHS-2 Nterminus was also obtained from Oxford Biomedical Research (PG-26). In addition, rabbit antisera were raised against deduced amino acid sequences of murine PGHS-2 using synthetic peptides conjugated to keyhole limpet hemocyanin via an N-terminal cysteine as antigens (15). IgG was purified from antisera by peptide-affinity chromatography using antigenic peptide and Sulfolink coupling gel (Pierce Chemical). Peptide sequences were selected because of their conservation across murine and chicken PGHS-2 (16-18) and their dissimilarity to ovine PGHS-1 (1, 2) as well as their predicted hydrophilicity (19). Preliminary experiments indicated that antisera (designated MH.745/6) from both rabbits immunized against one of these peptides exhibited selective reactivity with recombinant chicken and purified ovine PGHS-2 at high titer (1:50,000 dilution). Neither pre-immune sera nor antisera against other peptides exhibited PGHS reactivity. PCR-based amplification of ovine PGHS-2 mRNA. Total RNA was isolated from cultured oTECs (12) using guanidinium isothiocyanate lysis and centrifugation through cesium chloride (20). Poly(A)/ RNA was fractionated with a biotinylated oligo(dT) probe-streptavidin magnetic particle system (Promega) and was used to generate corresponding cDNA using MMLV reverse transcriptase. The cDNA product was amplified by PCR using an annealing cycle at 557C and Hot Tub DNA polymerase (Amersham). DNA primers were based on the sequences surrounding the active site Tyr385 (4) and the aspirin-acetylation site Ser532 in murine PGHS-2 (17, 18) which are conserved in ovine seminal vesicle PGHS-1 (1, 2) and chicken embryo fibroblast PGHS-2 (16) and were constructed to also contain extensions with either XhoI (CTCGAG) or SpeI (ACTAGT) restriction sites at their 5* ends. The downstream primer 5*-GATCACTCGAGATGGGATTTCCCATAAGTCCTTTCAAGGAGAA-3* contained an Xho1 site and nucleotides 1668-1698 of murine PGHS-2 which included Tyr385, and the upstream primer 5*-AATGCACTAGTGAATTCAACACACTCTATCACTGGCACCCCCT-3* contained an SpeI site and nucleotides 1219-1251 of murine PGHS-2 which included Ser532. Reverse-transcriptase PCR products were selected for further pursuit on the basis of size, Southern blotting against oligonucleotides containing internal PGHS-2 sequence, and Northern blotting against epithelial cell mRNA. PCR products were purified and ligated into XhoI/SpeI-digested pBlueScript II SK(//0) phagemid (Stratagene) with T4 DNA ligase. The resulting phagemid was used to transform E. Coli DH1 cells, and recombinant colonies were detected by white colony formation on IPTG/XGAL plates. Phagemid insert was subjected to DNA sequencing using the dideoxy chain termination method with the Sequenase Kit (U. S. Biochemicals) and primers based on flanking sequences of the T3 and T7 promoters in pBluescript. cDNA library construction and screening. Poly(A)/RNA from oTECs was fractionated by oligo(dT) affinity chromatography and converted to double-stranded blunt-ended cDNA (21). EcoRI(NotI) linker-adapters were added using the reagents contained in the Copy Kit (Invitrogen Corp.). After size selection by agarose gel electrophoresis, the cDNA was ligated into a phage cloning vector (lambda ZAP II, Stratagene) and then packaged in vitro (Gigapack II Gold, Stratagene). The library was amplified in E. Coli (host strain XL1-Blue, Stratagene) and was screened with 32 P-labeled cDNA generated from reverse transcriptase-PCR as noted above and then radiolabeled with [a-32P]dCTP by random primer synthesis using the Klenow fragment of DNA polymerase I (Multiprime, Amersham Corp.) to a specific activity of 109 cpm/mg. Phage from positive plaques was subjected to secondary screening and then rescued into pBluescript using f1 helper phage (R408, Stratagene). Insert size was determined by agarose gel eletrophoresis after digestion with NotI. Initial sequence was obtained using primers based on flanking sequences contained in pBluescript. Both strands of a full-length cDNA for ovine PGHS-2 (designated oPGHS-2) were sequenced using a linear sequencing strategy. Preparation of expression vectors and transfection of Cos-7 cells. For ovine PGHS-1, contiguous 0.5 kb and 1.5 kb cDNAs (2) were released from M13 phage by digestion with XbaI and EcoRI and then were gel purified and successively ligated into the EcoRI/XbaI-digested expression vector pECE (22) to form a 2.0 kb cDNA (oPGHS-1coding) containing the complete open-reading frame. In addition, this insert was released with KpnI/XbaI, blunt-ended with T4 DNA polymerase, and ligated into a new SmaI site in the expression vector pOSMLv. The parent vector 32
