COX-3, a Cyclooxygenase-1 Variant Inhibited by Acetaminophen and Other

Analgesic/Antipyretic Drugs: Cloning, Structure, and Expression

Author(s): N. V. Chandrasekharan, Hu Dai, K. Lamar Turepu Roos, Nathan K. Evanson,
Joshua Tomsik, Terry S. Elton and Daniel L. Simmons
Source: Proceedings of the National Academy of Sciences of the United States of America,
Vol. 99, No. 21 (Oct. 15, 2002), pp. 13926-13931
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3073509
Accessed: 17-09-2016 06:02 UTC

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 COX-3, a cyclooxygenase-1 v; 3riant inhibited by
acetaminophen and other ani algesic/antipyretic
drugs: Cloning, structure, an( I expression
N. V. Chandrasekharan, Hu Dai, K. Lamar Turepu Roos, Nathan K Evanson, Joshua Tomsik, Terry S. Elton,
and Daniel L. Simmons*

Department of Chemistry and Biochemistry, E280 Benson Science Building,

YoungBrigham
University, Provo, UT 84602

Communicated by John Vane, William Harvey Foundation, London, United Kingdon

i, August 5, 2002 (received for review April 17, 2002)

Two cyclooxygenase isozymes, COX-1 and -2, are known toiterials

cata-and
Mi Methods
lyze the rate-limiting step of prostaglandin synthesis and are
ilessthe Ur
otherwise stated all basic protocols used were from the
targets of nonsteroidal antiinflammatory drugs. Here we describe
nual on mEmolecular cloning by Sambrook and Russell (3).
a third distinct COX isozyme, COX-3, as well as two smaller
COX-1-derived proteins (partial COX-1 or PCOX-1 proteins).lation
COX-3 Isa and Construction of a cDNA Library. Isolation of
of RNA
and one of the PCOX-1 proteins (PCOX-la) are made fromIAthe andRI
library construction methods have been described (4).
COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins HL
Iman Multiple Tissue Northern blots (MTN) were purchased
additionally contain an in-frame deletion of exons 5-8 ofmthe fr
CLONTECH.
COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine antisense oligonucleotides to the first intron of huma
cerebral cortex and in lesser amounts in other tissues analyzed.
line In cai
COX-1 genes were synthesized and end-labeled u
human, COX-3 mRNA is expressed as an -5.2-kb transcript 32P]dATP.
and is [r_
most abundant in cerebral cortex and heart. Intron 1 is conserved
X canine cerebral cortex cDNA library was screened us
in length and in sequence in mammalian COX-1 genes. This intron
.0-kb ]canine COX-1 fragment previously cloned i
contains an ORF that introduces an insertion of 30-34 aa, depend-
oratorylat by reverse transcription-coupled (RT)-PCR.
ing on the mammalian species, into the hydrophobic signal peptide lib
rary was also screened with a 32P-labeled canine COX-1
that directs COX-1 into the lumen of the endoplasmic reticulum and 1 a
ntisense oligonucleotide. Two full-length clones were iso
nuclear envelope. COX-3 and PCOX-la are expressed efficiently in
npletely sequenced, and designated COX-3 and pa
insect cells as membrane-bound proteins. The signal peptide )X-la
is not (PCOX-la).
C( Both were derived from the canine C
cleaved from either protein and both proteins are glycosylated.
ie but retain intron 1. PCOX-la also has a 657-bp in-f
COX-3, but not PCOX-la, possesses glycosylation-dependentetion cy- despanning exons 5-8.
clooxygenase activity. Comparison of canine COX-3 activity with
murine COX-1 and -2 demonstrates that this enzyme is selectively
PCR ofRTCanine and Human Cerebral Cortex mRNA. Canine cerebral
inhibited by analgesic/antipyretic drugs such as acetaminophen, co
?tex cDNA was synthesized, and primers were designed for
phenacetin, antipyrine, and dipyrone, and is potently inhibited
'Rby pC
amplification. The sense primer (5'-CGGATCCGCCGC-
some nonsteroidal antiinflammatory drugs. Thus, inhibition of C
;AGAGCTATGAG-3') corresponded to nucleotides 15-32 of
COX-3 could represent a primary central mechanism by which these ca
line COX-3 sequence (submitted to GenBank under acces-
drugs decrease pain and possibly fever. si n no. AF535138), with the 3' end of the primer being two
