.1

. . .13‘ If... .

$49.91! .
13‘.

F .
1.». A
r E...
‘ in.

.3

t
«I z

23.. t...
. a.

mi. ....
W.‘ .11. \ l2
3. 3.2;:
.. . in}.
in:
an .....a

 

‘) . 1|

.1xiiL

A4393. :5

$1. 3:33....

.2... 11;.
tic-x
2.

Yin,
$.an

 

.
1:.
6

 

 

a
c

 

 

IND-{13d v

‘r:

3.
.2.
1...;
2H. 3..»

“-42.16% Ln.

      

6....
«.13.

 

:14
1:. .

.. z; .
m, .. .Wfiuu:€mum , .
V ‘ . . "$5,. ‘mvérw...r

 
 

f
31'.
(k) 53?
89

ll!“

 

 

 

 

IIIIIIII IIIIjI‘II IIIIIIIIII‘IIIII

 

LIBRARY
Mlchlgan State
Unlverslty

 

 

 

This is to certify that the

dissertation entitled

The Transcriptional Regulation of
Prostaglandin Endoperoxide Syntheses-1 and -2 by
2,3,7,8-Tetrachlorodibenzo-P-Dioxin

presented by

Stacey Anne Kraemer

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biochemistry

 

 

 

\\
124) 15 CA)
Major professor

”Md Deon

 

Date [21/13/517

MS U is an Affirmative Action/Equal Opportunity Institution 042771

 

 

 

PLACE" RETURN BOXtomw-uiudnckouflunwncord.
TO AVOID FINES Mum on or More data duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/EM Oppominlty Institution
WMJ

THE TRANSCRIPTIONAL REGULATION OF
PROSTAGLANDIN ENDOPEROXIDE SYNTHASES-l AND -2 BY
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
By

Stacey Anne Kraemer

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1996

ABSTRACT
THE TRANSCRIPTIONAL REGULATION OF PROSTAGLANDIN
ENDOPEROXIDE SYNTHASES-l AND -2 BY
2,3,7,8-TETRACHLORODIBENZO-P—DIOXIN
By

Stacey Anne Kraemer

Genomic clones for the entire PGH synthase-l (PGHS-l) gene and its 5’—flanking
region, and the 5’-flanking region of the PGH synthase-2 (PGHS-2) gene have been
isolated. The upstream elements of these genes have been analyzed to locate putative
regulatory sequences. The 5’-flanking region of both PGHS-1 and PGHS—2 contain
sequences with high similarity to the dioxin responsive element (DRE), a DNA sequence
found to control gene expression in response to exposure to halogenated aromatic
hydrocarbons. Using nuclear run-on assays to analyze the transcription of these genes,
and northern and western blotting to measure changes in PGHS-2 mRNA and protein
levels, we have examined the regulation of PGHS expression by 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) , the prototypical halogenated aromatic hydrocarbon,
using the Madin-Darby canine kidney (MDCK) cells as a model system.

TCDD treatment results in an increase in the rate of transcription of PGHS-2 in
these cells within one-half hour, followed by a transient elevation of PGHS-2 mRNA
levels after 2 hours, and subsequently, by increases in PGHS-2 protein levels after 4
hours. TCDD had no detectable effect on the transcription of the PGHS-l gene. Two
putative DREs were identified 165 base pairs and 1069 base pairs upstream from the

transcriptional start site of PGHS-2. Transient expression assays in MDCK cells,

Stacey Anne Kraemer

utilizing luciferase reporter plasmids containing the 5’-flanking regions of the PGHS-2
gene demonstrated statistically significant increases in luciferase expression from these
plasmids in response to TCDD treatment. Induction of PGHS-2 by TCDD may,
therefore, play a unique role in toxic responses to xenobiotic exposure. Increased PGHS-
2 may stimulate prostaglandin production leading to increased inflammation or altered
cell growth. Alternatively, elevated PGHS-2 levels may increase the oxidization of

xenobiotics to less or more toxic compounds.

ACKNOWLEDGMENTS

I owe a deep debt of gratitute to several people whose support and guidance so
significantly affected the direction and quality of this research project and dissertation
that a simple "thank you” seems inadequate. First and foremost among these are my
dissertation advisors Dr. David DeWitt and Dr. William Smith. I specifically would
like to thank Dr. David DeWitt for introducing me to the concepts and techniques of
molecular biology and transcriptional regulation with such infectious enthusiasm that I
soon altered my initial intentions to avoid molecular biology as much as humanly possible
in my graduate career to being entirely fascinated by it. To say that he taught me
everything I know about molecular biology would hardly be an exaggeration. I would
like to express my appreciation to Dr. William Smith for his lucid tutelage in the field
of prostanoids, for sharing his scientific insights and critical analyses on this project.