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(pOSML) lacking this SmaI site was obtained from W. Smith (Michigan State Univ.) and Genetics Institute, Inc (23). For the newly cloned PGHS-2, oPGHS-2 was released from pBluescript by digestion with NotI, blunt ended with the Klenow fragment of DNA polymerase I, and ligated into the SmaI site of pECE or pOSMLv. In addition, the coding region of ovine PGHS-2 (designated oPGHS-2-coding) was expressed in pECE and pOSMLv. To generate pECEoPGHS-2-coding, pECE was cut with BalI/XbaI (removing the 3*-UTR and a portion of the coding region of oPGHS2) followed by PCR to restore the full coding region. To generate pOSMLv-oPGHS-2-coding, the coding region was released from pECE with XbaI/HindIII, filled in with Klenow, and blunt ligated into the SmaI site in pOSMLv. Expression vector constructs (pECE- and pOSMLv-oPGHS-1-coding and pECE- and pOSMLv-oPGHS-2 and -oPGHS2-coding) were verified by agarose gel electrophoresis after digestion with PstI or PstI/XbaI, and all inserts were sequenced to insure proper orientation and sequence reproduction. The plasmids were grown in ampicillin-containing T broth, then purified through cesium chloride, and used to transfect Cos-7 cells (ATCC CRL-1651) by lipofectinmediated gene transfer (24). Northern analysis of recombinant and epithelial cell PGHS. Total cellular RNA was prepared from transfected Cos-7 cells and cultured oTECs and was subjected to electrophoresis in a 1.0% agarose gel containing 1 M formaldehyde. Equal amounts of RNA were loaded/lane on the basis of absorbance at 260 nm, and equivalency of sample amounts was initially verified by the intensity of ethidium bromide staining of the 28S and 18S rRNA bands. RNA was electroblotted to nylon membranes which were prehybridized for 15 min and then hybridized for 1 h at 687C in 10 ml of QuikHyb solution (Stratagene) with 32P-labeled cDNAs for ovine PGHS-1 or -2 prepared as described above. 32 P-labeled cDNAs were added to the hybridization mixture in 300 ml of 10 mg/ml salmon sperm DNA to achieve a final probe concentration of 1-2 1 106 cpm/ml. Blots were washed twice with 21 SSC plus 0.1% SDS for 15 min at 257C and then once in 0.11 SSC (SSC Å 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) plus 0.1% SDS for 30 min at 607C and then subjected to autoradiography. Immunoblotting of recombinant and epithelial cell PGHS. For recombinant PGHS, transfected Cos-7 cells were sonicated at 105 watts for 60 s in 50 mM Tris-HCl (pH 7.4) containing 5 mM EGTA, 1 mM EDTA, 1 mM phenol, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mM diethyldithiocarbamate (3, 12). The sonicated cell mixtures were subjected to differential centrifugation at 47C, and the 100,000 x g pellets (containing microsomes) were resuspended in sonication buffer (12). For immunoblotting, the mixture was subjected to SDS-PAGE using an 8% resolving gel followed by electrophoretic transfer to PVDF membranes (Immobilon-P, Millipore Corp.) and incubation with anti-PGHS Ab and anti-rabbit IgG conjugated to horseradish peroxidase. Binding of primary Ab was detected using enhanced chemiluminescence (Amersham). For epithelial PGHS, cultured oTECs were sonicated in 50 mM Tris-HCl (pH 7.4) containing 10 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mM diethyldithiocarbamate and the sonicate centrifuged at 15,000 1 g for 30 min at 47C. The resulting supernatant was diluted in extraction buffer and then treated with anti-PGHS Ab for 2 h followed by Protein A sepharose (Pharmacia) for 1 h at 47C. The immune complex was washed with 50 mM Tris-HCl (pH 7.4) in 0.9% NaCl containing 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 0.5% Triton X-100, and 0.1% SDS and then with the same buffer lacking detergents. Next, the immune-complex pellet was heated at 1007C for 10 min in reducing SDS-PAGE sample buffer. The mixture was centrifuged, and the resulting supernatant was subjected to SDS-PAGE and immunoblotting as described above. Assay of recombinant and epithelial cell PGHS activities. For recombinant PGHS activity, transfected Cos-7 cells (5 1 106 cells/condition) were incubated with or without 100 mM aspirin for 30 min at 377C and then with 20 mM [3H]arachidonic acid in Hepes-buffered HBSS (pH 7.4) for 5-15 min at 377C. Cell supernatants were extracted with acidified chloroform/propanol using PGB2 as an internal standard for product recovery, and the lipid extracts were analyzed by reverse-phase HPLC as described previously (12). For epithelial cell PGHS activity, cultured oTECs were switched to basal culture conditions (24 h culture in LHC-basal medium) and then treated with or without serum (20% fetal bovine serum for 1-24 h) in the presence or absence of actinomycin D (5 mg/ml) or cycloheximide (5 mg/ ml). Untreated and treated oTECs (1 1 106 cells/condition) were then incubated with 20 mM [3H]arachidonic acid, and cell supernatants were extracted and analyzed for arachidonate metabolites as described above.
