Imaging Cyclooxygenase2 ( Cox2 ) Gene Expression in Living Animals with a Luciferase Knock-in...

B Academy of Molecular Imaging 2006

Published Online: 24 March 2006 DOI: 10.1007/s11307-006-0034-7

Mol Imaging Biol (2006) 8:171Y187

RESEARCH ARTICLE

Imaging Cyclooxygenase-2 (Cox-2) GeneExpression in Living Animals with a LuciferaseKnock-in Reporter GeneTomo-o Ishikawa,1,2 Naveen K. Jain,1 Makoto M. Taketo,2 Harvey R. Herschman PhD2

1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, 341 Boyer Hall, 611 Charles E. Young Drive

East, UCLA, Los Angeles, CA 90095, USA2Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Abstract

The cyclooxygenase-2 (Cox-2 ) gene plays a role in a variety of normal and pathophysiologicalconditions. Expression of the Cox-2 gene is induced in a broad range of cells, in response tomany distinct stimuli. The ability to monitor and quantify Cox-2 expression noninvasively in vivomay facilitate a better understanding of the role of Cox-2, both in normal physiology and indifferent diseases. We generated a Bknock-in^ mouse in which the firefly luciferase reporterenzyme is expressed at the start site of translation of the endogenous Cox-2 gene. Correlationof luciferase and Cox-2 expression was confirmed in heterozygous Cox-2luc/+ mouse embryonicfibroblasts isolated from the knock-in mouse. In an acute sepsis model, following injection ofinterferon gamma and endotoxin, ex vivo imaging and Western blotting demonstratedcoordinate Cox-2 and luciferase induction in multiple organs. Using both paw and air pouchinflammation models, we can monitor repeatedly localized luciferase expression in the sameliving mouse. Cox-2luc/+ knock-in mice should provide a valuable tool to analyze Cox-2expression in many disease models.

Key words: Cyclooxygenase, Luciferase, Imaging, Knock-in mouse, Gene expression

Introduction

Prostanoids play a major role in many normal physio-

logical processes, including thermoregulation, platelet

aggregation, wound healing, luteinization, ovulation, partu-

rition, water balance, glomerular filtration, and hemostasis.

The key enzyme of the prostaglandin biosynthesis pathway is

cyclooxygenase (Cox), which catalyzes conversion of arachi-

donic acid to PGH2, the common intermediate for the

synthesis of the various prostanoids (prostaglandins, prosta-

cyclins, and thromboxanes [1]). All the commonly used

nonsteroidal anti-inflammatory drugs (NSAIDs) exert their

major pharmacological effects by inhibiting cyclooxygenase

activity and blocking prostanoid production. The effects of

the NSAIDs suggest major roles for cyclooxygenase in pain

perception, thermoregulation, chronic and acute inflamma-

tion, heart and blood vessel biology, epithelial cell biology, a

variety of cancers, and several neurodegenerative diseases.

There are two cyclooxygenase genes, constitutively

expressed cyclooxygenase-1 (Cox-1) and inducible cyclo-

oxygenase-2 (Cox-2) [2Y4]. Cox-2 expression is induced in

many kinds of cells in response to a wide range of

physiological inducers [5, 6]. Pharmacological studies that

used specific inhibitors and genetic studies using a Cox-2

knock-out mouse have been helpful in revealing the

function(s) of Cox-2 in disease. Although a role for Cox-1

in inflammation, cancer, and various neurodegenerative

diseases is also likely to exist, it is clear that exaggerated

Cox-2 expression plays a major modulatory, and perhaps

even causal, role in many cancers, in a variety of chronic and

acute inflammation conditions, and in various neurodegen-

erative diseases.

1

Correspondence to: Harvey R. Herschman PhD; e-mail: [email protected]

Elevated Cox-2 enzyme levels in response to inflamma-

tory stimuli and cancer progression results from transcrip-

tional activation from the Cox-2 gene, posttranscriptional

stabilization of the Cox-2 message, and regulation of Cox-2

mRNA translation [5Y7]. A number of cis-acting regulatory

elements have been observed in the 50 regulatory region of

the Cox-2 gene, including a cAMP response binding ele-

ment (CRE), several nuclear factor for interleukin 6 (NFIL6)

elements, an NFkB element, a sterol response element [8], an

NFAT response element [9], a PU.1/ets binding site [10],

and even a binding/transcriptional transactivation site for a

nuclear form of the erb-B2/HER2 receptor tyrosine kinase

[11]. The cis-acting regions, signal transduction pathways,

and transcription factors of the Cox-2 gene have been

analyzed primarily in cell culture studies [1, 5].

Recently, molecular imaging has brought new paradigms

to small-animal research [12]. Luciferase from the firefly

Photinus pyralis has been developed as a bioluminescent

reporter gene whose activity can be monitored noninva-

sively in vivo by systemic injection of substrate (luciferin)

and subsequent imaging with a sensitive charged-coupled

device (CCD) camera [13]. Faithful expression and nonin-

vasive, repeated monitoring in living animals of a luciferase

reporter from the regulatory region of the Cox-2 gene would

be of great use in investigating the role of Cox-2 in normal

and pathological processes. We previously demonstrated

that it is possible to monitor, repeatedly and noninvasively

in living mice, regulated luciferase expression from a Cox-2

promoter/luciferase transfected cell, using tumor xenografts

[14]. In this study, we describe a knock-in mouse in which

the endogenous Cox-2 chromosomal promoter drives ex-

pression of the firefly luciferase reporter.

Materials and Methods

Targeting Vector Construction, HomologousRecombination, Chimera Construction,and Germline Transmission to CreateCox-2luc/+ Mice

Mouse genomic DNA fragments of the Cox-2 50 proximal region

were isolated by screening a 129/Sv mouse genomic library in a

bacteriophage l vector (Stratagene) as described previously [15].

Genomic fragments of the Cox-2 30 distal region were isolated by

screening an RPCI-22 mouse BAC library (CHORI BACPAC

resource center). To construct the pCox-2-lucneo targeting vector

(Fig. 1A), a silent mutation was introduced at the ATG

translational start site to create an NcoI site in both the Cox-2 50

genomic fragment and in the firefly luciferase coding sequence

(from pRL vector, Promega). These fragments were cut with NcoI

and ligated so that the Cox-2 coding region portion of the Cox-2 50

genomic fragment is replaced at the start codon by the firefly

luciferase coding sequence. To the resulting plasmid, we added the

following fragments: a neomycin-resistant cassette flanked by loxP

sites [16], a 1-kb AccIYSacI Cox-2 genomic fragment (containing

exon 6, exon 7, and a part of exon 8) for recombination in the 30

region, and a diphtheria toxin (PGK-DT) selection cassette [16] to

create the pCox-2-lucneo targeting vector.

The pCox-2-lucneo targeting vector was linearized with NotI

and electroporated into LW1 embryonic stem (ES) cells by the

UCLA Embryonic Stem Cell Shared Resource. Homologous

recombinant ES cell candidates were screened by PCR (Fig. 1A).

To identify ES cells with the targeted Cox-2lucneo allele, we used

the following primer set: PGKRa in the PGK promoter (50-CTA

AAG CGC ATG CTC CAG ACT-30) and COX2R2 in exon 8 (50-GGA GTT GTT GTA GAG AAA CTG-30). Homologous recom-

bination in ES cells was verified by Southern hybridization,

using the genomic Southern (GS) probe shown in Fig. 1A.

The pTURBO-Cre vector, which expresses Cre recombinase

from the CAG promoter, was electroporated into one of the ES

clones carrying the targeted allele. Cre-loxP recombinant clones

in which the neomycin-resistant cassette is deleted were

identified by PCR. To detect the Cox-2luc allele in which the

neomycin selection cassette has been deleted (Fig. 1A), the

following primer set was used: LucF2 in luciferase coding

sequence (50-TTC TGG AGA CAT AGC TTA CTG-30) and

LoxR2 in the loxP flanking sequence (50-AGT GAA CCT CTT

CGA GGG ACC-30). Cre-loxP recombination in ES cells was

verified by Southern hybridization, using the same GS probe.

Chimeras were generated by injecting the ES cells into C57BL/6

blastocysts, and mice with the Cox-2luc allele transmitted to the

germline were obtained. Animal experiments were carried out with

Animal Research Committee approval at UCLA.

Genotyping of Mice and Cells

The Cox-2luc allele was detected by PCR with LucF2 and LoxR2

primers (described above). The wild-type Cox-2 allele was

detected by PCR with the following primer set: COX2E1F1 in

exon 1 (50-TCA GTC AGG ACT CTG CTC AC-30) and

COX2E2R1 in exon 2 (50-GTG TAG TAC AGT TTT CAC C-30).Mice with the Cox-2luc allele were also genotyped by luciferase

assay because basal luciferase expression can be observed in tail

tissue. Tail tips from each mouse were lysed overnight at 4-C in

passive lysis buffer (Promega). Lysates were centrifuged, and the

supernatants were assayed with the Luciferase Assay System

(Promega).

