derstanding of the important structure/function domains of this protein and its gene regulation.-. Funk, C. D.;. Funk, L. B.; Kennedy,. M. E.; Pong, A. S.; FitzGerald,.
Human
platelet/erythroleukemia
cDNA
cloning,
expression,
COLIN D. FUNK,’ LENA GARRET A. FITZGERALD Division
of Clinical
cell prostaglandin
B. FUNK,
Pharmacology,
Vanderbilt
and
MATTHEW University,
Nashville,
J.
phorbol ester
quantitative
by
a distinct
THE
enzyme
INCIDENCE
death
(6,
7).
Roth
and
Majerus
(8)
Tennessee 37232,
S. PONG,
synthase: assignment
AND
USA
ygenase reaction (II). In view of the possibility of significant species or cell-specific heterogeneity of PGG/H synthase cDNAs, we sought to characterize the eDNA derived from human platelets, the cellular target for aspirin action in cardiovascular disease.
METHODS Isolation clones
and
analysis
of PGG/PGH
synthase
eDNA
Oligonucleotide primers were designed based on the sheep PGG/H synthase (abbreviated throughout as COX) sequence (9, 10) and subsequently a human genomic sequence (12), and were used in polymerase chain reactions (PCRs) to isolate overlapping clones from platelet eDNA (Table 1). RNA was isolated from washed human platelets obtained from approximately 10 ml blood by the acid guanidinium thiocyan ate-phenol-chloroform extraction method (13). eDNA was synthesized (14) and PCR was performed as shown in Table I with approximately 25 pmol primers, 200 jaM dNTP, and 2.5 units Taq polymerase. PCR-amplified DNAs were platelet eDNA-derived (blood from three different donors) and not caused by genomic DNA contamination. PL-COX3 was “P-labeled and used as a probe for screening a Xgtll human erythroleukemia (HEL) cell eDNA library (kindly provided by Dr. G. J. Roth, Division of Hematology,
of Washington,
Veterans
Seattle)
Administration
Hospital,
by standard
hybridization
of human COX cDNA in COS-M6 cells
University
protocols.
expression
vectors
A 0.24-kb XmaI/EcoRI fragment from PL-COXI (XmaI site derived from polylinker of M13mp18) encoding the 5’ end of the COX eDNA was subcloned into pGEM 7Zf(-) (Promega, Madison, Wis.). This DNA construct was EcoRIcleaved, phosphatase-treated, and the 2.3-kb EcoRI insert of HEL-COXI3b was introduced. The correct orientation of this fragment was determined by AccI digestion. A 2.6-kb BamHI/SaII (both sites polylinker derived) fragment was introduced into the mammalian cell expression vector pcDNA-1 (Invitrogen, San Diego, Calif.) cleaved at BamHI
demon-
strated that aspirin irreversibly inhibited the cyclooxygenase reaction in human platelets by acetylation of a serine residue in human platelets. DeWitt and Smith (9) and Needleman et al. (10) had reported the sequence of cDNAs for PGG/H synthase obtained from sheep vesicular gland. Mutational analysis of a serine residue at position 530 suggested that the aspirin acetylation site was close to the active site for the cycloox-
2304
AMY
Construction and expression
of stroke, myocardial infarcin patients with cardiovascular disease (1). It also reduces the incidence of myocardial infarction in apparently healthy individuals (2). These effects have been attributed to the ability of aspirin to prevent formation of thromboxane (Tx)2 A,, a potent platelet agonist and vasoconstrictor (3, 4), by platelets (5). Aspirin exerts this effect by inhibiting the enzyme prostaglandin (PG) 0/H synthase, which by sequential cyclooxygenase and hydroperoxidase reactions transforms arachidonic acid to the cyclic endoperoxide intermediate, PGH,, from which TxA, is formed ASPIRIN REDUCES tion, arid vascular
chromosomal
E. KENNEDY,
ABSTRACT Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PCG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxidederived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a humanhamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and its gene regulation.Funk, C. D.; Funk, L. B.; Kennedy, M. E.; Pong, A. S.; FitzGerald, G. A. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB 5: 2304-23 12; 1991. Key Words: cyclooxygenase aspirin PCR chronic myelogerzous leukemia
gene
G/H
‘To whom correspondence should be addressed, Clinical Pharmacology, Vanderbilt University, 37232, USA. ‘Abbreviations: HEL, human erythroleukemia; ygenase;PG, prostaglandin; PDGF, platelet-derived EGF, PMA,
epidermal phorbol
growth factor; PCR, l2-myristate l3-acetate;
at: Division of Nashville, TN COX, cyclooxgrowth factor;
polymerase chain TX, thromboxane;
reaction; TLC,
thin layer chromatography.