RESULTS AND DISCUSSION
Molecular cloning and sequencing of ovine PGHS-2 cDNA. Reverse-transcriptase PCR amplification of poly(A)/RNA from cultured oTECs resulted in the generation of a 0.5 kb cDNA that selectively recognized a 4.0 kb (PGHS-2) mRNA in Northern blots of oTEC RNA. The 32P-labeled 0.5 kb cDNA identified three positive clones in a lambda ZAP II cDNA library prepared from cultured oTECs. Phage DNA from three plaques that strongly hybridized with the probe were rescreened at lower plating density, then released with NotI, and the products were analyzed by agarose gel electrophoresis. Two of these pPGHSov-2-S10 and pPGHSov-2S24 contained overlapping sequences and a third clone PGHSov-2-S21 encoded for the entire 501
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FIG. 1. Nucleotide and deduced amino acid sequence for ovine PGHS-2. Nucleic acid and amino acid positions are indicated on the far right. Critical residues for catalysis (Arg105, Glu509, Tyr340, Tyr370, Phe514, Ser515, Leu516, Lys517, Gly518), aspirin-acetylation (Ser515), N-glycosylation (Asn 52, 129, 395, and 579), heme coordination (His 192, 294, and 373), and inhibitor interaction (Arg120 and Val508) are each underlined and italicized. Each 3*-ATTTA motif is also underlined.
3.4 kb mRNA (Fig. 1). This insert (designated oPGHS-2) also selectively hybridized with a 4.0 kb (PGHS-2) mRNA whereas the cDNA for the coding region of PGHS-1 (oPGHS-1coding) selectively recognized a 2.8 kb (PGHS-1) mRNA in Northern analysis of oTEC RNA (see below). 502
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FIG. 2. Expression of ovine PGHS-1 and -2 assessed by Northern (A) and Western (B) blot analysis. In A, Cos7 cells were transfected with pOSMLv (Vector, lane 1), pOSMLv-oPGHS-1-coding (PGHS-1, lane 2) or pOSMLvoPGHS-2 (PGHS-2, lane 3), and then poly(A)/ RNA (2 mg/lane) from each condition was subjected to Northern blotting using 32P-labeled ovine PGHS-1 or -2 cDNA. Arrows indicate the positions of 3.0 kb PGHS-1 (left blot) and 4.4 kb PGHS-2 mRNA (right blot), each fused to the 1.0 kb DHFR mRNA. PGHS-2 mRNA also runs as an unfused 3.4 kb species (lower arrow in right blot). Control analyses for 0.8 kb GAPDH mRNA showed identical levels in each lane (lower arrow in left blot). Controls for transfection efficiency (using a pBH-RSV-luc luciferase-reporter plasmid) also showed identical levels in each condition (data not shown). In B, the same transfection conditions were used to prepare microsomal protein (25 mg/lane) for Western blot analysis using anti-PGHS-1 Ab (Oxford PG-16) or anti-PGHS-2 Ab (MH.746) and detection by enhanced chemiluminescence.
The predicted amino acid sequence of ovine PGHS-2 conserves each of the amino acid residues identified in ovine PGHS-1 as critical for catalysis (4, 6, 8-10), heme ligation (5), aspirin-acetylation (3, 6), interaction with carboxylic acid-containing inhibitors (25), and Nlinked glycosylation (7) (Fig. 1). In addition, ovine PGHS-2 contains an N-linked glycosylation site at Asn579 that may account for variable glycosylation and doublet formation during electrophoresis (see below) as has been suggested for murine PGHS-2 (7). Ovine PGHS-2 also differs from PGHS-1 at an active site residue (Val508) that confers selective inhibition by diarylheterocycle PGHS inhibitors (11). Primary sequence alignment indicates that ovine PGHS-2 is 80-88% homologous with PGHS-2 from mitogen-stimulated human vascular endothelial cells or transformed chicken and murine fibroblasts and 61% homologous with ovine seminal vesicle PGHS-1. 503
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FIG. 3. Effect of aspirin on the profile of arachidonate products generated by recombinant ovine PGHS-1 (A and B) and PGHS-2 (C and D). Cos-7 cells transfected with pOSMLv-oPGHS-1-coding or pOSMLv-oPGHS-2 were treated with vehicle (A, C) or aspirin (B, D) and then incubated with 20 mM [3H]arachidonic acid (2.2 1 105 dpm/ ml) in Hepes-buffered HBSS for 15 min at 377C. Products were extracted from cell supernatants (5 1 106 cells) and analyzed by reverse-phase HPLC. Major peaks of radioactivity coeluted with reference PGF2a (F), PGE2 (E), PGD2 (D), 15-HETE (15H), and arachidonic acid (AA). Results were identical for full-length (oPGHS-2) and coding-region (oPGHS-2-coding) constructs; control transfections with pOSMLv gave no detectable PG-forming activity (data not shown).