In Vitro Differentiation of ES Cells

ES cells were seeded at a density of 1 � 105 cells/ml in 10-cm

bacterial Petri dishes, in Dulbecco’s modified Eagle’s medium

(DMEM) with 10% serum, without either Lif or 2-mercaptoetha-

nol. The cells were cultured for 48 hours to allow aggregation. The

small embryoid bodies were then plated in tissue culture plates with

0.5% dimethyl sulfoxide (DMSO) to promote fibroblast differenti-

ation [17]. After four days, differentiated cells were cultured over-

night in DMEM with 0.5% serum. Cells were stimulated for four

hours with either tetradecanoylphorbol acetate (TPA, 50 ng/ml) or

epidermal growth factor (EGF, 10 ng/ml), harvested with passive

lysis buffer and assayed using the Luciferase Assay System.

Relative light units (RLU) for each sample were normalized by

protein assays (Bio-Rad).

172 Ishikawa et al.: In Vivo Imaging of Cox-2

Fig. 1. Construction of the Cox-2luc/+ knock-in mouse. (A) Targeting strategy showing the structures of the wild-type Cox-2allele, the targeting vector pCox-2-lucneo, the targeted Cox-2lucneo allele, and the Bneo-deleted^ Cox-2luc allele, in which theneomycin-resistant cassette is deleted by transient Cre recombinase expression in Cox-2lucneo/+ ES cells. Cox-2 exons areshown as filled boxes; 50 genomic DNA, intronic sequences, and 30 genomic DNA are solid lines. The firefly luciferase codingregion with SV40 polyA (ffluc), the PGK-neo (neo) cassette, and the PGK-DT (DT ) cassette are shown as open boxes, withtheir transcriptional orientations indicated by arrows. The PCR primers are shown as pairs of short arrows. The GS probe usedfor genomic Southern blots is the solid box below the wild-type allele. The XbaI (X) genomic fragments hybridizable to theprobe are also shown (9 kb for the wild-type Cox-2 allele, 3 kb for the targeted Cox-2lucneo allele, and 2 kb for the neo-deletedCox-2luc allele, respectively). (B) Southern blot verification of homologous recombination to produce ES cells containing theCox-2lucneo allele and subsequent Cre-loxP elimination of the neo-resistance cassette to produce ES cells containing the Cox-2luc allele. Extracted DNA samples were digested with XbaI, subjected to electrophoresis, transferred to membranes, andhybridized with the GS probe shown in (A). (C) Luciferase induction in differentiated Cox-2lucneo/+ ES cells and differentiatedCox-2luc/+ ES cells. Cells were treated with TPA (50 ng/ml) or EGF (10 ng/ml) for four hours, harvested, and extracts wereassayed for luciferase activity. Activity was attenuated in ES cells containing the targeted Cox-2lucneo/+ allele, when comparedwith ES cells containing the neo-deleted Cox-2luc/+ allele. Data are averages T SD of three cultures.

Ishikawa et al.: In Vivo Imaging of Cox-2 173

Fig. 2. Characterization of Cox-2 and luciferase expression in Cox-2luc/+ and Cox-2luc/luc embryonic fibroblasts. (A) Westernblot analysis of Cox-2 protein in wild-type Cox-2+/+, heterozygous Cox-2luc/+, and homozygous Cox-2luc/luc MEFs after fourhours’ stimulation with TPA (50 ng/ml). Antiserum to 14-3-3 was used as a loading control. (B) Western blot analysis ofluciferase protein in wild-type, heterozygous Cox-2luc/+, and homozygous Cox-2luc/luc MEFs after four hours’ stimulation withTPA. (C) Time course of induction of luciferase activity by TPA (50 ng/ml) in Cox-2luc/+ and Cox-2luc/luc MEFs. Data areaverages T SD for three independent cultures at each time point. (D) Cox-2 protein induction by various ligands in Cox-2luc/+

MEF cultures. Cells were treated with TPA (50 ng/ml), serum (20%), PDGF (10 ng/ml), EGF (10 ng/ml), or IL-6 (20 ng/ml) forfour hours. Lysates were prepared and supernatants were analyzed for Cox-2 expression by Western blotting. Antiserum to14-3-3 was used as a loading control. (E) Coordinate expression of Cox-2 protein and luciferase activity in Cox-2luc/+ MEFs.Cells were treated with PBS, TPA (50 ng/ml), EGF (10 ng/ml), PDGF (10 ng/ml), or 20% serum for four hours. Lysates wereprepared, and supernatants were analyzed for Cox-2 expression by Western blotting and for luciferase activity. Western blotintensity and luciferase activity were plotted for each set of cultures. Luciferase activity data are shown as averages T SD. ForWestern blotting, sets of treated cells were pooled and analyzed. (F) Dexamethasone inhibits both luciferase activity and Cox-2 protein expression in Cox-2luc/+ MEFs. Cells were treated with vehicle, TPA (50 ng/ml), dexamethasone (1 2M), or [TPA +dexamethasone] for four hours. Lysates were prepared, and supernatants were analyzed both for Cox-2 expression byWestern blotting and for luciferase activity. Luciferase data are averages T SD for three independent cultures. (G) Induction ofCox-2 and luciferase proteins in Cox-2luc/+ MEFs. Cox-2luc/+ fibroblasts were plated in multiwell slide chambers. Cells weretreated with vehicle (left), TPA (50 ng/ml) and 20% serum (center), or [TPA and 20% serum + dexamethasone] (right) for fourhours. The cultures were then washed, fixed, and stained for Cox-2 (green) and luciferase (red) immunoreactivity.


Isolation and Analysis of Embryonic Fibroblastsand Peritoneal Macrophages

To isolate mouse embryonic fibroblasts (MEFs), embryos (12.5

days postcoitus) were collected from the uteri of pregnant mice.

The head and liver regions were removed, and the carcasses were

minced and trypsinized twice. Tissue debris was discarded, and

fibroblasts from each embryo were collected and plated in DMEM

with 10% serum [18]. Genomic DNAs isolated from the embryo

heads were used to genotype each embryo by PCR. For Cox-2 and

luciferase induction, fibroblasts were plated at 1.6 � 105 cells/well

in 12-well tissue culture plates in DMEM with 0.5% serum. After

overnight culture, cells were treated with stimulants as described

in the BResults^ section.

To isolate peritoneal macrophages, mice were injected intra-

peritoneally with 3 ml of 3% thioglycolate medium (Sigma). After

two days, peritoneal macrophages were obtained by lavage of the

peritoneal cavity with phosphate-buffered saline (PBS). Cells were

washed, resuspended, and plated in DMEM containing 10%

serum. For Cox-2 and luciferase induction, cells were treated with

lipopolysaccharide (LPS, 1 mg/ml) for six hours.

Western Immunoblotting

Cell or tissue samples were lysed and homogenized in passive lysis

buffer or radioimmunoprecipitation assay buffer [RIPA; 1% NP-

40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate

(SDS) in 1 � PBS] containing a proteinase inhibitor cocktail

(Roche Diagnostics). After centrifugation to remove the insoluble

fraction, samples (30 mg protein for cell and 100 mg for tissue)

were separated in 10% SDS polyacrylamide gels and transferred to

nitrocellulose membranes. The membranes were blocked with

10% skim milk and incubated with polyclonal antibodies against

Cox-2 (Cayman Chemicals) at 1000-fold dilution, luciferase

(Rockland) at 1000-fold dilution, or 14-3-3 (Santa Cruz) at 500-

fold dilution. The ECL detection system (Amersham Pharmacia)

was used to detect the signals on X-ray film. The film was scanned

and analyzed for quantification using NIH image software.

Standard curves to quantify relative Cox-2 protein levels were

prepared by mixing extracts of induced and uninduced cells (data

not shown).

In Vivo and Ex Vivo Imagingof Luciferase Activity

Mice were anesthetized by intraperitoneal administration of a

ketamine (80 mg/kg, Phoenix Pharmaceutical) and xylazine (4 mg/

kg, Phoenix Pharmaceutical) mixture. Anesthetized mice were

injected intraperitoneally with D-luciferin (125 mg/kg, Xenogen)

and placed in the light-tight box of the IVIS imaging system

(Xenogen). Whole-body one-minute images were acquired repeat-

edly until the maximum peak of photon number was confirmed

during the one-minute scans. Data at the time point that gave the

highest photon number during one minute of scanning time were

used for quantfication. Collected photon number and images were

analyzed using Living Image software (Xenogen).

For ex vivo imaging of isolated tissues, mice were anesthetized

and injected with D-luciferin. Repetitive images of one-minute

duration were taken for each animal. After the photon accumula-

tion at one-minute intervals, examined by in vivo imaging, reached

a maximum, sustained level (usually õ ten minutes), the mice

were sacrificed and the tissues were rapidly excised. Tissues were

placed on culture dishes and imaged with the IVIS system.

Fig. 3. Cox-2 protein expression does not modulate tran-scriptional activation of the Cox-2 gene in mouse embryonicfibroblasts. (A) Cox-2luc/+ and Cox-2luc/j MEF cultures weretreated with TPA (50 ng/ml) for the times shown andharvested; lysates were assayed for luciferase activity. Dataare the averages T SD for triplicate cultures. (B) Lysates fromthe TPA-treated Cox-2luc/+ and Cox-2luc/j MEF cultureswere subjected to Western blotting for Cox-2 protein andfor luciferase protein. (C) Cox-2luc/+ and Cox-2luc/j MEFcultures were treated with TPA (50 ng/ml), serum (20%),PDGF (10 ng/ml), EGF (10 ng/ml), or IL-6 (20 ng/ml) for fourhours and assayed for luciferase activity. Data are theaverages T SD for triplicate cultures.