08926638I91I0005-2304I$01
.50. © FASEB
and Xhol sites. The construct (pcDNA-COX1) contained 39 bp before the COX ATG initiator codon (no other ATG sequence present), 1797 bp encoding human COX, including the putative leader peptide, and the complete 3’ untranslated region (751 bp). The serine residue at position 529 was changed to an asparagine by in vitro mutagenesis (Amersham kit) using the oligonucleotide ACCCTTGAGGTTAAAGGGAGCC (changed nucleotides are underlined) and the 2.6-kb BamHI/SalI COX fragment was cloned onto M13mp18. The mutated construct was checked by sequencing and restriction mapping and was cloned onto pcDNA-1 to yield pcDNACOXI (Asn529). Another expression vector pcDNA-COX2, containing only 18 bp 3’ noncoding sequence, was prepared from pcDNA-COX1 by cleavage at a BsmI site, a Klenow fragment fill-in reaction, isolation of a 1.85-kb BamHI/blunt end fragment, and subcloning into BamHI/EcoRV-cleaved pcDNA-1.
Expression constructs (12.5 jag) were introduced into COSM6 cells (1.2-3.0 x 106 cells/lO cm culture plate) by either calcium phosphate precipitation or lipofectin treatment (BRL protocol). Cells were harvested from four- to six plates 48-60 h after transfection and pooled. Cell pellets (300 x g) were resuspended in 2-3 ml 0.1 M Tris-HC1, pH 7.4, and sonicated for 3 x 3 s bursts at setting 3 with a Branson sonifier on ice. The sample was centrifuged for 5 mm at 10,000 x g, and the resulting supernatant was centrifuged for 1 h at 100,000 x g at 4#{176}C. The microsomal fraction was resuspended in 0.5 ml 0.1 M Tris-HC1, pH 7.4, to yield a protein concentration of 4 mg/mI. Cyclooxygenase Assays
were
A
panel
in
a 100-jal
final volume jaM epinephrine,
in
0.1
M 1 jaM
of human
available (BIOS
Corp.,
COX
gene
human/hamster New
Haven,
and
somatic Conn.)
was
cell
used
for
chromosomal determination of the COX gene by PCR detection of a 496-bp human cyclooxygenase DNA fragment (bp 1734-2230; see Fig. 2) contained within exon 11. PCR reactions were performed with the 25 somatic cell hybrid samples, plus control human and hamster DNAs, and a blank sample in a 100-jal volume with approximately 10 pmol primers (TTCCGTGTGCCGGATGCC and CTGGATCATCAGCCAGG) as mentioned previously for 35 cycles at 94#{176}C-50s, 54#{176}C-imm, and 72#{176}C-Imm. Amplified products were visualized by ethidium bromide staining of 1O-jal aliquots electrophoresed in a 3% NuSieve/1% Sea Kem agarose (FMC Bioproducts, Rockland, Maine) gel. Genomic DNA (20 jag) obtained from leukocytes of an individual
was
digested
to completion
with
selected
restriction
endonucleases and electrophoresed in a 0.7% agarose gel. The DNA was transferred to nylon membrane (Hybond-N, Amersham) and hybridized with “P-labeled PL-COX3 (specific activity of 10#{176} cpm/jag DNA) for 15 h at 65#{176}C in 6 x SSC/5 x Denhardt’s solution/0.5% SDS/iOO jag/ml salmon sperm DNA. The filter was washed briefly at room temperature in 2x SSC/0.1% SDS and twice at 65#{176}C with ix SSC/0.I% SDS for 15 mm/wash and exposed for autoradiography at - 70#{176}C.
Determination
medium
of COX mRNA
levels
in HEL
cells
cells were cultured in RPMI 1640 medium containing fetal calf serum and 10% adult bovine serum, sup-
plemented
out
localization analysis
commercially
hybrid
HEL 10%
assay carried
Chromosomal Southern blot
with
antibiotics.
18 h before
Cells
experiments.