Expression of recombinant ovine PGH Synthases in Cos-7 cells. Expression of recombinant ovine PGHS-2 (and PGHS-1) in Cos-7 cells was accomplished using pECE and pOSMLv expression vectors (Fig. 2). Higher levels of expression attained with pOSMLv may depend on including coding sequence for murine DHFR, since this sequence appears to enhance the stability of the hybrid mRNA (26). In that context, expression of PGHS-1 and several PGHS2 constructs (with and without the 3*-UTR) all gave similar levels of PGHS mRNA and protein (Fig. 2) and corresponding enzymatic activity (1.3-2.2 nmol PG/mg protein/15 min). Comparisons of activity in untreated to aspirin-treated Cos-7 cells expressing ovine PGHS-1 or -2 indicated that recombinant ovine PGHS-1 was completely inactivated by aspirin-acetylation, but PGHS-2 still converted arachidonate to 15-HETE (Fig. 3). This profile precisely matches PG- and 15-HETE-forming activities for endogenous PGHS isozymes in cultured ovine (and human) TECs (12, 27). Regulation of PGHS activity in cultured oTECs. Among a series of cytokines and growth 504
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FIG. 4. Selective serum-stimulation of PGHS-2 in cultured oTECs assessed at the level of mRNA by Northern blot (A), protein by Western blot (B), and enzyme activity by HPLC-based assay (C). In each case, cells were untreated (0) or treated (/) with 20% fetal bovine serum for 1 h (to detect mRNA) or 4 h (to detect protein and activity). In A, oTEC RNA (15 mg/lane) was hybridized with 32P-labeled ovine PGHS-1 or PGHS-2 cDNA under high stringency conditions (50% formamide, 687C). Control analysis for GAPDH mRNA showed identical levels in each lane (lower arrow). In B, oTEC protein (15 mg/lane) was immunoblotted against anti-PGHS-2 Ab (MH.746) and detected by enhanced chemiluminescence. In C, oTEC supernatants (1 1 106 cells) were extracted and analyzed by HPLC as described in the Fig. 3 legend.
factors (including PMA, IL-1, TNF, and LPS), serum was the most potent stimulus of PGHS2 mRNA levels in cultured oTECs (data not shown). Serum treatment caused little change in the level of a 2.8 kb PGHS-1 mRNA, but a 2-4x increase in the 4.0 kb PGHS-2 mRNA (Fig. 4) confirming that PGHS-2 is selectively regulated in this cell system (13). Serum treatment caused corresponding increases in 72-kDa PGHS-2 and a two-fold increase in PGE2-forming activity to a final level of 1.5 nmol/mg protein/15 min (Fig. 4). Serum stimulation of PGHS2 mRNA was completely inhibited by actinomycin D but was unchanged by cycloheximide treatment (data not shown), indicating dependence on mRNA transcription but not protein translation for the rapid serum effect. Thus, PGHS-2 expression may develop more rapidly at sites of epithelial inflammation (and apoptosis) than at other sites that depend on monocyte/ macrophage influx and activation (28). In summary, we have characterized a full-length cDNA for ovine epithelial PGHS-2 that conserves critical amino-acid residues used by ovine PGHS-1 for interaction with substrate, inhibitors, heme prosthetic groups, and carbohydrate moieties. The data provide direct evidence for ovine epithelial PGHS-2 that differs from PGHS-1 in its response to aspirin505
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acetylation and serum-stimulation, and the results form the basis for directed analysis of PGHS structure-function relationships in the context of mutagenesis and x-ray crystallographic data for ovine PGHS-1. ACKNOWLEDGMENTS This research was supported by Grants HL-40078, HL-07317, DK-38111, and HL/AI-51071 from the National Institutes of Health and the Alan A. and Edith L. Wolff Charitable Trust.
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