Systemic Cox-2 Induction in Mice by InterferonGamma and Endotoxin

Systemic Cox-2 expression was induced by treatment with

interferon gamma (IFN-g) and bacterial endotoxin (LPS). Each

mouse was injected intraperitoneally with IFN-g (1 mg/mouse,

Chemicon International) in 100 ml PBS, followed two hours later by

a 100-ml intraperitoneal injection of LPS (3 mg/kg dissolved in PBS,

Sigma). Control mice were injected with PBS. Six hours after LPS

injection, ex vivo tissue images were taken as described above.

Immnohistochemical Staining

The spleens from mice treated with LPS/IFN-g or PBS were

collected after six hours and frozen in OCT compound (Tissue-

Tek, Sakura Finetechnical) in liquid nitrogen. Cryosections (4 mm)

were fixed with acetone and treated with 0.3% H202 and 0.3%

normal goat serum (Vector Laboratories) in PBS to quench

endogenous peroxidase activity. The sections were blocked with

5% normal goat serum in PBS and incubated with goat anti-

luciferase (1:50, overnight at 4-C; Promega) or rabbit anti-Cox-2


(1:100, overnight at 4-C; Cayman Chemicals) in PBS with 5%

normal goat serum. The sections were incubated with biotinylated

rabbit anti-goat IgG or goat anti-rabbit IgG (1:300, at room

temperature, Vector Laboratories). After washing in PBS, sections

were processed with reagents from an ABC kit (Vector Labora-

tories). Slides were examined with a BX60 microscope (Olympus).

Images were captured with a Macrofire 599831 camera and

processed with Pictureframe software (Optronics).

Cox-2 Induction by Zymosan in Mouse Paw

Paw inflammation was induced by subplantar injection of 30 ml of

2.0% (w/v) zymosan A (Sigma) in PBS into the mouse right

(ipsilateral) hind paw [19]. The contralateral left hind paw, used as

a control, was injected with 30 ml of PBS. This experiment was

initially performed with C57BL/6 mice to establish the Cox-2

induction pattern, prior to in vivo imaging in Cox-2luc/+mice. For

Western blotting, mice were sacrificed, and the ipsilateral and con-

tralateral paws were immediately amputated and processed for

protein isolation. Extract supernatants were used for Western blot

analysis.

To examine the effect of glucocorticoids on zymosan-induced

luciferase induction, mice were injected intraperitoneally with

dexamethasone (Sigma, 10 mg/kg in corn oil [20, 21]) 24 hours

and again one hour prior to the zymosan injection.

Air Pouch Inflammation Model

Air pouches were raised on the dorsum by subcutaneous injection

of 1 ml of air on both shoulders of mice. After three days, the air

pouch on the left shoulder was injected with 1 ml of 2.0% (w/v)

zymosan suspension in PBS [22]. The air pouch on the right

shoulder was injected with 1 ml of PBS as control. For Western

blotting, mice were sacrificed at indicated time points after the

zymosan challenge, and pouch tissues were excised and homog-

enized with RIPA buffer. For immunocytochemistry, cell suspen-

sions were obtained by lavage of the air pouch with PBS. Cells

were washed and attached on chamber glass slides coated with

poly-L-lysine.

Immunofluorescent Staining

For immunofluorescence of embryonic fibroblasts, cells were fixed

in 10% formalin/PBS. Fixed cells were incubated with mouse anti-

Cox-2 (1:200, BD Transduction Laboratories) and rabbit anti-

luciferase (1:100; Cortex Biochem) overnight. Then cells were

washed and incubated with goat anti-rabbit IgG with Alexa Fluor

568 (1:100; Molecular Probes) and goat anti-mouse IgG with Alexa

Fluor 488 (1:100; Molecular Probes). Slides were examined by

bright-field (not shown) and by epifluorescence microscopy.

For immunofluorescence of cells from air pouches and from

peritoneal macrophages, cells were fixed in 10% formalin/PBS.

Fixed cells were incubated with rabbit anti-Cox-2 (1:100, 2 h at

room temperature; Cayman Chemicals), washed, and incubated

with goat anti-rabbit IgG labeled with Alexa Fluor 568 (1:100

dilution, 1 h at room temperature; Molecular Probes). Cells were

again washed, blocked, and incubated with goat anti-luciferase

(1:100, overnight at 4-C; Promega). Cells were washed again and

incubated with FITC-labeled donkey anti-goat secondary antibody

(1:100, 1 h at room temperature; Jackson Immunoresearch).

Results

Construction and Characterization of Cox-2luc/+

Embryonic Stem Cells

To monitor noninvasively Cox-2 expression in living ani-

mals, we constructed a knock-in mouse strain in which the

coding region of the firefly luciferase reporter protein is

expressed at the start site of translation of the endogenous

Cox-2 gene. The gene targeting strategy is shown in Fig. 1A.

Fig. 4. Cox-2 and luciferase induction in Cox-2luc/+ mice by interferon gamma and endotoxin. (A) Ex vivo images of luciferaseexpression in tissues of Cox-2luc/+ mice. Six experimental mice (three males and three females) were injected intraperitoneallywith IFN-g followed two hours later with LPS. Control mice (three males and three females) received saline injections. After sixhours the mice were anesthetized, injected with luciferin, and imaged in the IVIS camera to determine the optimal time ofluciferase expression, as described in BMaterials and Methods^. The mice were sacrificed, tissues were rapidly removed andplaced in culture dishes, and ex vivo tissue images were obtained. Representative images for tissues from control (left) and[IFN-g + LPS]-treated (right) female mice are shown. The color overlay on the images illustrates the photons per secondemitted from the tissues. Vas deferens from control and experimental male mice are shown in the insets. Pseudocolor scalesare shown next to the images; note the difference in scale for the vas deferens images. (B) Quantification of luciferase activity,determined by bioluminescent imaging, from tissues of male and female mice (n = 3 for each). Bioluminescent data wereanalyzed with Living Image software. A region of interest (ROI) was manually selected. The area of the ROI was kept constantfor each tissue; signal intensity from the ROI was measured and reported as photons per second. Data are the averages T SDfor tissues from three male and three female mice each, for both the control (indicated by Bj^) and for the [IFN-g + LPS]-treated (indicated by B+^) groups. (C) Luciferase activity in individual tissues, measured by luciferase assay of tissue extracts.The same tissues analyzed in B were homogenized, and supernatants were assayed for luciferase activity. Data are theaverages T SD for three male and three female mice, for control and [IFN-g + LPS]-treated groups. (D) Comparison ofluciferase fold induction data obtained by ex vivo bioluminescent imaging (B) and by enzyme activity measurements (C) formouse tissues, following induction by [IFN-g + LPS]. Values are fold increases for tissues of treated vs. control Cox-2luc/+ mice.(E) Western blot analysis of Cox-2 protein expression in tissues of control and [IFN-g + LPS]-treated mice. Lysatesused for luciferase assays (C) were also used for Western blotting. 14-3-3 antibody was used as a loading control.(F) Immunohistochemistry for Cox-2 and luciferase proteins in the spleens of control and [IFN-g + LPS]-treated Cox-2luc/+

mice. Spleen was chosen for this analysis because of the large fold induction observed in this tissue. Tissue fixation andstaining are described in BMaterials and Methods^. Scale bars: 100 mm.


We employed a knock-in strategy to make the Cox-2 fluc

reporter mouse because this strategy eliminates chromosom-

al position effects and copy number concerns that can occur

in transgenic mice following random integration of reporter

genes. In addition, with a knock-in reporter, luciferase

transcription will also be responsive to regulatory elements

that might lie at substantial distances from the coding region

of the endogenous Cox-2 gene. Because insertion of the

PGK-neo selection cassette into the genome by homologous

recombination can alter or disrupt surrounding gene

expression, leading to a hypomorphic or null allele [16,

23], the PGK-neo selection cassette, which we flanked by

loxP sites, was excised by transient Cre expression after the

homologous recombination in ES cells (Fig. 1A,B).


Before proceeding with blastocyst injection, we exam-

ined luciferase reporter gene activity driven by the endo-

genous Cox-2 promoter in the homologous recombinant

Cox-2lucneo/+ ES cells and in the neo-deleted Cox-2luc/+ ES

cells (Fig. 1C). Because wild-type ES cells are reported to

not express Cox-2 until they undergo differentiation [17],

Cox-2lucneo/+ and Cox-2luc/+ ES cells were treated with 0.5%

DMSO and differentiated into fibroblast-like cells prior to

TPA or EGF treatment and assay for luciferase activity

(Fig. 1C). Luciferase activity is induced with TPA or EGF

in Bfibroblasts^ from both ES cell lines. However, the

luciferase activity in Cox-2luc/+ cells is two to three times

that observed for Cox-2lucneo/+ ES cells following TPA or

EGF treatment, suggesting that the presence of the PGK-

neo selection cassette may attenuate expression of the

luciferase reporter gene. For this reason, we used Cox-2luc/+

ES cells to generate mice with the firefly luciferase reporter

protein coding sequence knocked into the Cox-2 gene at the

start site of Cox-2 protein translation.