were RNA
placed was
in serum-free extracted
from
5 x 10 cells of phorbol myristate acetate (160 ng/ml), Tris, pH 7.4 to 8.0, containing 500 PDGF (20 ng/ml), and thrombin (3 unit/ml)-treated or conhematin, 200 jag protein, and [1-14C]arachidonic acid (52.8 at various time intervals. RNA blot analysis was mCi/mmol; 19 jaM final concentration) for 15 mm at 37#{176}C. trol cultures performed as previously described (15) using the “P-labeled Samples were prewarmed at 37#{176}C for 2 mm before addition of substrate. Reactions were stopped by the addition of 10 jal PL-COX3 clone. I N HCI and 100 jal ethyl acetate. Thirty microliters of the COX mRNA was also detected by a quantitative PCR extracted organic phase was spotted directly onto a silica method (16) using a cRNA internal standard (17). Briefly, 1 plate and thin layer chromatography (TLC) was carried out jag total HEL RNA plus 9.25 x 106 molecules cRNA stanin a chloroform:methanol:acetic acid:water (90:8:1:0.8) soldard were reverse-transcribed as described previously with vent system. Plates were scanned for radioactivity using a Bioligo(dT) primer. PCR amplification of serial dilutions of oscan System 200 imaging scanner. Authentic prostaglandin the eDNA mixture was performed with 5 pmol primers and monohydroxy standards were run in parallel. Results (one “P-labeled, i0 cpm/assay) in a 50-jal volume and 1 are expressed as nanomole converted 20:4/mg protein. U Taq polymerase for 25 cycles at 94#{176}C-45 s, 62#{176}C-45 s, and
TABLE
1. PCR
amplification
5’ oligonucleotide
of platelet
DNA
and 3 oligonucleotide
clones
DNA
PCR
conditions
CTTGGA TCCGCGCCATGAGCCGGAGTC AGCGCATGCGTGAAGTGTTGTGCAAAGAA
PL-COX
AAGTGGA TCCCTGACCCTCAAGGCA AGTAGCCCCGGGTAGAATTCC
PL-COX2
94#{176}C-45 s 45#{176}C-45
TGGAATTCTACCCGGGGCTACTTC
PL-COX3
94#{176}C-imm
1
94#{176}C-45 s 50#{176}C-i mm 15 s 72#{176}C-i mm 30 s
72#{176}C-i mm TGAAGCT7CCTTCAGAGCTCTGT
37/50#{176}C-i mm 45 s 72#{176}C-3 mm
are displayed 5’ to 3. All PCR reactions were for 30 cycles except for PL-COX3 which had 3 previous cycles at the lower annealing Nucleotides in italics represent restriction sites incorporated into the primers or found naturally in the sheep sequence (EcoRl). Underlined represent differences between the ovine and human sequences.
Oligonucleotides
temperature. nucleotides
HUMAN
PLATELET CYCLOOXYGENASE
2305
IJo
0
t
U
U
“
HEL-COXI 3b
Expression
____ ____ _____ _______ _______
PL-COXI
PL-COX3
4
-k.
4
COS-M6
4
4
kb 0
0.5
1.0
1.5
2.0
2.5
Figure 1. Partial restriction map and cloning strategy for human platelet/erythroleukemia cell PGG/PGH synthase eDNA. Direction and extent of sequencing determinations are indicated by arrows. The open box indicates the protein coding region.
72#{176}C-I mm. Standard curves were constructed as described in ref i6. Samples were also checked for expression of cytoplasmic j-actin using an analogous procedure. The HEL cell-amplified eDNA was 451 and 320 bp for the amplified cRNA standard. The primer sequences CTCATAGGGGAGACCATCAAG (nts 1003-1023; Fig. 2) and CCTThTCTCCTACGAGCTCCTG (nts 1431-1452) were mRNA-specific crossing exons 8/9 and exons 10/li (12).
RESULTS Cloning synthase
of human cDNA
platelet/erythroleukemia
cell
PGG/H
At the onset of this study, only the sequence for ovine seminal vesicle COX was known. To clone the human platelet COX eDNA, oligonucleotide primers based on this sequence were designed and used to amplify DNA starting from estigial platelet RNA (Table i). Two clones, PL-COX2 and PL-COX3 (Fig. I), were isolated and sequenced and found to contain sequences with high homology to the ovine sequence. We were unable to obtain a DNA clone encoding the 5’ end of COX using sheepderived oligonucleotides. Therefore, we chose to screen a eDNA library constructed from a cell line displaying megakaryocytic/platelet properties (18; HEL, human erythroleukemia). Using “P-labeled PL-COX3, two clones, HEL-COXi3a and HEL-COXi3b (Fig. 1), were isolated after screening -3 x 10’ clones from a PMA-stimulated HEL cell eDNA library constructed in the XgtIi vector. When digested with EcoRI, inserts of 2.15 kb plus 0.24 kb and 2.3 kb were obtained from the two clones, respectively. Clone HEL-COXi3b contained nearly the complete coding sequence and 751 bp of 3’ noncoding sequence, including a polyadenylation signal AATAAA (Fig. 2). However, no poly(A) tail was present. Recently, a human genomic cyclooxygenase sequence was reported (12). To isolate the missing 5’ DNA coding sequence of platelet COX, new primers were synthesized based on the genomic sequence and were used in a PCR with platelet eDNA template. PL-COX1 was isolated and sequenced (Fig. 1 and Fig. 2). The complete composite platelet sequence was identical to the human erythroleukemia cell sequence at all locations. However, four differences involving three amino acid changes, were seen between the human platelet/erythroleukemia cell sequence and the genomic sequence (Fig. 2 legend). Notable characteristics of the deduced protein structure include a putative 23 amino acid leader peptide, a 576 amino acid mature protein displaying an EGF2306
like domain (residues 33-71), four potential glycosylation sites (amino acids 67, 103, 143, and 409), and the putative aspirin acetylation site at serine 529 (cf. refs 9-12).