nu cleotides downstream of the initiating methionine. The anti-
A cetaminophen is often categorized as a nonsteroidalise
anti- sei (5 '-cgccatcctggtgggggtcaggcacacgga-3') corre-
primer
inflammatory drug (NSAID), even though in clinical )nded
prac-tosp(
nucleotides 1865-1894, located 32 nucleotides up-
tice and in animal models it possesses little antiinflammatory
eam ofstr
the stop codon.
activity (1). Like NSAIDs, however, acetaminophen inhibits
Northern blot analysis of human tissues with an intron 1 probe
pain and fever and is one of the world's most popular analgesic/
:ected ande=5.2-kb mRNA similar in size to one previously
antipyretic drugs. Despite acetaminophen's long use and popu- rel Marathon-ready human cerebral cortex cDNA
iorted (5).
larity it lacks a clear mechanism of action. Flower and Vane (C was amplified by PCR (CLONTECH, Advan-
LONTECH)
showed that acetaminophen inhibited cyclooxygenase ;e (COX)
2 PCRtal
enzyme system), using 5' and 3' primers, and an
activity in dog brain homogenates more than in homogenates ;
.2-kb amplified fragment was recovered and found to contain
from spleen (2). This gave rise to the concept that variants of the
, entire coding region of human COX-1 with intron 1 retained.
COX enzymes exist that are differentially sensitive to this drug
and that acetaminophen acts centrally. Yet, even though iression
two ExI of COX-3 and PCOX-la in Baculovirus. Both COX-3 and
isozymes of COX are known, neither isozyme is sensitive OX-lato p( cloned into the baculovirus expression vector
were
acetaminophen at therapeutic concentrations of the drug in 4.5/V5-His
lueBac pB (Invitrogen). Sf9 cells (=1 x 106) were
whole cells or homogenates. Instead, COX-1 and -2 in homog- inf viral stocks at a multiplicity of infection (moi) of
ected with
enates frequently exhibit the paradoxical property of)rbeing 3f of COX-3, PCOX-la, mouse COX-1, and mouse
expression
stimulated by submillimolar concentrations of acetaminophen
)X-2 (6).C(
and inhibited by supermillimolar levels of the drug (1). This
In some cases, tunicamycin was added to a final concentration
finding suggests that neither isozyme is a good candidate for the
site of action of acetaminophen.
In analyzing COX-1 and -2 RNA expression in dog tissues, our COX,
reviations: Abt cyclooxygenase; NSAID, nonsteroidal antiinflammatory drug; PCOX-1,
laboratory observed that the cerebral cortex of dog brain par ial COX-1; RT, reverse transcription-coupled.
contains two distinct RNAs that hybridized to a canine COX-1 deposition:
Dat The sequences reported in this paper have been deposited in the GenBank
cDNA. One RNA was =2.6 kb in size and the other was = 1.9 kb dat ibase (accession nos. AF535138 and AF535139).
in size, and analyses of these RNAs suggest that they encode see commentary on page 13371.
previously uncharacterized COX-i-related proteins. *To
whom correspondence should be addressed. E-mail: dan-simmons@byu.edu.

13926-13931 I PNAS I October 15,2002 I vol. 99 I no. 21 www.pnas.org/cgi/doi/10.1073/pnas.162468699

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of 10 /ag/ml to insect cells 1 h after infection, and cells were A
cultured and harvested after 48 h. Activity of intact cells was i 1 2 B 2 3
determined by RIA (7).
IS .'1Kb
Detection of 60-, 53-, and 50-kDa COX-1-Related Proteins in Human ~~~~~~18S k~1.2
Aorta Tissue. Total protein (20 jag) from human aorta was
analyzed by Western blotting, using COX-1 mAb (Cayman
Chemical, Ann Arbor, MI) and COX-3 antipeptide polyclonal
antibodies (pAb). Primary antibodies were either preincubated
with a mixture of human and mouse COX-1 intron 1 peptide
Kb Kb 1 2
C Cc Md Sc Op FI T1 Pu Am Cn Co Hi B Th
(described below) for 1 h at 4?C, or left unblocked. Blots were 7.5

processed with appropriate rabbit-anti-mouse secondary anti-

body (1:2,000) or goat-anti-rabbit secondary antibody (1:10,000) 1.4
from Sigma. Densitometry of the autoradiographic image was
performed using the AlphaImage 2000 Documentation and 2:4 "
Analysis System (Alpha Innotech, San Leandro, CA). Ht B P Lu Li M K Pn B Lu Li K
7.5