Luciferase and Cox-2 Expression in EmbryonicFibroblasts from Cox-2+/+, Cox-2luc/+,and Cox-2luc/luc Mice

Cox-2luc/+ ES cells were injected into blastocysts and mice

with germline transmission of the Cox-2luc allele were ob-

tained. Cox-2luc/+ mice are heterozygous for luciferase and

for Cox-2. To examine the correlation between gene dosage

and gene expression, wild-type Cox-2+/+, heterozygous

Cox-2luc/+, and homozygous knock-in Cox-2luc/luc MEF

cultures were established from embryos of a Cox-2luc/+

intercross.

The Cox-2 gene was originally identified in our labora-

tory as a gene induced by TPA in 3T3 mouse fibroblasts [2].

When treated with TPA, wild-type MEFs demonstrate sub-

stantial Cox-2 protein expression (Fig. 2A) and no lucifer-

ase protein (Fig. 2B). In contrast, homozygous Cox-2luc/luc

MEFs express no Cox-2, but have substantial amounts of

immunologically detectable luciferase. Heterozygous Cox-

2luc/+ MEFs express intermediate amounts of both Cox-2

and luciferase proteins, suggesting a gene dosage effect at

the Cox-2 locus.

As expected for a gene dosage effect, Cox-2luc/luc MEFs

have about a twofold higher baseline luciferase activity

relative to Cox-2luc/+ MEFs (Fig. 2C). This difference is

maintained throughout the time course of TPA induction.

These results indicate that both Cox-2 and luciferase

expression from the endogenous Cox-2 gene regulatory

region are gene-dosage-dependent in cultured MEFs.

Cox-2 expression is induced by a variety of inducers,

including TPA, serum, EGF, platelet-derived growth factor

(PDGF), and other ligands, in fibroblasts [2]. In Cox-2luc/+

MEFs, Cox-2 protein is strongly induced by 20% serum

treatment and by TPA treatment (Fig. 2D). PDGF and EGF

also induce Cox-2 protein; however, the induction is less

than that observed for TPA or serum. IL-6 does not induce

Cox-2 protein expression in Cox-2luc/+ MEFs. When

extracts used for Cox-2 quantitative Western blots are also

used to assay luciferase activity in Cox-2luc/+ MEFs induced

with these ligands, the correlation coefficient R for lucif-

erase activity and Cox-2 protein level is 0.99 (Fig. 2E),

demonstrating coordinate induction of the luciferase and

Cox-2 alleles in Cox-2luc/+ MEFs.

Cox-2 transcriptional induction in 3T3 cells and in many

other cell lines and tissues is inhibited by glucocorticoids

[24]. Luciferase expression from the Cox-2luc allele, like

Cox-2 protein expression from the Cox-2+ allele, is in-

hibited by dexamethasone in Cox-2luc/+ MEFs (Fig. 2F).

Transcriptional activation of the Cox-2luc allele appears to

be identical to transcriptional activation of the wild-type

Cox-2+ allele in Cox-2luc/+ MEFs. We also used immu-

nofluorescence to confirm the induction of luciferase and

Cox-2 proteins and the inhibition of their expression by

dexamethasone in Cox-2luc/+ fibroblasts (Fig. 2G).

Cox-2 Protein Expression Does Not ModulateTranscriptional Activation of the Cox-2Gene in MEFs

The construction of the Cox-2luc/+ mouse permits us to

isolate cells in which no Cox-2 protein is produced, but

in which transcriptional activation from the endogenous

Cox-2 gene regulatory region can be analyzed. Heterozy-

gous Cox-2luc/+ luciferase knock-in mice were crossed with

Fig. 5. Cox-2 and luciferase expression following intraplantar zymosan injection in Cox-2luc/+ mice. (A) Cox-2 expression inwild-type C57BL/6 mice following intraplantar zymosan (2%) injection. Paw samples were collected, homogenized, andanalyzed for Cox-2 protein by Western blotting. 14-3-3 antibody was used as a loading control. (B) Cox-2 protein expressionin paw of control (Vehicle) and dexamethasone-treated mice (Dex) at nine hours. Zymosan-injected (Z) and saline-injected (S)paw samples were analyzed by Western blotting. (C) In vivo imaging of Cox-2luc/+ mice following intraplantar zymosaninjection. Four mice were injected with zymosan in the right rear paw (left side in the pictures) and saline in the left paw (rightside in the pictures). The mice were injected with luciferin and imaged (ventral view) at 0, 3, 6, 9, 12, 24, and 48 hours followinginjection. (D) Quantification of luciferase activity of the paw regions, using Living Image software. Bioluminescentmeasurements of luciferase activity from the Cox-2luc allele are shown for both the zymosan-injected and the saline-injectedpaws for each mouse. (E) Dexamethasone inhibits luciferase expression from the Cox-2luc allele in response to intraplantarzymosan injection. One group of three Cox-2luc/+ mice was pretreated with dexamethasone (10 mg/kg) both at 24 hours and atone hour prior to the zymosan injection. The second group of three mice did not receive dexamethasone. At nine hours afterzymosan injection, the mice were injected with luciferin and imaged. Data are averages T SD. Data were analyzed by Student’st test; p values are shown.


heterozygous Cox-2+/j knockout mice (Ishikawa et al., un-

published data), and MEFs were established from Cox-2luc/+

and Cox-2luc/j embryos. Cox-2luc/+ and Cox-2luc/j MEFs

were treated with TPA and assayed for luciferase activity

(Fig. 3A). Protein samples used for luciferase assay were

also analyzed both for Cox-2 protein expression and for

luciferase protein expression by Western blotting (Fig. 3B).

Cox-2 protein is not detectable in Cox-2luc/j MEF samples;

the Cox-2luc knock-in allele, like the Cox-2j allele, disrupts

the Cox-2 coding region. In contrast, Cox-2 protein is in-

duced in response to TPA in Cox-2luc/+ MEFs. Despite the

absence of any Cox-2 protein expression in Cox-2luc/j

MEFs, the time course and magnitude of luciferase

expression in TPA-treated Cox-2luc/+ and Cox-2luc/j MEFs


is identical (Fig. 3A). When treated with serum, PDGF, or

EGF, the magnitude of induced luciferase expression in

Cox-2luc/+ and Cox-2luc/j MEFs is also identical (Fig. 3C).

Luciferase Activity and Cox-2 Expressionin the Organs of Cox-2luc/+ Mice Followinga Systemic Inflammatory Stimulus

This set of experiments is designed to address the question

of coordinate luciferase and Cox-2 expression in Cox-2luc/+

mice. Many murine tissues respond to IFN-g and LPS in-

duction by transcriptional activation of the Cox-2 gene. To

examine the relationship between luciferase and Cox-2

expression at times relatively early after a systemic inflam-

matory stimulus, Cox-2luc/+ mice were injected with IFN-g(1 mg/mouse i.p.), followed two hours later by LPS (3 mg/

kg i.p. [20]). Six hours after LPS injection, the mice were

anesthetized, injected with luciferin, and imaged in the IVIS

apparatus. When peak overall bioluminescence was ob-

served, the IFN-g- and LPS-treated mice and control Cox-

2luc/+ mice were killed. Organs were removed and imaged

with the IVIS apparatus (Fig 4A,B), then homogenized and

used for luciferase activity assay measurements (Fig. 4C)

and Cox-2 analysis by Western blotting (Fig. 4E).

Basal luciferase signal can be detected in many tissues

(Fig. 4A, left). The greatest signal in control, untreated

Cox-2luc/+ mice is observed in the male mouse vas deferens

(Fig. 4A, inset). This result is consistent with the high level

of constitutive Cox-2 expression in vas deferens reported by

Lazarus et al. [25]. Uninduced luciferase activity is inter-

mediate in brain, thymus, lung, stomach, pancreas, kidney,

intestine, uterus, and skin, and lowest in liver, spleen, and

heart. Following [IFN-g + LPS] injection, luciferase activity

is induced in a wide range of tissues (Fig. 4A, right). Using

Living Image software, we quantified the luciferase expres-

sion from each tissue of untreated and treated Cox-2luc/+

mice from the optical imaging data (Fig. 4B). Luciferase

enzymatic activity was then measured, by conventional

luciferase assay, in lysates from each tissue of these same

untreated and treated Cox-2luc/+ mice (Fig. 4C). Good cor-

relations are observed in the fold-induction values of brain,

lung, heart, and kidney when luciferase expression by tissue

image analysis and luciferase activity in tissue extract as-

says are compared (Fig. 4D). The spleen and liver have

particularly low basal Cox-2 expression levels (Fig. 4AYC).

The difference in the fold-induction values for luciferase

image analysis and luciferase biochemical assay compar-

isons of spleen and liver from untreated and [IFN-g + LPS]-

treated Cox-2luc/+ mice may be due to the accuracy with

which the very low levels of basal expression in these or-

gans can be measured.

The same tissue lysates used for conventional luciferase

assays (Fig. 4C) were also analyzed for Cox-2 protein

expression by Western blotting (Fig. 4E). Substantial Cox-2

protein expression is observed in brain, lung, spleen, kid-

ney, and vas deferens, the same tissues in which elevated

luciferase is observed (Fig. 4AYC).

Induction of luciferase activity by [IFN-g + LPS] is

observed in essentially all tissues of the Cox-2luc/+ mouse.

Differences in the relative levels of Cox-2 and luciferase

protein products, suggested for some of the tissues, may be

the result of posttranscriptional regulation of Cox-2 gene

expression. This possibility is considered more extensively

in the BDiscussion^.