Vol. 5
June 1991
of native
and
mutant
human
COX
in
cells
A hybrid platelet/HEL cell COX eDNA construct (pcDNACOXI) under control of the cytomegalovirus promoter was introduced into simian COS-M6 cells by calcium phosphatemediated transfection or lipofectin treatment. COX activity in microsomal fractions was monitored by the conversion of radiolabeled arachidonic acid into PGG/H decomposition products (PGE, PGD, PGF, HHT, and other rearranged products)
using
TLC
radioscanning.
Microsomal
fractions
from pcDNA-COX1 transfected cells produced readily detectable levels of products (Fig. 3), up to 40% [1-’4C]204 being metabolized in a 15-mm incubation (0.19 nmol 20:4/mg protein, range: 0.20-0.12; n=4). Boiling this fraction abolished all COX activity. Microsomes from mocktransfected COS cells produced barely detectable products ( 10% of pcDNA-COXI expressed levels; 0.017 nmol/mg protein). Preincubation of samples for 3 mm with indomethacm, aspirin, or sodium salicylate (all 100 jaM) inhibited COX activity 80-95% (0.038-0.010 nmol 20:4/mg protein; n=4), 30% (0.13 nmol 20:4/mg protein; n=3), and 4% (0.18 nmol 20:4/mg protein; n=I), respectively. Longer preincubation times and millimolar concentrations of aspirin were needed to inactivate COX completely, as was demonstrated for the expressed sheep enzyme (Ii). The expression levels reported here are much lower than those reported previously for the ovine seminal vesiele COX ( 200 nmol 20:4.min.mg protein’), which was also expressed in COS cells albeit under the control of an SV-40 promoter. Removing most of the 3’ noneoding sequence (pcDNA-COX2), a potential
site
of translational
repression
(19),
did
not
enhance
activity (0.14 ± 0.02 nmol 20:4/mg protein for pcDNACOX1 vs. 0.10 ± 0.03 for pcDNA-COX2; n=2). The role of Ser529 in controlling COX activity was investigated. Mutagenesis to an asparagine residue and subsequent expression of the mutated construct pcDNA-COXI (Asn529) in COS cells (Fig. 3) revealed markedly diminished enzyme activity (90-97% reduction, 0.034-0.022 nmol 20:4/mg protein; n = 2), although mRNA levels in the transfected cells were approximately equal to those in pcDNA-COX1 transfected cells (not shown). The relatively low levels of expression precluded accurate measurement of hydroperoxidase activity. Chromosomal Southern blot
assignment analysis
of human
COX gene and
A panel of 25 human/hamster somatic cell hybrid DNAs was analyzed by PCR screening for a 496-bp human COX DNA fragment. The fragment was amplified in a normal human genomic DNA sample and in hybrid clones 732, 862, and 968, and was absent in the remaining 22 hybrid and hamster DNA samples (Fig. 4). Analysis for concordance allowed us to assign unambiguously the cyclooxygenase gene to human chromosome 9. Hybridization of human genomic DNA with an exon Ii specific probe, containing the highly conserved region around the aspirin acetylation site and subsequent washing under moderately stringent conditions, revealed single bands in the EcoRI, BamHI, EeoRI/BamHI, and BgIII digested samples (Fig. 5). Three bands were detected in the MspI digested sample (two MspI sites lie within the probe sequence).
The FASEB Journal
FUNK
ET AL.
GCGcC
-1 120 40
240 CinHisGinGlyl
leCysVaiArqPheGlyLeuA3pA.rgTyrGlnCysAspCysThrArgThrGlyTyrSerGlyProAsnCysThrI
ieProCiyL.uTrpThrTrpLauArqA.inS.r
80 360
120
AsnLeuI
leProSarProeroThrTyrAsnSarAlaHtsAspTyrl
leSerTrpGI
uSerPheSerAsnVaiSarTyrTyrThrArql
leL.uProserVaiProLysAspCysproThr
480 160 600 200 720 240 840 280 960 320
ThrrrpGlyAspGluGlnLeuPheGlnThrThrArqLeuIleL.uIlsGlyGiuThrIieLysIlaVa
1.G1uG1u?yrVa1G1nG1nL.uSerG1ytyrPh.LeuG1nLuuLy.Ph.
1080 360 1200 400 1320 440
HiaHial
leLeuHisVaiAlaVa
lAspVaiIleArgGl
uSer
1440 480
rqGiuZ4.tArqLeuGlnProPheAsnGluTyrArqLyMrgPheGlyM.tLysProtyrthrSerph.GInGIuL.u
lePheGlyG1uS.r
1560 520 1680 560 1800
A1aThrLeuLysLysLeuVa1CysLeuAsnThrtysThrCysProTyrVa
lSerPheArgVa
1ProAsp1aSerG1nAspAspG1yProA1aVa1G1uArgProSerThrG1uL.uZnd
599
1920 2040 2160 2280 2400 2520 crrTCrCATGAAGCmA’rAAAATTCGCCC
2549
Figure 2. Composite nucleotide sequence of human platelet and erythroleukemia cell PGG/H synthase eDNA and the predicted amino acid sequence. Nucleotides and amino acids are numbered beginning with the ATG initiator codon. Nucleotides to the 5’ side are designated by negative numbers. The genomic sequence are at nucleotide the GenBank database (accession
Regulation
of
consensus polyadenylation position 36 (G/Phe), 338 number M59979).