Drug Inhibition Assays. Sf9 cells were infected with high titer viral
stocks (moi = 3) and cultured for 48 h. Cells were preincubated
with drug for 30 min at 25?C, arachidonic acid (100 al, final 1.4 '
concentration 5 or 30 ILM) was then added for an additional
3 4
10-min incubation at 37?C. Supernatant was assayed for COX
activity by RIA for PGE2. Assays were performed multiple times Fig 1. North
in triplicate. Inhibition curves were constructed and IC50 values cer ebral cort
were determined using PRISM 3.0 (GraphPad, San Diego). car line COX-
cle otide to in
Production of Polyclonal Anti-COX-3 Antibodies. Peptides corre- Lai ie 1, ethid
)X-la cont
sponding to the first 13 aa of human (MSRECDPGARWGC) PC
ron 1; lan
and mouse (MSREFDPEAPRNC) COX-3, as predicted by int ise oligon
genomic clone sequences, were synthesized and coupled to ser )be. (C) Hu
keyhole limpet hemocyanin. Peptides (a 50:50 mixture) were lab eled huma
injected into New Zealand White rabbits. The resulting poly- wa s detected
clonal antibodies were then affinity purified using the above B, )rain; C, c
peptides immobilized on a Sulfolink coupling gel (Pierce) ac- Ht, heart; K,
cording to the manufacturer's instructions, cat idate nucl
spi nal cord;
Results

Two distinct mRNA species (=2.6 and -1.9 kb) were detected
on a Northern blot with a canine COX-1 coding region cDNA ls simpler,
)NA clo
probe using RNA isolated from canine cerebral cortex (Fig. 1A, CT
lane 1). To further investigate these transcripts, a canine cerebral CC rrespon
cortex cDNA library was constructed and the nonamplified na ted par
library was screened as described above. Eleven clones were RT-PCR
isolated and subsequently characterized by automated DNA Ni )rthern
sequencing. All of the eleven clones were found to contain br ain regi
canine COX-1 cDNA sequence. However, three clones harbored an d data n
an insertion of 90 nucleotides at, or near, the 5' end of their sti ated tha
respective cDNAs, which showed 75% sequence identity to
intron 1 of either human or mouse COX-1 genes (data not
shown). This extra sequence also contained 5' and 3' consensus
dl m d2 c ....
splice sites indicative of a retained intron. In addition to the
retention of intron 1, one of the three clones had a 657-bp
COX-1 1 1 I
in-frame deletion corresponding to exons 5-8 of the COX-1
message (sequence submitted to GenBank under accession no. i s
AF535139).
To determine whether the two detected COX mRNA tran- COX-3
scripts (i.e., =2.6 and -1.9 kb) harbored intron 1, the Northern
blot experiment was repeated using a radiolabeled antisense
canine COX-1 intron 1-specific oligonucleotide probe (Fig. 1A,
lane 2). Both the l1.9-kb and the =2.6-kb transcripts were PCOX-la 1 Deleted
hz[,, I..
detected, suggesting that multiple intron-l-containing splice
variants were indeed expressed in canine cerebral cortex. The aa 119-337

COX encoded by the =2.6-kb cDNA clone with nonspliced Fic

. 2. Structure of COX-3 and PCOX-1a. A schematic representation of th
intron 1 has been designated as COX-3. We have named thisofdCOX-3 and PCOX-1 in comparison to COX-1. s, signal peptide; dl
mains
newly discovered enzyme COX-3, even though it derives from di, domain/EGF-like domain 1; d2, dimerization domain 2; m, mem-
nerization
the same gene as COX-1, because experience has shown thatmethe br domain; c, catalytic domain; i, 90-bp sequence encoded by
binding
important differences between COX-1 and -2 are pharmacolog- inl
ron 1. PCOX-lb is identical to PCOX-1 a except that PCOX-1 b lacks intron 1
ical rather than genetic. Furthermore a numerical nomenclature Ar
lino acid numbering is according to residues in sheep seminal vesicle COX-1