I would like to convey my thanks to the members of my graduate committee, Dr.
Michael Denison, Dr. Jerry Dodgson, Dr. Robert Roth, Dr. Lee Kroos, and Dr. Zachary
Burton for their guidance. Especially, I would like to thank Dr. Denison for many acute
analyses, one of which notably provided the initial impetus to explore a possible role of
TCDD in the regulation of PGHS. In addition, I would like to thank him for generously
sharing his material and facilities for such studies to transpire.

My deepest appreciation goes to my friends and collegues in the Smith and

iv

DeWitt laboratories for their help and support. In particular, I am greatly indebted to
Dr. Elizabeth Meade, whose scientific contributions, particularly in mapping the PGHS-1
gene, were indispensable to the project, and whose camaraderie was invaluable. Thanks
to Virginia Leykam for her support in managing the technical aspects of TCDD. I
would like to thank my many friends, who most generously tolerate my "venting"
monologues, among my other endearing qualities. Special thanks go out to my "sanity
patrol": Beta and Chris Meyer, Virginia and Joe Leykam, and Marty Regier, whose
encouragement enabled me to obtain greatly needed perspective and strength to overcome
difficult times.

Lastly, I would like to express my deepest gratitude to my parents and sister,
Karen, for all of their love and support. I specifically want to thank my father for his
no-nonsense (and his occassional humorous nonsensical) guidance, and for always
encouraging my competitive drive, which is so vital in this business. I thank my mother
for her poise in her constant balancing act between "Mom" and "Dr. Kraemer"-- between
providing comfort and professional advice, and for always furnishing me with a

professional role model without ever having to look further than home.

TABLE OF CONTENTS

LIST OF TABLES ....................................

LIST OF FIGURES ....................................

ABBREVIATIONS .....................................

CHAPTER 1: LITERATURE REVIEW

PGH Synthases ...................................
TCDD .........................................

CHAPTER 2: GENE STRUCTURE, TRANSCRIPTIONAL START SITE
ANALYSIS, AND ANALYSIS OF THE 5’-FLANKING SEQUENCES
OF THE PGHS-1 GENE

Introduction .....................................
Methods .......................................
Results ........................................

CHAPTER 3: INDUCTION OF PGHS-2 AND PGHS-1 BY 2,3,7,8-
TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN MADIN-DARBY
CANINE KIDNEY (MDCK) CELLS

Introduction .....................................
Methods .......................................
Results ........................................
Discussion ......................................

CONCLUSION .......................................

BIBLIOGRAPHY ......................................

vi

vii

viii

Table 1

Table 2

Table 3

Table 4.

Table 5.

Table 6.

LIST OF TABLES

Factors which affect PGHS-1 expression in various cell lines and
tissues ....................................

Factors which positively regulate PGHS-2 in various cell lines and
tissues ....................................

Corticosteroids which inhibit PGHS-2 stimulation in various cell
lines and tissues ..............................

Exon/Intron borders of mouse PGHS-1 ................

Oligonucleotides used to sequence the 5’-flanking region of
PGHS-2 ...................................

Assessment of the relative AhR binding affinity for the putative
PGHS-2 DREs ...............................

vii

LIST OF FIGURES

Figure 1. Biosynthetic pathway for prostaglandin formation .......... 2
Figure 2. Mechanism of PGHS oxidation of arachidonic acid ......... 5
Figure 3. Comparison of the amino acid sequences of mouse PGHS-1 and
PGHS-2 ................................... 8
Figure 4. Mechanism of PGHS-mediated co-oxidation of xenobiotics . . . . 19
Figure 5. Structures of examples of polycyclic aromatic hydrocarbons . . . . 22
Figure 6. Mechanism of TCDD/AhR activation ................. 24
Figure 7. Alignments of DREs from the cytochrome P4SOIA1 and
glutathione-S-transferase genes ...................... 30
Figure 8. EcoRI restriction map of mouse PGHS-1 and PGHS-2 cDNAs . . 35