COX expression
in HEL
signal is underlined. Differences between (T/Leu), 393 (T), and 1133 (C/Thr). This
cells
The effects of PDGF, phorbol ester, and thrombin on steadystate COX mRNA levels in cultured HEL cells were investigated. PDGF induced an approximate 3- to 3.5-fold increase in COX mRNA, reaching a maximum approximately 4 h after addition, as assessed by quantitative PCR (Fig. GA). At 8 h, the levels had decreased, and by 24 h levels had returned to basal conditions. In contrast, PMA induced a sustained increase in COX mRNA (two- to threefold at 24 h by PCR analysis and even greater at 48 h by Northern analysis (Fig. 6A and Fig. 6B). Thrombin, a potent platelet activator, did not markedly affect COX mRNA levels (not shown).
HUMAN
PLATELET CYCLOOXYGENASE
this sequence and the human sequence has been deposited in
DISCUSSION Platelet PGG/H synthase (cyclooxygenase) is the key cellular target for aspirin in the treatment of cardiovascular disease. In the present study we cloned both the human platelet and human erythroleukemia cell PGG/H synthase cDNAs, confirmed their identity within the coding region, and expressed the human enzyme in COS cells. The platelet/HEL PGG/H synthase primary sequence is 91% identical to the ovine vesicular gland sequence (9, 10), 89% identical to the mouse 3T3 fibroblast enzyme (11), and virtually identical to the predicted coding sequence of a human genomic sequence (12) (4 nucleotide differences/3 amino acid changes; Fig. 2
2307
pCDNA-COX
translocation between chromosomes 9 and 22 often occurs in pluripotent stem cells (including megakaryocytic cell types) of human chronic myelogenous leukemia patients (23). However, we were unable to detect any abnormalities in COX mRNA expression by our RT-PCR analysis (not shown) in preliminary experiments. Urinary excretion of the cyclooxygenase products, thromboxane A2 and prostacyclmn, in patients with chronic myelogenous leukemia had previously been found to be unaltered (I. A. G. Reilly, and G. A. FitzGerald, unpublished results). The human platelet/HEL COX 3’ untranslated sequence reported here is remarkably well conserved across species boundaries (70% identical to ovine sequence, 47% identical to murine sequence, and 65% identical to the human embryonic lung eDNA sequence; Fig. 7). Studies from other genes with highly conserved 3’ untranslated regions indicate that this region is important for the posttranscriptional regulation of gene expression (19). We expressed COX cDNAs both with the complete 3’ untranslated sequence (751 bp) and with only 18 bp, yet were unable to detect any major differences in expression. However, it is possible that the COX 3’ untranslated region participates in a translational
-2000
ii P60 PGF
boil ed
cpm
JL
-o r4000 cpm
10 -3200
regulatory
cpm
1 ndomethocin
role
in other
cell
types
that
possess
specific
regula-
tory proteins. Because COX is expressed only at very low levels in COS cells, it might be expected that these cells do not express potential COX translational control proteins. DeWitt and Smith (11, 20) have reported extensively on the aspirin-sensitive serine residue at position 530 of ovine vesicular gland COX (position 529 in the human platelet/HEL sequence) by mutational analysis. Changing this residue to an alanine (11) had no effect on COX activity whereas introduction of an asparagine (20) virtually abolished activity.
#{149}0
1L ________________ L
pCDNA-COX 1 (Asn529)
1 2 3 4 5 6 7 8 9 10
cpm
3200
;
#{232} 12
1018-
16
Dlst#{248}nce (cm)
Figure 3. Thin layer chromatograms of cyclooxygenase products from pcDNA-COX1 and peDNA-COXi (Asn529) transfeeted COS-M6 cells. Microsomal fractions from sonicated cells were assayed for cyclooxygenase activity as described in Methods. The migration
of prostaglandin
standard
compounds
ings were from one representative experiment. obtained from two additional experiments.
is indicated.