Chandrasekharan et a/. PNAS I October 15, 2002 I vol. 99 I no. 21 I 13927

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 COX-3 PCOX-la Mouse COX-1 1 2 3 4 5 6 7 8
COX-I MAb - - ++ - -
Tu - + - + - + COX-3 PAb + + - - + +
COX-1 intron 1 Peptide - - + - +
RAM secondary Ab - - + +
GAR secondary Ab + + + +

6, - + -
16 + -.+ ,0, 47 kDa

l12__l
4

6 S 60 0 k

40 40
Fig. 3. Expression in insect cells. Western blots showi
COX-3, PCOX-la, and COX-1 in 20 insect
20~ cells
n/dtreated wit
tunicamycin (Upper). Arrows indicate glycosylated form
65 kDa 53 kDa 50 kDa 65 kDa 53 kDa 50 kDa
not present in cells treated with tunicamycin. Polyclonal
and mouse COX-1 intron 1 COX-Isequence were
MAb blocked used
with Peptide to PA
COX-3 p
PCOX-la blots, whereas a monoclonal antibody to ovi
. 4.the
Chemical) was used to probe Western blot of
mouse human aorta
COX-1 blot.lysate
CO
expressing COX-3, tibodies.
PCOX-la, and (A) Western blot
COX-1 (lanes 3-8, Cells
(Lower). 20 JLg
and without (-) tunicamycin. prc )bed with primary, secondary, or blocked a
ho rizontal arrow indicates the 65-kDa protein
53. kDa proteins, and an upward diagonal soli
)tein. A single asterisk denotes unglycosylat
actually a mixture of two mRNAs that differed in size by =90 prc ast erisk denotes unglycosylated canine PCOX-l
nucleotides (Fig. 1B). One of these mRNAs was PCOX-la and 65t ,53-, and 50-kDa proteins. Percent relativ
the other (PCOX-lb) was identical to PCOX-la except that it we re calculated by comparison to the signal fr
lacked intron 1. PCOX-la and -lb are expressed in equal bo dies. The 50-kDa protein is not detected (n
amounts in brain cortex (Fig. 1B). cc X-3 polyclonal antibody (pAb).
To determine whether previously uncharacterized COX-1-
related mRNA transcripts were also expressed in human tissues,
human Northern blot experiments were :OX-la completely
performed lacked detecta
using a P
,wer).
human intron-l-specific (HCI) probe. These COX demon-
results activityLCin cells treated
olished, indicating
strated the existence of previously uncharacterized =5.2- and abthat N-linked glyc
=2.8-kb mRNA transcripts (Fig. 1C). Faint)X activity of
hybridization COX-3.
signals C(
RNA
were also seen around 1.9 kb (data not shown). studies in
Hybridization ofhuman tissues indi
)X-3
HCI to the =5.2-kb form was tissue-specific, message
with highest to be C?
levels in the cerebral c
present in the cerebral cortex, followed )tby
analysis of These
the heart. human bl' aorta (Fig. 4)
observations differ from the characterized)noclonal
expression antibody
patterns oform' COX-3 antipep
COX-1 mRNA (8). de tected the presence of distinct 65-
COX enzymes are intralumenal oteins. Additionally,
residents of the the
COX-1endbu
reticulum and depend ontected a 69-kDa
N-linked protein, corresp
glycosylation f
)X-1, as of
folding and activity. Retention well as a 50-kDa
intron protein, w
1 theoretic
prevent COX-3 and PCOX-la 3teolytic fragment
expression byof preventin
COX-1 or PC
the 65- and
of these mRNAs from the nucleus or 53-kDa proteins was
by targeting the
eincubation of the
to another subcellular compartment, antipeptide glyc
preventing sera
Lereas
Therefore, insect cells (Sf9) weredetection of the
infected same recom
with protei
baculovirus expressing COX-3, PCOX-la,
nal antibody and COX-1
was unaffected by this
homogenates were assayed Common analgesic/antipyretic
for protein expression by drug
W
blotting. Antibodies specific
r their for
abilitythe conserved
to inhibit am
activity of C
sequence (MSREXDPXA) predicted
re done in the topresence
be encoded
of exogenby
in mammals were used to id probe
at 30- for COX-3
and 5-/M and PCOX
concentrations.
analysis demonstrated that both COX-3
n of substrate, and
only COX-3 PCO
was inh
efficiently expressed in ig.insect cells. No
5A). Moreover, COX-3detectabl
was signif
resulting from removal of intron 1 than
etaminophen by splicing
either COX-1wereor
immunologically or by RT-PCR
ncentration
analysis
(Fig. 5B).
of RNAAcetaminoph
extra
infected Sf9 cells. Moreover,
IC50the signal
value of 64 peptide,
/M when donewhich in
achidonic acid,
and PCOX-la contains an additional whereas1IC50
intron values f
encoded
was not removed by signal d peptidase as it
92.4-fold higher, is in COX-
respectively.
Posttranslational N-linked glycosylation
Acetaminophen of COX-
is considered to be
PCOX-la was compared with that
enacetin, of popular
a once COX-1 by usin
analgesic/an
mycin to inhibit core glycosylation.
iger extensively Immunoblot
used because of ana the
onstrated a decrease in or )binemia, renal toxicity, and suspected
disappearance of renalglycosyla
and bladder
of COX-3, PCOX-la, and COX-1
rcinogenesis (9,(Fig. 3 isUpper;
10). Phenacetin Left,
rapidly O-deethylated in the C
Right, respectively). Expression systems
dy to form acetaminophen assayed
and is further for
metabolized to other
duction found COX-3 to nor be =20%
but toxic compounds. ofThus,that ofof COX-1
only small levels phenac-