The greatest difference in Cox-2 protein levels in tis-

sues from untreated vs. [IFN-g + LPS]-treated Cox-2luc/+

mice (i.e., the greatest fold induction) is in the spleen

(Fig. 4E). We used spleen tissue from these mice to

demonstrate the [IFN-g + LPS] induction of both luciferase

and Cox-2 protein in Cox-2luc/+ mice by immunocytochem-

istry (Fig. 4F).

Luciferase Induction from the Cox-2luc AlleleDuring Zymosan-Induced Paw Inflammationin Cox-2luc/+ Mice

Zymosan-induced paw edema in rodents is a classic local-

ized inflammatory model, albeit more commonly used in

rats than in mice. This model is very useful for initial

characterization of inflammatory response measurement

by optical imaging in the Cox-2luc/+ mouse; the inflam-

mation is localized, anatomically distinguishable, and in a

tissue where light absorption is minimal. We first op-

timized the zymosan dose to identify a concentration that

Fig. 6. Cox-2 and luciferase expression in the zymosan-injected air pouch in Cox-2luc/+ mice. (A) Cox-2 expression in thezymosan-induced air pouch in wild-type C57BL/6 mice. Zymosan (2%, 1 ml) or PBS (1 ml) was injected into 1-ml air poucheson wild-type C57BL/6 mice. Air pouch samples were collected, homogenized, and analyzed by Western blotting for Cox-2protein. (B) In vivo imaging of Cox-2luc/+ mice during zymosan-induced air pouch inflammation. Three mice were injected withzymosan (2%) in air pouches on their left shoulders; PBS was injected into similar air pouches on their right shoulders. Themice were injected with luciferin and imaged (dorsal view) at 0, 6, 12, and 24 hours following injection. (C) Quantification ofluciferase activity of the zymosan-injected and saline-injected air pouches, using Living Image software. Bioluminescentmeasurements of luciferase activity from the Cox-2luc allele are shown for both the zymosan-injected and the saline-injectedair pouch for each mouse. (D) Colocalization of Cox-2 and luciferase proteins in inflammatory cells present in the zymosan-induced air pouch of Cox-2luc/+ mice. Pouch exudates were removed at six hours and attached to glass chamber slidestreated with poly-L-lysine. The cultures were then washed, fixed, and stained sequentially for Cox-2 (red) and luciferase (green)immunoreactivity. Merged images demonstrate colocalization of Cox-2 and luciferase proteins.


gives a robust Cox-2 induction in wild-type C57BL/6 mice

(data not shown). Subplantar injection of 30 ml of a 2%

zymosan suspension in PBS results in detectable Cox-2

protein accumulation at two hours, with peak Cox-2 ac-

cumulation between 6 and 12 hours (Fig. 5A). We also

determined an appropriate schedule and dose for dexa-

methasone inhibition of zymosan-induced Cox-2 accumu-

lation (Fig. 5B).

After determining optimal zymosan concentrations and

times of Cox-2 accumulation, we injected zymosan into the

right rear paws of four Cox-2luc/+ mice. Saline was injected

into the left rear (contralateral) paws. Baseline luciferase

activity was measured noninvasively by whole-body optical

imaging before zymosan administration (Fig. 5C; 0 hours).

Luciferin was readministered, and whole-body images were

taken at 3, 6, 9, 12, 24, and 48 hours (Fig. 5C, D).

Significantly higher Cox-2 gene-directed luciferase activity

is observed in the ipsilateral, zymosan-injected paw,

compared with the control saline-injected contralateral

paw in all four mice three hours after injection. Cox-2

promoter-driven luciferase signal is further increased at six

hours and reaches a maximum level at nine hours for all

four mice.

The signal from two mice decreased to baseline values at

12 hours, but remained elevated in the other two mice. By

24 Y 48 hours, the luciferase signal returned to the baseline

level in all mice, despite substantial remaining edema in the

ipsilateral paw. Injection of the contralateral paw with saline

induces either a nondetectable or a very small elevation in

luciferase expression from the Cox-2luc allele. These data

illustrate the variability of individual animals with regard to

Cox-2 transcriptional responses to inflammatory stimuli and

the consequent value of repeated noninvasive imaging of

individual animals. Pretreatment of mice with dexametha-

sone caused significant inhibition (p = 0.002) of luciferase

signal at the nine-hour peak following zymosan injection

(Fig. 5E).

Luciferase Induction from the Cox-2luc AlleleDuring Zymosan-Induced Air PouchInflammation in Cox-2luc/+ Mice

The air pouch model, in which an inflammatory stimulus is

injected into an air pouch on the back of rodents, is an

additional well-established inflammation model. In this case,

the sequential recruitment of inflammatory cells in acute

immune reactions can be studied. Like the zymosan footpad

model, the zymosan-induced air pouch inflammation model

is a good one in which to study the Cox-2-gene-mediated

luciferase induction response from the Cox-2luc allele of

Cox-2luc/+ mice because the inflammation is localized and

the optical signal should not be substantially attenuated in

shaved mice. The air pouch inflammation model also makes

subsequent isolation and characterization of the participat-

ing inflammatory cells more accessible. We first established

two 1-ml air pouches on C57BL/6 mice to compare the

zymosan-stimulated Cox-2 and luciferase expression with

an unstimulated control. Seventy-two hours after air

injection to both shoulders, a 2% zymosan suspension in

PBS (1 ml) was injected into the left air pouches, and PBS

(1 ml) was injected into the right air pouches. Cox-2

protein was present at six hours, increased at 12 hours, and

remained elevated at 24 hours (Fig. 6A). No Cox-2 protein

expression was detected in the PBS-injected air pouches.

We next prepared two air pouches on three Cox-2luc/+

mice. Baseline luciferase activity was measured by nonin-

vasive optical imaging before zymosan injection (Fig. 6B; 0

hours). Images were taken at 6, 12, and 24 hours after

zymosan and PBS injection into the paired air pouches

(Fig. 6B, C). Zymosan injection elicited substantially higher

Fig. 7. LPS-induced Cox-2 and luciferase expression in peritoneal macrophages from Cox-2luc/+ mice. (A) Luciferaseinduction in peritoneal macrophages, measured by bioluminescent imaging. Peritoneal macrophages were cultured in four-well chamber slides. PBS was added to some cultures, LPS (1 mg/ml) to others. After six hours, luciferin was added to theculture wells, and luciferase activity was measured by bioluminescent imaging. (B) Colocalization of Cox-2 and luciferaseproteins in LPS-treated peritoneal macrophages. The LPS-stimulated cells were washed, fixed, and stained sequentially forCox-2 (red) and luciferase (green) immunoreactivity. Merged images demonstrate colocalization of Cox-2 and luciferaseproteins. Uninduced cultures showed little or no fluorescent staining for either anti-Cox-2 or anti-luciferase antibody (notshown).


induction of luciferase activity at six hours, compared with

the control air pouches, in all three mice. The bioluminescent

signals start to decline at 12 hours and have nearly returned

to baseline values at 24 hours. In the air pouch model, both

resident cells of the pouch lining tissue and cells that are

recruited into the pouch in response to zymosan injection

express Cox-2 [26, 27]. Pouch cells were collected by la-

vage six hours after zymosan injection and stained with

both antibody to Cox-2 (Fig. 6D, left) and antibody to lu-

ciferase (Fig. 6D, center). As shown in the merged pattern

(Fig. 6D, right), essentially identical expression patterns for

Cox-2 and luciferase proteins are observed at the cellular

level.

It is of interest to note that Cox-2 levels remain high at

24 hours after zymosan injection in the air pouch (Fig. 6A),

whereas transcriptional activity from the Cox-2luc allele is

reduced to near-background levels (Fig. 6C). This result is

considered further in the BDiscussion.^

Luciferase and Cox-2 Induction from the Cox-2luc

and Cox-2+ Alleles of Peritoneal Macrophagesfrom Cox-2luc/+ Mice in Responseto LPS Treatment

Although the data from the zymosan-induced air pouch

experiment illustrates well the colocalized expression of

Cox-2 and luciferase in individual cells from Cox-2luc/+

mice, the cell populations are changing during the course of

this experiment, and it is difficult to identify the individual

cell types. To more extensively colocalize luciferase and

Cox-2 expression following an inflammatory stimulus in

cells from Cox-2luc/+ mice, we isolated peritoneal macro-

phages, placed them in multiwell slide chambers, stimulated

them with LPS, and characterized the expression of both

Cox-2 and luciferase by immunofluorescence. To determine

the level of luciferase induction, the luciferase activity in

untreated and LPS-induced Cox-2luc/+ peritoneal macro-

phage cultures was measured by optical imaging six hours

after LPS addition (Fig. 7A). As observed for TPA-treated

Cox-2luc/+ MEFs, LPS-treated Cox-2luc/+ macrophages dem-

onstrate a substantial induction in luciferase activity from

the Cox-2luc allele. The cells were then fixed and sequen-

tially stained with antibodies to detect Cox-2 protein and

luciferase protein (Fig. 7B). The induced Cox-2luc/+ macro-

phages express both Cox-2 and luciferase. Moreover, we

observed essentially identical patterns of expression from

the Cox-2luc and the Cox-2+ alleles of Cox-2luc/+ macro-

phages in response to LPS treatment when the immunofluo-

rescence images are merged.