Similar
Vol. 5
June 1991
394-
Trac-
results were
legend) and a partial length sequence from human umbilical vein endothelial cells (seven amino acid changes) (20). Southern blot analysis using PL-COX3 (Fig. 5), which contains the highly conserved sequences surrounding the aspirin-sensitive serine residue as a probe, indicates a single COX gene we have assigned to chromosome 9 by PCR mapping of a panel of human/hamster hybrid DNAs. Although this observation suggests the presence of a single COX gene, a human embryonic lung cell eDNA that displays more than 90 differences at the amino acid level has been reported recently (21). Dissociation between COX enzyme activity and mRNA induction in PDGF-stimulated NIH-3T3 cells raises the possibility of other COX-related genes (22). A reciprocal
2308
516/506
Figure 4. Chromosomal localization of the human COX gene. Human/hamster somatic cell hybrid DNA samples were subjected to PCR analysis using two gene specific oligonucleotides. The products from the reaction were electrophoresed in a 3% NuSieve/i% SeaKem agarose gel and stained with ethidium bromide. Lanes: 1, 10, 1 kb ladder standard (BRL); 2, human genomic DNA (positive control); 3,6,7, positive hybrid clones 732, 862, and 968, respectively; 4,5,8, three negative hybrid clones 803, 750, and 423, respectively (an additional 19 negative samples are not shown); 9, no DNA template (negative control). Analysis of the samples indicates the presence of the COX gene on chromosome 9.
The FASEB Journal
FUNK
ET AL.
I-I
z
0
1 I-I
Qr1 (I)
Origin
U H
HI
I-I
t5
D
I-I
0
fi
U
(0
Ed
20
-J
LU
HI
15 (0 In
0
U)
C.) Q)
5
0
E >< 00
C)
0
4
8
12
16
20
24
Time (h)
B 0 +
26S
cox
las Figure 5. Southern blot analysis of human genomic DNA. Restriction enzyme-digested DNA (20 jag) was electrophoresed in a 0.7% agarose gel, transferred to nylon membrane, and hybridized with f”P]PL-COX3. After washing, the membrane was subjected to autoradiography for 4 days at -70#{176}C. The small hybridizationpositive bands in the MspI lane are somewhat diffuse owing to the depurination step prior to transfer.
28S
The present data indicate that the human platelet/HEL COX probably behaves similarly. Thus, substitution of a bulky functional group likely reduces the ability of 20:4 to bind properly at the active site. A single 2.9-kb mRNA was observed in PMA-
las-
stimulated
HEL
cells.
However,
occasionally
we
detected
a
band at 5 kb (slightly above the ribosomal 28S subunit). mRNA of these two sizes was also seen in platelet and placenta samples, as examined by Northern blot analysis (24), and could represent splice variants. Bailey et al. (25) reported the presence of 3.1- and 2.8-kb COX mRNA transcripts in human lung fibroblasts. Holtzmann and colleagues (26) observed the presence of a 4.0-kb mRNA, in addition to the 2.8-kb COX mRNA, in sheep airway epithelial cells by using Northern analysis under low stringency wash conditions but not under high stringency. Further research will be
HUMAN
PLATELET CYCLOOXYGENASE
actin
Figure 6. A) The effect of PDGF and phorbol ester on the steadystate COX mRNA levels in HEL cells. mRNA levels were measured by quantitative PCR. B) Northern blot analysis of HEL RNA before and after 48 h phorbol ester treatment (160 nM). Human poly(A)4 RNA (0.5 jag) was electrophoresed in a 1% agarose gel containing 0.22 M formaldehyde, blotted Onto nitrocellulose, and hybridized with the “P-labeled PL-COX3 clone. The blot was stripped and reprobed with a rat fl-actin probe to verify approximate equivalence of sample loading and blot transfer.
2309
hPL hLU oVG 3T
TGAG GGGCaGGAAACCACCATrCTCGA GGGgAGAGCTTTCT TGAg GGGC CCGgA&agCtATTCTCGTCGeTAGAGCT CT TCAa.;GGGCtGGGCAGCAGCAT1CTGGLT GCTAG&GCTTCCT TCACgGaGCtGGAAAGCACC c1CTGCA GGGagGAGtTTrCTtcctgatgaag*caatccttgacgcgggc:ttcgcgQC