Figure 9. Sequence of the mouse PGHS-1 cDNA, showing the location of
the Oligonucleotides used for probes in Southern blots and primer
extension analysis ............................. 38

Figure 10. Construction of the plasmid used to produce an antisense mouse

genomic PGHS-1 probe used for RNase protection analysis . . . . 42
Figure 11. Construction of the luciferase reporter plasmid containing the 5’-

flanking region of the mouse PGHS-1 gene .............. 47
Figure 12. Construction of the luciferase reporter plasmid containing the 5’-

flanking region of the mouse PGHS-2 gene (PGHS-2mm) ...... 50
Figure 13. Sequence of the PGHS-2,,hon insert ................... 53

Figure 14. Restriction endonuclease map of the mouse PGHS-1 gene, and the
AFIXII clones from which it was derived ............... 57

viii

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Figure 25.

Figure 26.

Figure 27.

Figure 28.
Figure 29.

Figure 30.

Sequence of the mouse PGHS-1 gene

Mapping of the transcriptional start site of the mouse PGHS-l gene
using primer extension analysis .....................

Mapping of the transcriptional start site of the mouse PGHS-1 gene
using RNase protection analysis .....................

Sequence of the 5’ flanking region of the mouse PGHS-l gene,
showing location of putative responsive elements ...........

Luciferase activities observed upon serum induction in transfected
3T3 cells ...................................

Correlation of the mouse PGHS-1 exons with specific functions of
the PGHS enzyme .............................
control

plasmids, pGL2-Basic and

Luciferase reporter
pGUDLUCLl

Construction of the luciferase reporter plasmids containing the 5’-
flanking region of mouse PGHS-2

Sequence of the PGHS-2,0,8 insert, showing locations of putative
responsive elements ............................

Western blot analysis of TCDD induction of PGHS-2 in MDCK
cells ......................................

Northern blot analysis of TCDD induction of PGHS-2 in MDCK
cells ......................................

Transcription of the PGHS-2 and PGHS-1 genes as determined by
nuclear run-on assays ...........................

Luciferase activities Observed upon TCDD induction in transfected
MDCK cells .................................

Sequence of events in TCDD-stimulation of PGHS-2 expression
Speculative roles for PGHS-2 in TCDD-induced toxic effects . . .

Models for thromboxane Az-mediated thymocyte negative selection
under normal conditions, and after exposure with TCDD ......

ix

65

69

71

75

78

100

106

109

113

118

121

128

132

AHH
AhR
ARE
Amt
CAMP
APC
CAT
DME
DRE
DTT
EGF
EpRE
FSH
HAH
hCG
IL-lB

LDso
LH

LPS
MDCK
N F KB
GAPDH
nGRE
N SAID
PAH
PAI-2
PAS
PCB
PCR
PDGF
PGHS-1
PGHS-2
TBS
TCDD
TGF-oz
TPA

ABBREVIATIONS

aryl hydrocarbon hydroxylase
aromatic hydrocarbon receptor
aromatic responsive element

AhR nuclear translocation protein
cyclic adenosine 5’-3’-monophosphate
antigen presenting cell
chloramphenicol acetyl transferase
Dulbecco’s modified Eagle’s

dioxin responsive element
dithiothreitol

epidermal growth factor
electrophilic responsive element
follicle stimulating hormone
halogenated aromatic hydrocarbon
human chorionic gonadotropin
interleukin 16

lambda bacteriophage

lethal dose 50

leutinizing hormone
lipopolysaccharide

Madin-Darby canine kidney
nuclear factor KB
glyceraldehyde—3-phosphate dehydrogenase
negative glucocorticoid responsive element
non-steroidal antiinflammatory drug
polycyclic aromatic hydrocarbon
plasminogen activator inhibitor-2
Per-Amt-Sim (homology domain)
polychlorobiphenyl

polymerase chain reaction
platelet—derived growth factor
prostaglandin G/H synthase-l
prostaglandin G/H synthase—2

tris buffered saline
2,3,7,8—tetrachlorodibenzo—p-dioxin
transforming growth factor-0:
lZ-O-tetradecanoyl l3-acetate