Discussion

Because Cox-2 is inducible by a wide range of stimuli and

plays an important role in a wide range of pathophysiol-

ogies, the repeated, quantitative, noninvasive monitoring of

Cox-2 expression in living animals would have significant

utility. In this study, we generated a knock-in mouse in

which the coding region of a luciferase reporter gene is

placed at the beginning of the coding region of the en-

dogenous Cox-2 gene. The Bknock-in^ strategy provides

what should be the most reliable model for monitoring

reporter gene expression from the Cox-2 regulatory region

because both the luciferase coding region integrated within

the transcribed region of the endogenous Cox-2 gene and

the endogenous Cox-2 coding region should be transcrip-

tionally regulated in the same manner, without chromosom-

al positional effects that may occur in transgenic mice.

Luciferase reporter gene expression from the Cox-2luc

knock-in allele was first compared with Cox-2 expression in

mouse embryonic fibroblasts cultured from Cox-2luc/+

embryos. Cox-2 was first isolated as one of the Bprimary

response^ genes induced by TPA in Swiss 3T3 fibroblasts

[2]. Cox-2 is also induced by mitogens and tumor promoters

in mouse embryonic fibroblasts [28]. There is an excellent

correlation between luciferase activity and Cox-2 protein

expression in Cox-2luc/+ MEFs following a six-hour expo-

sure to TPA, elevated serum, PDGF, or EGF.

Glucocorticoid inhibition of mitogen-induced Cox-2

expression was first observed in fibroblast cell lines [4, 24]

and is also a property of MEFs [29], as well as many other

cell types and tissues. Despite many attempts using trans-

fection experiments with Cox-2/luciferase plasmids, gluco-

corticoid inhibition has been difficult to demonstrate with a

luciferase reporter. In contrast, glucocorticoids inhibit lu-

ciferase induction in Cox-2luc/+ MEFs and macrophages,

suggesting that endogenous chromatin conformation, and

not the reporter gene, accounts for the lack of inhibition in

transfection experiments.

Induction of both Cox-2 protein and luciferase demon-

strate a gene dosage effect in unstimulated Cox-2+/+, Cox-

2luc/+, and Cox-2luc/luc MEFs, and in MEFs treated with

mitogen. This result indicates that we can accurately monitor

Cox-2 transcriptional activation by examining luciferase

activity of Cox-2luc/+ heterozygous cells or mice. We have

also demonstrated that luciferase activity, following induc-

tion in Cox-2luc/+ fibroblasts, is elevated coordinately with

unprocessed Cox-2 transcripts, using an intron-PCR ap-

proach previously developed in our laboratory [30] (data not

shown). Although Cox-2+/j heterozygous mice have differ-

ent tumor susceptibility [15, 31], they have a mostly normal

phenotype. We anticipate that by using Cox-2luc/+ hetero-

zygous mice, it will be possible to monitor noninvasively

Cox-2 expression in a wide range of pathophysiological

conditions.

Because the Cox-2luc knock-in allele also serves as Cox-2

Bknockout^ allele with regard to Cox-2 protein expression,

we could investigate whether the Cox-2 protein or any of

the prostanoid products resulting from Cox-2 induction

affect transcriptional activation of the Cox-2 gene by using

Cox-2luc/j cells or mice. A number of studies have focused

on the feedback control of Cox-2 gene expression; however,

the results of these studies have been varied and dependent


on the cell type. Studies using human synovial fibroblasts,

monocytes, and a prostate cancer cell line show positive

effects of PGE2 on Cox-2 expression [32Y34]. 15d-PGJ2, a

metabolite of PGD2, is reported to down-regulate Cox-2

expression in macrophage-like differentiated cell line

(U937) and rheumatoid synovial fibroblasts (RSFs) [35,

36], but not in the BAEC bovine endothelial cell line or in

human umbilical vein endothelial cells [35]. In mouse lung

fibroblast cells, PGE2, 15d-PGJ2, 6-keto PGF1�, and

PGF2� each enhanced Cox-2 protein expression [37]. In

our experiments, we do not see a difference in luciferase

induction when comparing Cox-2luc/j and Cox-2luc/+ het-

erozygous fibroblasts. Cox-2luc/j mice and other primary

cell cultures from these mice will be of great use in studying

the relationship between Cox-2-mediated prostanoid pro-

duction and transcription from the Cox-2 gene.

Ex vivo analysis shows that basal luciferase activity is

expressed in many tissues of the Cox-2luc/+ mouse. Highest

constitutive luciferase expression from the Cox-2luc allele is

observed in the vas deferens. Constitutive high Cox-2 ex-

pression in vas deferens was reported in rats [38], humans

[39], and mice [25]. In rat, castration severely depletes Cox-

2 and androgen replacement after castration restores Cox-2,

indicating that Cox-2 expression in the vas deferens is an-

drogen dependent [38]. Intermediate levels of luciferase

activity were observed in brain, thymus, lung, stomach,

pancreas, kidney, intestine, uterus, and skin in the Cox-2luc/+

mouse; lowest constitutive luciferase activity occurs in liver,

spleen, and heart. These results are consistent with a pre-

viously reported RTYPCR analysis of Cox-2 mRNA expres-

sion in mouse tissues under normal, uninduced conditions

[40].

Cox-2 induction is as an important early response to in-

fection. LPS stimulates Cox-2 expression by initiating

several signaling pathways, activating multiple transcription

factors to initiate Cox-2 mRNA expression [41, 42]. LPS

also induces other pro-inflammatory cytokines, including

IFN-g and interferon regulatory factors (IRFs), which are

important in the overall regulation of Cox-2 expression in

response to LPS [43]. Moreover, exogenously added IFN-ginduces Cox-2 expression in synergy with LPS [44]. To

obtain greater Cox-2 expression in systemic inflammation

experiments, we pretreated mice with IFN-g before LPS

injection. Cox-2 protein levels were higher in all tissues in

mice treated with [IFN-g + LPS] than in mice treated only

with LPS (data not shown). As demonstrated both by ex

vivo imaging of tissues and by luciferase assay of tissue

extracts, [LPS + IFN-g] treatment induces luciferase ex-

pression in all the tissues examined. There are no gender

differences in induction of luciferase activity.

In the acute inflammation study with LPS + IFN-g, we

see some differences in the relative levels of Cox-2 protein

and luciferase activity in tissues from Cox-2luc/+ mice. The

most likely explanation for this discrepancy in Cox-2 pro-

tein levels and luciferase activity is posttranscriptional regu-

lation of Cox-2 mRNA stability (reviewed in [7, 45]). The

Cox-2 30 untranslated region (UTR) contains 22 BARE^elements, which regulate the stability and translation of a

number of mRNAs whose expression is modulated by ex-

tracellular stimuli [46]. The Cox-2luc allele does not contain

the 30 UTR of the Cox-2 gene; the luciferase allele has an

SV40 30 UTR instead of Cox-2 30UTR. The activation of the

p38 MAPK pathway stabilizes Cox-2 message [47, 48]; in-

hibition of the p38 pathway leads to a decrease in Cox-2

message [49, 50]. Both stability of the Cox-2 message and

its translation are regulated by proteins that bind to the Cox-

2 AREs. HuR [51, 52], AUF1 [53], CPF-A [54], and TTP

[55] are reported to regulate message stability, and TIAR,

TIA-1 [56, 57], and CUGBP2 [58] are reported to regulate

Cox-2 message translation. Furthermore, activation of cells

with LPS results in modification of these RNAYprotein

complexes [59] and concomitant modulation of their

abilities to mediate Cox-2 mRNA stability and translation.

Although essentially all the studies of Cox-2 message sta-

bility and translational regulation have been performed in

cell culture, posttranscriptional mechanisms are certain to

play a role in regulation of Cox-2 expression in tissue-/cell-

type-specific manners in living animals. In Cox-2luc/+ mice,

luciferase expression reflects Cox-2 promoter activity. We

are currently developing murine strains to monitor, repeat-

edly and noninvasively in living animals, posttranscriptional

effects on Cox-2 mRNA stability and translation. In this

model, a transgenic mouse will be created in which the

coding sequence for the luciferase reporter gene will be

driven by a constitutive promoter and will be followed by the

30 UTR of the Cox-2 mRNA. To isolate the studies of Cox-2

mRNA stabilization to specific target tissues, a loxP flanked

STOP cassette will be used to restrict transgene expression to

tissues in which Cre recombinase is expressed.

Zymosan or carrageenan injections into the mouse paw

provoke inflammatory reactions [60, 61]. NSAIDs inhibit

edema in both zymosan- and carrageenan-induced inflam-

mation, and Cox-2 mRNA and protein expression has been

analyzed in the carrageenan-induced paw inflammation

model [62, 63]. We examined, by Western blotting, Cox-2

protein levels in both the zymosan- and carrageenan-

induced paw inflammation models in wild-type mice.