G. TTCtC ATTCCACAGtGCTCA ggCcaGCC GC;TTGCCATTCC.GA ATGgCCcCGCCCTG CC TTCCC ATTCCCA ATG CCaCCGGGTG TTG4C Afl gtGACaGCTCAtgctcacatttgaaac
cow
TCAgGgGCtgG-aAcegcatTCTCGAtgGgteGAGcTtt-T--
GC-TTGcC-ATTccaGA-gc
hPL hLU
T5aTGGTCTTA CACTTGTCTT GA TTCTCTT
tga-a
72 70 70 115
cg-cc-cggggc-
T I
83 81 80 235
CON
-a-t-GTCTTa-
hPL hLU oVG 3T
aATGCTCA tTrrerc GrrTcCcaTgGtGAGtTtgCGCTTOAC&TTTAGAAcT tCUgTCTCACCC ATtaTCTCGALTA TTCTG AttCTCTTr aTTcTTCcAG GATGTT GGGTTTCTGcATTTCCTGTC AGAG tcatGTGTCGACGTTTA AACTCT gctaCCATC AAT&CtTCTGlCCTTCTTrCgtTcCTTgaAG GATGTr GGG1TrCTC ATTTCGTGTCGAGAGcaTcaGTGTCCACGTTTAGAACTCTacc rCTCACCCCATGTCTGGAATAc TCTCTTCCTTG1TC TTGTTCtAG gcaaCtCAgaaagccagaflTClC GIl GaTrtggAaIataggCItaaaAccrtatAttatAgggIagGGtTggTTCcacAcaCctTaatcccagcaCttG
186 175 187 336
CON
gatg-Ica
hPL hUi oVG i3T
AATGCTCAACTCCIlCTtARCCCT TCAGATTGTrAGGAG TGCIl CtCArrtGGTcTGCCACAAtACTCCGTTC TTaGCTCACaACCTACAATGTCACATTrCTgGTTGATttG aATG ecAaaCgctGTAAACCkTaTGAGAA GSgtGGAGC*TGGTTATCC c caaccGCCAG cAgcGGGTTCCacgGaTGACCAC TAGCATGTCA t rggrtcTc tc rcc t AATGCTCAACTCCT5CTAAACCAT TGAGAATGTTACGAG TGGTTATCCCtTcaGcaCTCCCAGAAcACTGGGTTCC TGCgTGACCACCTAGAATGTCACATTTCTaGTTGATCCG
oVC m3t
:99 287 302
CON lIP!. lILU
oVG
-
AAc.8CAGtCATTCTAggAIgTCC AGCTACTGATGAAATCtgCTaGAAAGTrA GCAfl1AGtCACTCTGAA TATCGctaA CECCT ATGGAATCAT ttAcgGTGA GAAtTrAG5CACTCTCAAATATCG A CTCCTGATCGAATCATCTgGAAAGTCA
i3T
GGGGG GGCGG GGGGG
TTCTT ATTTTGCATrC CAGAATc ITT AT1TrGCA TCTAGAATTCTCGGTGGC tCAGAATG TITTI ATTITCCATTCTAGAATTCTCGCTCGCCCtCCAGAATG tTTCTTcctagaITT ggeggccccttCAGAAlg
CON
gaAt-Agtca-rc
lIP!. lILU oVG uu3T
TCGACTTC GACTGGTTATCCCGAATGTTCTG CTCCCAGTTGC agICCACAACAGTGGtTCGCAT c CaATCAGT CTGATCCCAATGTCTAGA GTGTgccAGAATTCATr cCC TCCACrITCTCACTGGTTATCCGGAATGTTGTG CTCCGAGTTGCTGATCCAGAACAGTGGCTGGCATtCtAgATCAGTCCTCATCCGAATGTCTAGA GTGT CgAGAATTCAITrTCC rrCACTaTCTGAcaGGTGAcTCAgA.agGIccIGTTCC TGGtcSATGATCCACAACA TgggCCAAAaCaCTCccaAcCTGaaTGTCTAGAATGTGGAaITGgTTCATTTTCC
CON
CCC
hPL hL.U oVG n3T
t-uAIetgg-t-agct-ctgAtg-AAtcatcttgAa.ICT-A-5GCGG-
TT-t t-ATTTtcattctagaettctgggtggc-ct
380 380 402 560
cCAGAATg
TGTTCAGTGAGA TaCCACaGAGACCGAG ACCCTAagGTCCAACAAGAATGCATT cCCTGAATCTCTGCCTGCAcCGAGAGGGCAAGGAATGGGGTGTTC TGTTCAGTGccAgtcCacCGGAGCAGGAGGATCTCGTaTCCTACAAGAtacCATTgGCCTGGATCTG CCTGC TGGAGAGGGC CaaXCGTGGGCTGTTC TCTTCAGTGAGAcaCCACGCAGCAGGAGGA TCTCCTGTCCTACAAGAAcGCATT CCCTCGATCTGTCCCTCCATCGAGAGGGCAAGGAAGTGCGGTGTTCstc TGTTCAGTGA AaTggacecaiaACa&A&gMCCcAgtGTCCAgCLAGAATtccI CgCccAMCctacgtcCacgccMAGGCCAAGGeAGTaaGGTG
TTCT TGGCACC cc sgCAGTGGGgaC CTCTCAGTGGGACC TTCT TCGGAgC
500 492 519 671
611 606 637 780
CON lIP!. lILU oVG a3T
CCCACTaACACCCTGCTCCgAgGATCTaGAGAGAACAGGT GCCcTgtATtcAcGCCLTTCGTTGGAAGC CACCACAGCT CTATCCCCATCCAGGTCTTgACTCATGG CAGCTCTIT CCTG TCAGACCCTG agZggaGAGAGAACAGGTcGGCTTT TCCAcGatATTGGtGCAAGCCgAccAGAGCTasrCTCCTATCCAGGTCCcACTCACGGtCAGCTC1Tr CCTCaTgAGACCCTGG ATaTgG&GAGAACAGGT GCCITrcTCCAG GCCATTGGTTGGAAGCC ACCAGAGCT CTGTCCTCATCCAGGTCTcaACTCACGG CAGCTGTTI CaCACTCAGAC tcttECcaMGATCTgCAGgGAACAG aCGGactcATct.ACGacTTGGIlGGAAaC cACCAcAGCT CIATCCCCATCCAGaTCTTC gCTCgIGG CAGCTGTTT
CON
Cc--e
hPL lILU oVG 531
CTCATG AAGCT AATAAAATTCgccc I CATGcAAGTTccCTAAAATGgTaTTCC*aa* TTCATG AAGTT AATAAAATGCTtTTCCcg CTCATG AAGCT AATAAAATTC
CON
-
rtAGACccCggcc-e-.!eIgt-GAGeGAACAG#{128}t-gG-t
ttc-ca-cG--aTTCGTTGCAAgCc-ACCAgAGcT-cT-TCC-cATCCAGgTCP
727
718 894
C .aCtca-GG-CAGCTCTTT
754 750 776 915
CCATG-AAG-T-aaTh.A.AAT-Ct-tt-
Figure