CHAPTER 1

LITERATURE REVIEW

PGH Synthases

Prostaglandins are a family of autocoids which regulate biological processes such
as vascular homeostasis, kidney function, maintenance of pregnancy, and initiation of
parturition, bone resorption, and inflammation [1-9]. Prostaglandins are not stored, but
rather are synthesized de novo, from fatty acid precursors, primarily arachidonic acid,
in response to an external stimulus, such as circulating hormones, cytokines, or proteases
[10-15]. Prostaglandins are rapidly metabolized, and therefore are thought to be active
only near their site of synthesis; hence, they have been termed local hormones [1].
Prostaglandin production is regulated acutely by the activation of phospholipases that
release arachidonic acid from cellular phospholipid stores. Arachidonate is next
converted to PGH2 by the action of one of two PGH synthase (prostaglandin
endoperoxide synthase, cyclooxygenase) isozymes (Figure 1). Both enzymes catalyze
two identical reactions: an oxygenation and a peroxidation. The cyclooxygenation
reaction adds one molecule of oxygen to the C-9 and C-11 positions forming a cyclic
oxygen bridge, and a second molecule of oxygen at the C-15 position, ultimately yielding

the hydroperoxide PGGz. The peroxidation reduces the C-15 hydroperoxide in P662 to

Figure 1. Biosynthetic pathway for prostaglandin formation. Prostaglandin
production is acutely regulated by the activation of phospholipases which release
arachidonic acid from cellular phospholipid stores. PGH synthase first bis-oxygenates
arachidonate to form the hydroperoxide P062, and subsequently reduces P662 to form
PGHZ. Tissue-specific isomerases and reductases catalyze the conversion of PGH2 to the
biologically active prostaglandins and thromboxanes.

 

 

 

 

 

PATHWAY FOR PROSTANOID SYNTHESIS

CIRCULATORY HORMONE
GROWTH FACTOR
CYTOKINE

 

PHOSPHOLIPASE
ACITIVATION

— m
(ZZZ, momma
ACID

CYCLOOXYGENASE ’ °’
ACTIVITY

PGHS C00"
00!

\ X”

 

PEROXIDASE
AcnvrrY
OOOII
K322 :: : PGH 2
_ h
SPECIFIC

PROSTANOID SYNTHASES

 

 

first” 5 mm

 

 

4
form a C-15 hydroxyl, yielding PGH2 (Figure 2) [16]. All of the biologically active

prostaglandins and thromboxanes are derived from PGH2, by tissue-specific isomerases
and reductases (Figure 1) [1]. The observation that PGH synthase isozymes self-
inactivate during catalysis suggests the level of expression of these enzymes may limit
the synthesis of prostaglandins [17-21]. While the two PGH synthase isozymes are
catalytically similar, their expression is differentially regulated. PGHS-l is constitutively
expressed in differentiated cells, although its level may vary among cell types [22-27].
PGHS—2 is highly regulated; it is not expressed in most tissues, but can be induced by
a number of stimuli, such as growth factors, hormones, cytokines, and tumor promoters
[22, 26, 28-36].

The PGH synthases are the sites of pharmacological inhibition by non-steroidal
anti—inflammatory drugs (NSAIDs), such as aspirin, ibuprofen and indomethacin. The
NS AIDs reversibly or irreversibly are bound by PGH synthase, and competitively or non-
competitively inhibit arachidonate binding, thereby inhibiting the cyclooxygenase
reaction. Aspirin is unique among NSAIDs in that it not only binds to PGH synthase,
but also covalently modifies the enzyme. Acetylation by aspirin of a single specific
serine sterically blocks the arachidonic acid binding and irreversibly inhibits the
cyclooxygenase activity [37-40 ].

The anti-inflammatory properties of glucocorticoids are, in part, mediated by their
effects on expression of PGHS-2. Glucocorticoids have been shown to inhibit LPS-
induced PGHS-2 expression and subsequent prostaglandin production in peritoneal
macrophages isolated from adrenalectomized rats [41], as well as in synovial tissues in

patients with rheumatoid arthritis [42, 43]. In vitro, dexamethasone inhibits serum

Figure 2. Mechanism of PGHS oxidation of arachidonic acid to PGGZ. The
cyclooxygenase activity of PGHS catalyzes the initial removal of the 13-pro-S hydrogen
of arachidonic acid, followed by addition of two molecules of 02: one to the C-11
position, which followed by the formation of a cyclic oxygen bridge at the C-9 position,
and the other to the C-15 position, ultimately yielding the hydroperoxide 13662. Figure

reproduced from [16].