Because peak Cox-2 protein was higher in the zymosan-

induced model than in the carrageenan-induced model (data

not shown), we used the zymosan model for in vivo

imaging with Cox-2luc/+ mice. In wild-type mice, zymosan

induced substantial Cox-2 protein at both 6 and 12 hours

after injection. Local administration of zymosan into the

paw triggers both luciferase and Cox-2 protein induction

only in the zymosan-treated paw. However, the luciferase

signal is attenuated substantially before Cox-2 protein levels

are reduced (Fig. 5). The half-life of both firefly luciferase

and Cox-2 protein are estimated to be three to four hours

[64, 65]. Although these measurements were performed in

cell culture, it is likely that the two proteins have close to

the same half-lives in vivo as well. Consequently, we suggest

that the differences in Cox-2 protein and luciferase protein


accumulation in the paw of zymosan-injected Cox-2luc/+

mice reflects the additional posttranscriptional regulation

conferred by the 30 UTR region of the Cox-2 mRNA, as

discussed previously. Although cell culture experiments

show a strong correlation between luciferase activity and

transcriptional activation of the endogenous Cox-2 gene in

Cox-2luc/+ MEFs, similar experiments in Cox-2luc/+ mice

need to be done to confirm the extrapolation of this ex-

planation to in vivo inflammatory responses.

Dexamethasone, the most widely used anti-inflammatory

glucocorticoid in animal model experiments, inhibits Cox-2

expression in vitro and in vivo [24, 26]. Dexamethasone

inhibits both luciferase induction and Cox-2 protein accu-

mulation in the zymosan paw inflammation model. Howev-

er, luciferase inhibition by dexamethasone is not as

extensive as inhibition of Cox-2 protein accumulation. The

difference in dexamethasone sensitivity could also be

caused by the differences in 30 UTR regions for the Cox-

2+ and Cox-2luc alleles of Cox-2luc/+ mice; glucocorticoids

inhibit p38 MAPK function and destabilize Cox-2 mRNA

[66]. Noninvasive in vivo studies of the regulation of Cox-2

mRNA by inflammation and other stimuli await the de-

velopment of appropriate mouse models.

To demonstrate the potential utility of Cox-2luc/+ mice

in examining synovial inflammation, we examined Cox-2

and luciferase expression in the air pouch inflammation

model. Mechanical disruption of subcutaneous tissue by

air injection provides a structure closely resembling

synovial tissue [67]. Moreover, Cox-2 protein accumula-

tion is induced by injection of zymosan into the air pouch

[22]. Cox-2 expression is observed in both resident cells of

the pouch lining tissue and the recruited cells, predomi-

nantly polymorphonuclear neutrophils [27]. We modified

the original protocol, making two smaller air pouches on

individual mice, to compare Cox-2 and luciferase induc-

tion by zymosan and saline on same mouse. Cox-2 protein

accumulation and luciferase induction occur only in the

zymosan injected air pouch. We isolated cells from the air

pouch of zymosan treated Cox-2luc/+ mice, and demon-

strated Cox-2 and luciferase coexpression in essentially all

positive cells. Colocalization of Cox-2 and luciferase

expression was also confirmed in LPS-stimulated peritoneal

macrophages isolated from Cox-2luc/+ mice. These colocal-

ization studies, from both inflammatory challenges in the

whole animal and in isolated cells, demonstrate the validity

of surrogate luciferase expression as a measure of endog-

enous Cox-2 gene transcription in the Cox-2luc/+ heterozy-

gous mouse model.

In conclusion, we have established a murine strain with

a Cox-2luc knock-in allele and demonstrated the monitoring

of Cox-2 endogenous promoter-driven luciferase expres-

sion, noninvasively and repeatedly, in living animals. The

Cox-2luc/+ mouse should become a useful tool to analyze

noninvasively Cox-2 expression in a variety of pathophys-

iological conditions, including inflammation, neurodegener-

ative diseases, and cancer.

Acknowledgments. We thank Art Catapang and Raymond Bascontillo fortechnical assistance, Masanobu Oshima for genomic fragments, AlexGarcia and Hong Wu for assistance with immunohistochemistry andimmunofluorescence, and Shankkar Pattabhiraman and Hong Wu forhelpful discussions and comments. We also acknowledge the importantassistance of the UCLA Jonsson Comprehensive Cancer Center SharedResource for embryonic stem cell homologous recombination, and fortransgenic/knockout mouse production. These studies were supported bygrants R01 CA84572 and P50 CA86306 (H.R.H., principal investigator).

References1. Herschman HR, Talley JJ, Dubois R (2003) Cyclooxygenase 2 (COX-

2) as a target for therapy and noninvasive imaging. Mol Imaging Biol5:286Y303

2. Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR(1991) TIS10, a phorbol ester tumor promoter-inducible mRNA fromSwiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxyge-nase homologue. J Biol Chem 266:12866 Y12872

3. Xie W, Chipman JG, Robertson DL, Erikson RL, Simmons DL (1991)Expression of mitogen-responsive gene encoding prostaglandin syn-thase/cyclooxygenase homologue. Proc Natl Acad Sci U S A 88:2692Y2696

4. O’Banion MK, Sadowski HB, Winn V, Young DA (1991) A serum-and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclo-oxygenase-related protein. J Biol Chem 266:12866 Y12872

5. Herschman HR (2003) Historical aspects of COX-2: Cloning andcharacterization of the cDNA, protein and gene. In: Harris RE (ed)COX-2 blockade in cancer prevention and therapy. Clifton, NJ:Humana Press, pp 13Y32

6. Herschman HR (2004) Regulation and function of prostaglandinsynthase 2/cyclooxygenase. In: Curtis-Prior P (ed) The eicosanoids.New York: John Wiley & Sons, Ltd

7. Espel E (2005) The role of the AU-rich elements of mRNAs incontrolling translation. Semin Cell Dev Biol 16:59Y67

8. Smith LH, Petrie MS, Morrow JD, Oates JA, Vaughan DE (2005) Thesterol response element binding protein regulates cyclooxygenase-2gene expression in endothelial cells. J Lipid Res 46:862Y871

9. Duque J, Fresno M, Iniguez A (2005) Expression and function of thenuclear factor of activated T cells in colon carcinoma cells. J BiolChem 280:8686Y8693

10. Joo M, Park GY, Wright JG, et al. (2004) Transcriptional regulation ofthe cyclooxygenase-2 gene in macrophages by PU.1. J Biol Chem279:6658Y6665

11. Wang S-C, Lien H-C, Xia W, et al. (2004) Binding at and trans-activation of the COX-2 promoter by nuclear tyrosine kinase receptorErbB-2. Cancer Cell 6:251Y261

12. Herschman HR (2003) Molecular imaging: Looking at problems,seeing solutions. Science 302:605Y608

13. Contag PR, Olum IN, Stevenson DK, Contag CH (1998) Biolumines-cent indicators in living mammals. Nat Med 4:245

14. Nguyen JT, Machado H, Herschman HR (2003) Repetitive, noninva-sive imaging of cyclooxytenase-2 gene expression in living mice. MolImaging Biol 5:248Y256

15. Oshima M, Dinchuck JE, Kargman SL, et al. (1996) Suppressionof intestinal polyposis in ApcD716 knockout mice by inhibition ofcyclooxygenase 2 (COX-2). Cell 87:803Y809

16. Ishikawa T, Tamai Y, Qin L, Oshima M, Taketo MM (2003)Requirement for tumor suppressor Apc in the morphogenesis ofanterior and ventral mouse embryo. Dev Biol 253:230Y246

17. Zhang X, Morham SG, Langenbach R, Bagg RB, Young DA (2000)Lack of cyclooxygenase-2 inhibits growth of teratocarcinomas in mice.Exp Cell Res 254:232Y240

18. Hogan B, Beddington R, Costantini F, Lacy E (1994) Manipulating themouse embryo. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress

19. Tegeder I, Del Turco D, Schmidtko A, et al. (2004) Reducedinflammatory hyperalgesia with preservation of acute thermal noci-ception in mice lacking cGMP-dependent protein kinase I. Proc NatlAcad Sci U S A 101:3253Y3257

20. Zhang N, Weber A, Li B, et al. (2003) An inducible nitric oxidesynthaseYluciferase reporter system for in vivo testing of anti-inflam-matory compounds in trasgenic mice. J Immunol 170:6307Y6319


21. Zhang N, Ahsan MH, Zhu L, et al. (2005) NF-kB and not the MAPKsignaling pathway regulates GADD45b expression during acuteinflammation. J Biol Chem 280:21400 Y21408

22. Posadas I, Terencio MC, Guillen I, et al. (2000) Co-regulation betweencyclo-oxygenase-2 and inducible nitric oxide synthase expression inthe time-course of murine inflammation. Naunyn Schmiedebergs ArchPharmacol 361:98Y106

23. Lewandoski M (2001) Conditional control of gene expression in themouse. Nat Rev Genet 2:735Y755

24. Kujubu DA, Herschman HR (1992) Dexamethasone inhibits mitogeninduction of the TIS10 prostaglandin synthase/cyclooxygenase gene.J Biol Chem 267:7991Y7994

25. Lazarus M, Munday CJ, Eguchi N, et al. (2002) Immunohistochemicallocalization of microsomal PGE synthase-1 and cyclooxygenases inmale mouse reproductive organs. Endocrinology 143:2410Y2419

26. Masferrer JL, Zweifel BS, Manning PT, et al. (1994) Selectiveinhibition of inducible cyclooxygenase 2 in vivo is antiinflammatoryand nonulcerogenic. Proc Natl Acad Sci U S A 91:3228Y3232

27. Cadieux J-S, Leclerc P, St-Onge M, et al. (2005) Potentiation ofneutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatorysignal. J Cell Sci 118:1437Y1447

28. Reddy ST, Herschman HR (1994) Ligand-induced prostaglandinsynthesis requires expression of the TIS10/PGS-2 prostaglandinsynthase gene in murine fibroblasts and macrophage. J Biol Chem269:15437Y15480