13928 | www.pnas.org/cgi/doi/10.1073/pnas. 162468699

Chandrasekharan et al.

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 A B
160 120

100

80 - ' ......

Cicoo ID 40- 1 | t | ^-X.^--

40 EZ EZFic

4 ..........X-........ 1000
20 -0
- COX.3
-1 460
2 3 4
0I

Log[AcM for acetaminophen tested under sitamilar conditiophon]s. As with pM Log [Acetaminophen] ILM

C D
120-
120 -

100 .......... ........ > 100 ..-

5 80 80

60 ..... 60 -

S 40o- + ? 40 - COX- 1 350

COX- 2 >1000
inhibition of COX-2 by dipyrOX-ne was observed below 1 mM. 000
20- COX. 3 102 20 - coX- 3 52

0- ' ' ' ... I .......I . ... ' ' .. . ......

-2 -1 0 1 2 3 4 -1 0 1 2 3 4
etin circulate in the blood. Interestingly, however, phenacetin 1'

Log[Phenacetin] iLM
Log[Dipyron

icantly more 5.
Fig. potent at inhibiting COX-3inhibition
Drug than either COX-1 or ac n studies.
(C), and The effect
dipyron
insect cells. COX ogenous
activity was 5 jLM by
measured (B)th
are expressed as mean ? SEM (n = 6-9).

uM and COX-1 at a 6.6-fold higher concentration. No detectable

etin circulate in the blood.