 

 

 

:00

 

1000

2000

:00

:000

1000

 

7

stimulation of PGHS-2 expression in mouse 3T3 fibroblasts [44, 45 ], phorbol ester-
stimulated PGHS-2 expression in Swiss 3T3 cells [46, 47], and human monocytes [48,
49], LPS-induced PGHS-2 expression in human monocytes [30, 50], IL—l stimulated
synthesis of PGHS-2 in human dermal fibroblasts [51] and rat aortic smooth muscle [52],
as well as v-src induction of PGHS-2 in NIH3T3 fibroblasts [53].

PGHS-1 and PGHS-2 have been localized to the endoplasmic reticulum and
perinuclear membranes, and PGHS-1 has further been shown to reside on the lumenal
surface of these membranes [54, 55]. Both isozymes contain a single heme [56], are
glycosylated, and have molecular weights of approximately 69-74 kilodaltons, (depending
on the extent of glycosylation) [57]. PGH synthases are homodimers [56, 5 8]. PGHS-1,
was first cloned in 1988, and is coded for by a 2.7-3.0 kb mRNA in sheep seminal
vesicles [37, 38, 59], mouse fibroblasts [39], rat peritoneal macrophages [60], and human
platelets [24, 61, 62]. A second isozyme, PGH synthase-2, coded for by a 4.8 kb RNA
was cloned first in 1991. cDNAs for this second gene have now been isolated from
mouse [22], chicken [63], human [64], and rat [60]. PGHS-1 and PGHS-2 are products
of separate genes; the PGHS-1 gene is located on chromosome 9 in humans [24], and
chromosome 2 in mice [65], while the PGHS-2 gene is located on chromosome 1 in
mouse and in humans [28, 29]. There is also some evidence that the human PGHS-2
gene is polymorphic in approximately 5% of the population [29].

The deduced amino acid sequences of mouse PGHS-1 and PGHS-2 are 63.9%
identical, and 79% similar (allowing for conservative amino acid substitutions) (Figure
3). Amino acids required for activity in PGHS-1 are preserved in PGHS-2, including

the heme binding site [56, 67], the aspirin acetylation site [39], the active-site tyrosine

Figure 3. Comparison of the amino acid sequences of mouse PGHS-1 and PGHS-2.
The deduced amino acid sequences between mouse PGHS-1 and PGHS-2 are 63.9%
identical, and 79% similar (allowing for conservative amino acid substitutions).
Conserved features between PGHS-1 and PGHS-2 are an EGF-like region, (defined by
the cysteines indicated by V), the proximal and distal heme binding sites (underlined, not
bold), the aspirin acetylation site serine (CI), and the active site tyrosine (O), 3
glycosylation sites (bold). Mouse PGHS-l contains 17 additional amino acids in the
amino-terminal signal peptide not found in mouse PGHS-2, while PGHS-2 contains an
18-amino acid C-terminal insertion, in comparison to PGHS-1 (underlined and bold).

 

 

 

 

PGHS-1 1
PGHS-2 1
51
34
101
84
150
134
200
184
250
234
300
284
350
334
400
384
450
434
500
484
550
534
587

584

v v v
MSRRSLSLWFPLLLLLLLPPTPSVLLADPGVPSPVNPCCYYPCQNQGVCV
.I. . .II I. II I I
'I°°°’ 'I l° I-- "ll llll'l :-
...............MLFRAV..LLCAALGLSQAANPCCSNPCQNRGECM
v.1 .v

RFGLDNYQCDCTRTGYSGPNCTIPEIWTWLRNSLRPSPSFTHFLLTHGYW

STGFDQYKCDCTRTGFYGENCTTPEFLTRIKLLLKPTPNTVHYILTHFKG

LWEFVNA. TFIREVLMRLVLTVRSNLIPSPPTYNSAHDYISWESFSNVSY

VWNIVNNIPFLRSLTMKYVLTSRSYLIDSPPTYNVHYGYKSWEAFSNLSY

YTRILPSVPKDCPTPMGTKGKKQLPDVQLLAQQLLLRREFIPAPQGTNIL

III II I.IIIIIIIIII.||I ooolIlI'II'II' I..
III II'I’°IIIIIII°II'I°III’°°°°IIIIIIII’III'I"

YTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRREFIPDPQGSNMM

FAFFAQHFTflQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLF

FAFFAQHFTHQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLF

KDGKLKYQVLDGEVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPG

IIIIIIIII..|IIIIII. IIIII.I I.IIIIIIIII.II
IIIIIIIII°°III|II°I°°°I I III’I" I'IIIIIIIII'II

KDGKLKYQVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPG

LMLFSTIWLREHNRVCDLLKEEHPTWDDEQLFQTTRLILIGETIKIVIEE

II..IIIIIIIIIIII.|I.IIII.IIII|IIIIIIIIIIIII|II.
II’°'IIIIIIIlllII'II‘III'I'IIIIIII‘IIIIIIIIIIIIIl'

LMMYATIWLREHNRVCDILKQEHPEWGDEQLFQTSRLILIGETIKIVIED
Q . O

YVQHLSGYFLQLKFDPELLFRAQFQYRNRIAMEFNHLYHWHPLMPNSFQV

YVQHLSGYHFKLKFDPELLFNQQFQYQNRIASEFNTLYHWHPLLPDTFNI

GSQEYSYEQFLFNTSMLVDYGVEALVDAFSRQRAGRIGGGRNFDYHVLHV

.IIII.III.II.I..I. .I. III III..IIII
00'I.'OO'I'O'OIO'OOO'OOOOI:O'OI' |||00||||ooo

EDQEYSFKQFLYNNSILLEHGLTQFVESFTRQIAGRVAGGRNVPIAVQAV

AVDVIKESREMRLQPFNEYRKRFGLKPYTSFQELTGEKEMAAELEELYGD

I.I I I IIIIII.IIIIIII.IIIIIIIIIIl
’ I"IIII"I‘:IIIIIII’IIIIIII’IIIIIIIIIIII°‘II°I

AKASIDQSREMKYQSLNEYRKRFSLKPYTSFEELTGEKEMAAELKALYSD
O a O

IDALEFYPGLLLEKCQPNSIFGESMIEMGAPFSLKGLLGNPICSPEYWKP

II.I.II.II.II I. IIII I.I.IIIIIIIII.IIIIII|.IIII
II"I'II°I|'II"I:'IIII'I'I'IIIIIIIII’IIIIIII‘IIII

IDVMELYPALLVEKPRPDAIFGETMVELGAPFSLKGLMGNPICSPQYWKP

STFGGDVGFNLVNTASLKKLVCLNTKTCPYVSFRVPD....... ..... .

IIIII.III..I|II. I.IIIII.IIII
IIIII'III"°IIII'°'I°I I‘I'Il"II'l‘I

STFGGEVGFKIINTASIQSLICNNVKGCPFTSFNVQDPQPTKTATINAfiA

....YPGDDGSVLV.RRSTEL 602

SHSRLDDINPTVLIKRRSTEL 604

100
83

149
133
199
183
249
233
299
283
349
333
399
383
449
433
499
483
549
533
586

583

betv

 

SifU

cart

leng

m‘I

n
u

isr

syr

 

[6%

EX
DI

 

 

10
[68], and three N—linked glycosylation sites [57]. An EGF-like domain is also conserved

between species which is thought to be involved in dimerization [56, 66]. The primary
structural differences between the PGHS amino acid sequences lie in the amino— and
carboxyl- termini. The two proteins have dissimilar signal peptides of slightly different
lengths, 17 amino acids for PGHS-2 and 23-26 amino acids for PGHS-1. A second
major difference is at the C-terrnini; PGHS-2 contains an 18 amino acid sequence that
is not present in PGHS-1. Recent studies have demonstrated that while both PGH
synthase isozymes have similar catalytic properties, they are pharmacologically distinct
[69, 70].

The most noteworthy difference between the two PGHS isozymes is their
differential expression and regulation. PGHS-1 is expressed in most differentiated cells
and tissues. Although small elevations in PGHS-1 mRNA have been detected [24, 26,
31, 33, 44, 49, 71, 72] in response to external stimulation, for the most part, PGHS-l
levels do not change and are maintained constant [22, 29, 46, 64] within a given cell type
(Table 1). Because PGHS-l does not demonstrate a high degree of regulation and is
expressed constitutively, it is thought to be responsible for the production of
prostaglandins in response to hormonal stimulation that are needed instantaneously to
regulate "house-keeping events" such as stomach or kidney function, or to regulate
vascular homeostasis. In contrast, PGHS-2 message and/or protein levels are rapidly and
dramatically increased by factors such as phorbol esters [22, 26, 28, 30, 46, 49, 64],
serum [22, 28, 44]; PDGF [28]; hCG [32]; forskolin [22, 46]; EGF [73] calcium
ionophore [74]; interleukin-1 [29]; cyclic AMP [28]; and lipopolysaccharide [29, 30].