29. Gilbert RS, Reddy ST, Kujubu DA, et al. (1994) TransformingGrowth factor b1 augments mitogen-induced prostaglandin synthesisand expression of the TIS10/prostaglandin synthase 2 gene both inSwiss 3T3 cells and in murine embryo fibroblasts. J Cell Physiol159:67Y75

30. Reddy ST, Gilbert RS, Xie W, Luner S, Herschman HR (1994) TGF-beta inhibits both endotoxin-induced prostaglandin synthesis andexpression of the TIS10/prostaglandin synthase 2 gene in murinemacrophages. J Leuk Biol 55:192Y200

31. Tiano HF, Loftin CD, Akunda J, et al. (2002) Deficiency of eithercyclooxygenase (COX)-1 or COX-2 alters epidermal differentiationand reduces mouse skin tumorigenesis. Can Res 62:3395Y3401

32. Faour WH, He Y, He QW, et al. (2001) Prostaglandin E2 regulates thelevel and stability of cyclooxygenase-2 mRNA through activation ofp38 mitogen-activated protein kinase in interleukin-1 beta-treatedhuman synovial fibroblasts. J Biol Chem 276:31720 Y31731

33. Hinz B, Brune K, Pahl A (2000) Cyclooxygenase-2 expression inlipopolysaccharide-stimulated human monocytes is modulated bycyclic AMP, prostaglandin E2, and nonsteroidal anti-inflammatorydrugs. Biochem Biophys Res Commun 278:790Y796

34. Trandrawinata RR, Hughes-Fulford M (1997) Up-regulation of cyclo-oxygenase-2 by product-prostaglandin E2. Adv Exp Med Biol407:163Y170

35. Inoue H, Tanabe T, Umesono K (2000) Feedback control of cyclo-oxygenase-2 expression through PPARg. J Biol Chem 275:28028Y28032

36. Tsubouchi Y, Kawahito Y, Kohno M, et al. (2002) Feedback controlof the arachidonate cascade in rheumatoid synoviocytes by 15-deoxy-D12,14-prostaglandin J2. Biochem Biophys Res Commun283:750Y755

37. Vichai V, Suyarnsesthaforn C, Pittayakhajonwut D, Sriklung K,Kirtikara K (2005) Positive feedback regulation of COX-2 expressionby prostaglandin metabolites. Inflamm Res 54:163Y172

38. McKanna JA, Zhang M-Z, Wang J-L, Cheng H-F, Harris RC (1998)Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am JPhysiol 275:R227YR233

39. Kirschenbaum A, Liotta DR, Yao S, et al. (2000) Immunohistochem-ical localization of cyclooxygenase-1 and cyclooxygenase-2 in thehuman fetal and adult male reproductive tracts. J Clin EndocrinolMetab 85:3436Y3441

40. Oshima M, Oshima H, Taketo MM (2005) Hypergravity inducesexpression of cyclooxygenase-2 in the heart vessels. Biochem BiophysRes Commun 330:928Y933

41. Smith WL, DeWitt DL, Garavito RM (2000) Cyclooxygenases:Structural, cellular, and molecular biology. Annu Rev Biochem 69:145Y182

42. Hwang D (2001) Modulation of the expression of cyclooxygenase-2 byfatty acids mediated through toll-like receptor 4-derived signalingpathways. FASEB J 15:2556Y2564

43. Zhang S, Thomas K, Blanco JCG, Salkowski CA, Vogel SN (2002)The role of the interferon regulatory factors, IRF-1 and IRF-2, in LPS-induced cyclooxygenase-2 (COX-2) expression in vivo and in vitro.J Endotoxin Res 8:381Y390

44. Blanco JCG, Contursi C, Salkowski CA, et al. (2000) Interferonregulatory factor (IRF)-1 and IRF-2 regulate interferon g-dependentcyclooxygenase 2 expression. J Exp Med 191:2131Y2144

45. Dean JLE, Sully G, Clark AR, Saklatvala J (2004) The involvement ofAU-rich element-binding proteins in p38 mitogen-activated proteinkinase pathway-mediated mRNA stabilisation. Cell Signal 16:1113Y1121

46. Saklatvala J, Dean J, Clark A (2003) Control of the expression ofinflammatory response genes. Biochem Soc Symp 70:95Y105

47. Srivastava SK, Tetsuka T, Daphna-Ikem D, Morrison AR (1994) IL-1beta stabilizes COX II mRNA in renal mesangial cells: Role of 30-untranslated region. Am J Physiol 267:F504YF508

48. Cok SJ, Morrison AR (2001) The 30-untranslated region of murinecyclooxygenase-2 contains multiple regulatory elements that altermessage stability and translational efficiency. J Biol Chem276:23179Y23185

49. Gou Q, Liu CH, Ben-Av P, Hla T (1998) Dissociation of basalturnover and cytokine-induced transcript stabilization of the humancyclooxygenase-2 mRNA by mutagenesis of the 30-untranslatedregion. Biochem Biophys Res Commun 242:508Y512

50. Dean JL, Brook M, Clark AR, Saklatvala J (1999) p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stabilityand transcription in lipopolysaccharide-treated human monocytes.J Biol Chem 274:264Y269

51. Dixon DA, Tolley ND, King PH, et al. (2001) Altered expression ofthe mRNA stability factor HuR promotes cyclooxygenase-2 expressionin colon cancer cells. J Clin Invest 108:1657Y1665

52. Sengupta S, Jang B-C, Wu M-T, et al. (2003) The RNA-bindingprotein HuR regulates the expression of cyclooxygenase-2. J BiolChem 278:25227Y25233

53. Lasa M, Mahtani KR, Finch A, et al. (2000) Regulation of cyclo-oxygenase 2 mRNA stability by the mitogen-activated protein kinasep38 signaling cascade. Mol Cell Biol 20:4265Y4270

54. Dean JLE, Sully G, Wait R, et al. (2002) Identification of a novel AU-rich-element-binding protein which is related to AUF1. Biochem J366:709Y719

55. Sawaoka H, Dixon DA, Oates JA, Boutaud O (2003) Tristetraprolinbinds to the 30-untranslated region of cyclooxygenase-2 mRNA. J BiolChem 278:36157Y36162

56. Cok SJ, Acton SJ, Morrison AR (2003) The proximal region of the 30-untranslated region of cyclooxygenase-2 is recognized by a multimericprotein complex containing HuR, TIA-1, TIAR, and the heterogeneousnuclear ribonucleoprotein U. J Biol Chem 278: 36157Y36162

57. Dixon DA, Balch GC, Kedersha N, et al. (2003) Regulation ofcyclooxygenase-2 expression by translational silencer TIA-1. J ExpMed 198:475Y 481

58. Mukhopadhyay D, Houchen CW, Kennedy S, Dieckgraefe BK, AnantS (2003) Coupled mRNA stabilization and translational silencing ofcyclooxygenase-2 by a novel RNA binding protein, CUGBP2. MolCell 11:113Y126

59. Cok SJ, Acton SJ, Sexton AE, Morrison AR (2004) Identification ofRNA-binding protein in RAW 264.7 cells that recognize alipopolysaccharide-responsive element in the 3-untranslated regionof the murine cyclooxygenase-2 mRNA. J Biol Chem 279:8196 Y8205

60. Tarayre JP, Delhon A, Aliaga M, et al. (1989) Pharmacological studieson zymosan inflammation in rats and mice. 1: Zymosan-induced pawoedema in rats and mice. Pharmacol Res 21:375Y384

61. Calhoun W, Chang J, Carlson RP (1987) Effect of selectedantiinflammatory agents and other drugs on zymosan, arachidonicacid, PAF and carrageenan induced paw edema in the mouse. AgentsActions 21:306Y309

62. Fernandez P, Guillen MI, Ubeda A, et al. (2003) A novel indazolo-triazolo-benzotriazepine exerts anti-inflammatory effects by inhibitionof cycloozygenase-2 activity and nitric oxide synthase-2 expression.Naunyn Schmiedebergs Arch Pharmacol 368:26Y32

63. Posadas I, Bucci M, Roviezzo F, et al. (2004) Carrageenan-inducedmouse paw oedema is biphasic, ageYweight dependent and displaysdifferential nitric oxide cyclooxygenase-2 expression. Br J Pharmacol142:331Y338


64. Leclerc GM, Bookfor FR, Faught WJ, Frawley LS (2000) Develop-ment of a destabilized firefly luciferase enzyme for measurement ofgene expression. Biotechniques 29:190Y598

65. Kujubu DA, Reddy ST, Fletcher BS, Herschman HR (1993)Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells. J Biol Chem268:5425Y5430

66. Lasa M, Mahtani KR, Finch A, et al. (2001) Dexamethasonedestabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activatedprotein kinase p38. Mol Cell Biol 21:771Y780

67. Edwards JC, Sedgwick AD, Willoughby DA (1981) The formationof a structure with the features of synovial lining by subcutaneousinjection of air: An in vivo tissue culture system. J Pathol 134:147Y156


Imaging Cyclooxygenase2 ( Cox2 ) Gene Expression in Living Animals with a Luciferase Knock-in...

Documents

Transcript of Imaging Cyclooxygenase2 ( Cox2 ) Gene Expression in Living Animals with a Luciferase Knock-in...