)revents Interes
transl
was more potent er,
at inhibiting
both COX
COX-
(Fig. 5C). Under Ils
substrate
retain condit
the i
inhibited COX-3 at an
nslatedIC50 value3)
(Fig. o
/iM for acetaminophen
"OX-la tested unde
include
acetaminophen,
acetaminophen, phenacetin preferentially inhibited COX-3. tic phenacetin
,nally prefe
different
Another analgesic/antipyretic
intron 1 dru
provid
icantly more potent at inhibiting
terized COX en
-2 (Fig. 5D). Dipyrone
sues.inhibited CO
Consistent
AMB and COX-1 at a 6.6-fold higher concentration. No detectable portant in cre
inhibition of COX-2 by dipyrone was observed below 1 mM.i ding that the D
Dipyrone is a pro-drug that spontaneously breaks down in M >use COX-1 gen
aqueous solutions to structurally related pyrazolone compounds
that differ in their potency as analgesic/antipyretic agents.
Antipyrine and dimethylaminopyrene are related to dipyrone, Ta ble 1. ICso value
and possess markedly reduced therapeutic potency and inhibi- aa d NSAIDs
tion of COX-3 (Table 1). However, these compounds, like other
IC50, p,M
analgesic/antipyretic agents, preferentially inhibit COX-3.
COX-3 was also found to differ in its sensitivity to inhibition Dr jg COX-1 COX-2 COX-3
by a selection of NSAIDs. Diclofenac was the most potent -
etaminophen > 1,000 >1,000 460
inhibitor of COX-3 tested and diclofenac, aspirin, and ibuprofen Ac
linopyrine* > 1,000 >1,000 688
preferentially inhibited COX-3 over COX-1 and -2. Thalidomide Ar
tipyrine > 1,000 >1,000 863
and caffeine, both of which have been described as having Ar
pirin 10 >1,000 3.1
analgesic properties, did not inhibit COX-3. The overall results As
:lofenac 0.035 0.041 0.008
indicate that COX-3 possesses COX activity that differs phar- D
)yrone 350 > 1,000 52
macologically from both COX-1 and -2, but is more similar to Dl
COX-1. lb iprofen 2.4 5.7 0.24
Inc
Ins lomethacin 0.010 0.66 0.016
Discussion Ph enacetin >1,000 >1,000 102
ffeine > 1,000 >1,000 > 1,000
Both COX-3 and
Th
PCOX-la ar
alidomide > 1,000 > 1,000 >1,000
poorly understood form of alternative splicing. We have previ-
ously shown that COX-2 in chicken is regulated by intron 1 All assays were carried out in the presence
retention (11). In the case of chicken COX-2, retention of intron *4 dimethylaminoantipyrine.