7. Alignment of the highly conserved 3’ untranslated regions from four prostaglandin G/H synthase sequences. Sequences were aligned and a consensus sequence was generated using the GENALIGN program of Intelligenetics software. Sequences start with the ‘ItA termination codon. hPL, human platelet/erythroleukemia sequence (Fig. 2); hLU, human embryonic lung sequence (ref 21); oVG, ovine vesicular gland sequence (ref 9); m3T, murine Swiss 3T3 fibroblast sequence (ref 20); CON, consensus sequence. For CON, capital letters represent nucleotides found in all four sequences, small letters represent nucleotides found in two or three sequences, and dashes represent regions where no clear consensus is evident. required to elucidate the significance and mechanisms for generation of heterogeneity of COX mRNA transcripts. The COX transcript in HEL cells detected by PCR was present at relatively low levels (estimated five copies/cell) under basal conditions and appears to increase with cell culture age (unpublished results), as demonstrated recently in human umbilical vein endothelial cells (27). An exceedingly complex picture on the regulation of COX expression is emerging. Interleukin-1 (28, 29), PDGF (this study and ref 22), lipopolysaccharide (30), serum (31), EGF (32), phorbol esters (this study and ref 33), oncogenic transformation (34), and progesterone (35) have been reported to induce either transient or persistent COX expression in several different cell types. The induction can occur at the transcriptional, translational, and possibly posttranslational
2310
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June 1991
levels (or combinations) the vestigial quantities surprising regulation.
if it were However,
depending of mRNA subject regulation
to
on the stimulus. Given in platelets, it would be physiologically of megakaryocyte
important COX ac-
tivity might be of critical relevance to the rate of recovery of thromboxane biosynthesis as new platelets emerge into the circulation after ingestion of aspirin. In this regard, Pash and Bailey (32) have demonstrated that EGF accelerated the recovery of COX activity in vascular smooth muscle cells after their exposure to aspirin. In the present studies, using HEL cells as a model for megakaryocytes we demonstrated that PDGF stimulates an increase in steady-state COX mRNA levels. Message levels were differentially regulated by the phorbol ester, PMA, which has been shown to enhance formation of arachidonic acid metabolites in other cellular
The FASEB Journal
FUNK ET AL.
systems
(33).
Thrombin,
a potent
platelet
agonist,
did
not
appear to regulate COX mRNA levels although HEL cells have been shown to possess thrombin receptors (36). In summary, we have cloned and expressed eDNA for the cellular enzyme that is the target for aspirin when this drug is used in the treatment of cardiovascular disease. Further studies
zyme lated. The
should
and
authors
and Alan stimulating National G.A.F. apeutics.
characterize
the
the mechanisms
wish
Brash
functional
by which
to thank
Lawrence
domains
of this
its expression
J. Marnett
for help with the cyclooxygenase
and colleagues, assay and for
discussion. This work was supported by grants Institutes of Health (HL30400) and Daiiehi is the
William
Stokes
Professor
of
en-
is regu-
from the Seiyaku.
Experimental
Ther-
C. D., Rdmark, 0., Fu, J. Y., Matsumoto, T., Jornvall, H., Shimizu, T., and Samuelsson, B. (1987) Molecular cloning and amino acid sequence of leukotriene A4 hydrolase. Proc. Nail. Acad. Sci. USA 84, 6677-6681
15. Funk,
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of
the
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RNA isolation phenolehloroform
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Res. 3, 8-16 26. Rosen,
J.
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T. M.,
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Received for publication January Accepted for publication
The FASEB Journal
April
FUNK
21, 1991. 9, 1991.
ET AL.