Chandrasekharan et al. PNAS | October 15, 2002 | vol. 99 | no. 21 | 13929

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fact intron 1 is more conserved in these species than is exon 1. (2z , 25), and some new COX-1-selective inhibitors, such as
COX-1 and -2 genes differ in the placement of intron 1. COX-1 SC -560, have proven ineffective at inhibiting LPS-induced fever
has ten introns, whereas COX-2 has nine. The additional intron in animal models (26, 27). Clinically, rofecoxib, a COX-2-
in the COX-1 gene is intron 1, which is retained in COX-3. sel ective inhibitor, inhibits naturally occurring fever (28) and
Highly conserved elements of intron 1 may also regulate intron als o inhibits the maintenance of fever in animal models. Yet
retention. as} firin, a COX-1 preferential inhibitor, is one of the most
COX-3 shares all
ective antipyretic NSAIDs, and inhibits of th
fever at doses ranging
structural features
m 5-15 mg/kg (29), far below the 60-80 mg/kgof C
used to treat
intron 1 two amino
lammatory disease acid
(30, 31). Furthermore, nimesulide, a
thionine would
)X-2 preferential inhibitor, was result
found to be antipyretic in dogs
peptide. Despite
[y at plasma concentrations having
that would also inhibit COX-1
sequence retained,
t). Thus, a role for COX-1 in fever may exist. CO
PAGE gels. It
rhe mechanism also appear
of action of acetaminophen has been un-
where it is own glycosylated
and postulated to be through inhibition of a brain COX
activity. In insect
it has never cell
been identified (1, 2). Northern blot analysis and
the activity of
NA cloning show thatCOX-1,
COX-3 is expressed in canine brain. w
activity of)X-3 also COX-2. CO
appears from Northern blot studies to be expressed
show equivalent
specific regions of the human express
brain, in particular cerebral
lowered ability of
rtex (Fig. 1C). Moreover, our studiesinsec
using ectopically ex-
COX-2 may -ssed be
COX-3 in insect due
cells demonstrate thattoCOX-3 is t
lationally process COX-
nificantly more sensitive to acetaminophen than COX-1 or -2.
COX-3 also e exhibits
steady-state concentrations of acetaminophen thiafter thera-
COX-1. Subcellular loc
utic dosage are approximately 100 tM, at which concentration
centrifugation
ly COX-3 is appreciablydemonst
inhibited (Fig. SB). Thus, inhibition
brane-bound (data not shown). of COX-3 in brain and the spinal cord may be the long
Retention of intron 1 could alter ight-after
folding and may
mechanism affect
of action so
of acetaminophen. This pro-
dimerization and the active site. Thesesed effects could be through
mechanism of action also appears to extend to pyrazolone
structural changes or altered protein igs such as dipyrone and COX-1
targeting. site-aminopyrine
related compounds d and
directed mutagenesis of either Cys-313 or -540,
tipyrine. Dipyrone'sboth of which
active breakdown an 4-methylami-
product,
are more than 25 A from the heme iron, was observed to reduce
antipyrine, reaches concentrations of 104 FM and 86 tM in
the activity of the enzyme by 80-90% (12). Therefore, although p,
isma and the central nervous system, respectively (33). Thus,
COX-3 contains all of the COX-1 sequence, the retained intron
)X-3 inhibition occurs at known physiological concentrations
sequence could significantly alter its pyrazolone enzymatic drugs.
properties. In- of
hibition studies of COX-3 indicate this to be the case.
Analgesic/antipyretic drugs penetrate the blood-brain barrier
Our studies show COX-3 to be sensitive to drugs that are
11 and accumulate in the CNS at high enough concentrations
analgesic/antipyretic, but which have low antiinflammatory ac- tw
inhibit COX-3. Carboxylate-containing NSAIDs, on the other
tivity. Pain and fever have many etiologies that employ complex h?
nd, cross the blood-brain barrier poorly. Still, central anal-
cellular and biochemical pathways. The finding that COX-3 is
sic mechanisms of action for carboxylate NSAIDs have been
sensitive to analgesic/antipyretic drugs suggests that the COX-1 ge
)posed in brain or spinal cord (34, 35). Because COX-3 is so
gene plays an integral role in pain and/or fever. Depending on pr
isitive to some carboxylate NSAIDs, COX-3 in the CNS may
the physiological context, pain pathways involve products from se
an essential target of both analgesic/antipyretics and standard
either the COX-1 or -2 genes. COX-2-selective drugs, for be
;AIDs. Furthermore, the sensitivity of COX-3 to analgesic/
example, are clinically useful in inhibiting inflammatory pain in N
tipyretic drugs and NSAIDs observed in these studies (Fig. 5,
humans (13) and are more potent than COX-1-selective an
ble 1) suggests that highly selective inhibitors can be made for
NSAIDs at inhibiting pain induced by proinflammatory agents
)X-3.
(e.g., carrageenan) in some paw inflammation assays in rodents
Human COX-3 is mainly expressed as an -5.2-kb mRNA and
(14, 15). COX-1-selective drugs, in contrast, are superior to
COX-2-selective agents at inhibiting visceronociception causeds ha
a tissue-specific pattern of expression (Fig. 1C). This =5.2-kb
RNA is an alternatively polyadenylated human COX-1 mes-
by a variety of chemical pain stimulators (16-18). Moreover, m
Ballou and colleagues (19) found that visceronociception was ;e reported
sa and partly characterized in its 3' region (5). It
pears,
greatly decreased in COX-1 but not COX-2 knockout mice. Both af therefore, that the retention of intron 1 may influence
COX-1 and -2, on the other hand, have been implicated in- site
th at which the mRNA is polyadenylated. This finding
nociception models that measure analgesia outside the gut, such ggests
Su that the 3' untranslated regions of the mRNA may play
as in formalin and urate crystal tests (18-20). A role for COX-1unctional
a i role in expression of COX-3 and perhaps PCOX-la.
in pain is further supported by the fact that COX-1-selective Tl te functional significance and the mechanism by which intron
NSAIDs [e.g., as identified by Warner and colleagues (21)]- :ention
re and alternative polyadenylation are coordinated need
such as aspirin, ketorolac, ketoprofen, ibuprofen, and supro- be to elucidated. It is also interesting to note that the =5.2-kb
fen-are clinically important analgesic agents in humans and RNA mhas been shown to be regulatable (36) and hence may be
animals. Despite their relative exclusion from the brain, these regulated in response to physiological stimuli and signal trans-
drugs may reach sufficient concentration to effect COX-3 in the ction.
du Indeed, the levels of COX-3 mRNA in human and
brain. Furthermore, the analgesic effects of these drugs often nine
ca cerebral cortex are relatively low. This may be due to cell
occur at significantly lower doses than those needed to inhibitpe-specific
tyl expression such as has been shown for COX-1
inflammation (22). Clinical and experimental association of munoreactive
in protein in a subpopulation of putative nocicep-
COX-1 and pain may be functionally explained by the finding r neurons
to (23). However, COX-3 in human will require further
that COX-1 is a marker for subpopulations of putative nocicep- perimentation
ex because some of the published sequences differ
tor neurons in the dorsal root ganglion (23). by one nucleotide in intron 1 and hence are out of frame. These
With regard to pyresis, COX-2 but not COX-1 knockout ty constitutemicegenuine
m polymorphisms or sequencing errors.
demonstrate reduction in LPS- and interleukin-l-induced fevers A: ternatively, intron 1 may be out of frame in humans, requiring

13930 | www.pnas.org/cgi/doi/1 0.1073/pnas. 162468699 Chandrasekharan et a/.

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other mechanisms such as ribosomal frame shifting to produce Although the peroxidase activity of cyclooxygenase is needed
a functional COX-3 protein. to create the protein radical used in the cyclooxygenase reaction,
We have immunologically identified ntinued peroxidase activity is not
a 65-kDa essential for in
protein continued
cc
human aorta, which we postulate is COX-3, and 53 kDa cy clooxygenase activity (39). Because only one turnover of the
proteins, which we postulate to be PCOX-la. These proteins are roxidase
pC active site is required for cyclooxygenase activity in
detected by both COX-3 antipeptide polyclonal antibody and a Cl)X-1 and -2, there may be enough residual peroxidase activity
COX-1 monoclonal antibody and appear to be present at about in PCOX-1 proteins to prime them.
25% of the level of COX-1. The 65-kDa protein is smaller than We have previously found that cyclooxygenases bind to
cleobindin (40). Nucleobindin is a candidate for binding to
would be predicted if the protein is glycosylated to the same nu
extent as COX-1, suggesting that hypoglycosylation or other 'OX-1
P proteins as well. Additionally, a form of COX-1 has
differences exist between the 65-kDa protein and COX-1. The be described that colocalizes with prostacyclin synthase in
en
53-kDa proteins are present as a doublet, and are of a higheramentous structures of cultured endothelial cells (41). This
fil
molecular weight than that predicted by the PCOX-la protein amentous form of COX-1 has no cyclooxygenase activity, and
primary sequence. This suggests that, like canine PCOX-la a candidate for being a PCOX-1 protein.
We previously identified an acetaminophen-inhibited COX
expressed in insect cells, the human protein may be glycosylated,
zyme activity in a murine macrophage cell line (J774.2)
and that different glycosylation states may exist, giving rise to the tr
'ated with diclofenac (42). We proposed that this activity was
doublet observed. A 50-kDa protein is also detected only by the
variant of COX-2, because a protein immunoreactive with
COX-1 monoclonal antibody, and is a candidate for being ar ti-COX-2 sera was co-induced with the activity. However, this
PCOX-lb. It appears to be present at about 15% of the level of aC
tivity was insensitive to aspirin and showed reduced sensitivity
COX-1. to diclofenac, indomethacin, and flurbiprofen-properties not
PCOX-la isared with
identical to
COX-3. This finding suggests that additional distinct
in the catalytic domain
etaminophen-inhibitable COX forms exist that are likely
5-8. It lacks rived from detectable
COX-2.
inability to make pros
deleted portion contain
e thank and acknowledge the assistance of Marcus Rampton, John
and part of H6
nger, Jenny defined
Taylor, Joel Wilson, and Eme Ekpo for assistance with
and H5 formastern blots and radioimmunoassays.
part We thank Kenneth
of D. Westover t
of the lack suggestingof the acronym PCOX. H2 We gratefully acknowledge
and the
fin
ancial support of the National Institutes of Health (Grant AR46688),
detectable peroxidase activity. In this way they are similar to th ' Brigham Young University Cancer Research Center, the Brigham
plant pathogen-induced oxygenase (PIOX) enzymes and Gaeu- Yt )ung University Technology Transfer Office, the Office of Research
mannomyces graminis linoleate diol synthase, which also lack an d Creative Work, and the College of Physical and Mathematical
peroxidase activity (37, 38). Sc ences.

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