A list of the tissue and cell lines that PGHS-2 has been shown to be regulated in is

 

 

 

 

present
after 11
implice
express

anUInI

restrict
potenti
mMm
in prod
funcrio

Inhibits

[2351f OI:

11

presented in Table 2. PGHS-2 expression is also increased in chicken embryo fibroblasts
after transformation with rous sarcoma virus [75] (Table 2). PGHS-2 has been
implicated in the inflammatory, and possibly the mitogenic, response due to its exclusive
expression in stimulated cells, its induction in inflammed tissues, and its inhibition by
anti-inflammatory agents, such as the glucocorticoid dexamethasone (Table 3).

Because of the importance of inflammation in human disease, the pharmacology
of the PGHS isozymes has undergone extensive investigation. NSAIDs are the most
frequently used class Of medicinal drugs and are used for their anti-inflammatory, anti-
pyretic and analgesic properties. Unfortunately, the deleterious side effects of NSAIDs,
including gastrointestinal bleeding, or complications in patients with kidney problems,
restrict their long-term application [76, 77]. It was proposed that these difficulties
potentially can be limited by using an NSAID which selectively inhibits PGHS-2 and its
production of pro-inflammatory prostaglandins, but not PGHS-1, which may be involved
in producing prostaglandins involved in protective stomach mucous secretion, or kidney
function. Recently, a novel analgesic and antipyretic drug, NS398, which preferentially
inhibits PGHS-2, was discovered, and initial animal experiments suggests that it lessens
gastrointestinal complications usually associated with NSAIDs [69, 78, 79].

More recently, aspirin or other NSAIDs have been examined for their usefulness
in other medical problems. Many of the beneficial effects of aspirin are due to aspirin’s
ability to inhibit thrombosis by selectively inhibiting platelet thromboxane synthesis.
Because restoration of PGHS after inhibition requires de novo protein synthesis, recovery
of platelet PGHS requires the production of new platelets. In contrast, active PGHS is

resynthesized in the surrounding nucleated endothelial cells in several hours, tipping the

12

Table 1. Factors which affect PGHS-1 expression in various cell lines or tissues.
(+, PGHS-1 is positively regulated by factor; -, PGHS-l is negatively regulated by
factor; NE=factor did not demonstrate any effect on PGHS-1.)

 

 

 

 

 

13

 

I... ma .. mu m

. ,scxp.

 

 

promonocytic cells (THP-l, + + 49,72
U937 cell lines)

 

HEL + 24

 

TPA human lung fibroblasts + 169

 

rat tracheal epithelium NE + 26
(EGV6)

 

mouse 3T3 NE + + 44

 

serum
human lung fibroblasts + 169

 

PDGF HEL + 24

 

IL-l HUVEC + 31

 

human lung fibroblasts + 169

 

TGF-B human lung fibroblasts + 45,191,
192

 

TN F-a human lung fibroblasts + 169

 

v-fes NIH3T3 + 45
trans form.

 

cAMP mouse osteoblastic cells + 193
(MC3T3-E1)

 

development ovine pulmonary arterial + + 163
endothelium

 

dexamethasone U937 49

TPA'

 

HBGF-l HUVEC - - 194

 

 

 

 

 

 

 

 

 

L? serr ser'f

rotein leveI changeSTin response to factor, demonstrated by western or immunoprecipitation
t’mRNA level changes in response to factor, demonstrated by northern analysis or quantitative PCR analysis
“Transcription rate changes in response to factor, demonstrated by run-on analysis.

*Transient expression assays utilizing regions of the PGHS-l gene in reporter plasmids demonstrate regulation in
response to factor.

°Stimulation by TPA is inhibited in response to factor.

fStimulation by serum is inhibited in response to factor.

14

Table 2. Factors which postively regulate PGHS-2 in various cell lines and tissues.
+, PGHS-2 is positively regulated by factor.

‘Protein level changes in response to factor, demonstrated by western or immunoprecipitation.

t’mRNA level changes in response to factor, demonstrated by northern analysis or quantitative PCR analysis.
“Transcription rate changes in response to factor, demonstrated by run-on analysis.

dTransient expression assays utilizing regions of the PGHS-2 gene in reporter plasmids demonstrate regulation in
response to factor.

 

 

effector

scmm

 

 

 

phcrbol

CSICI'S

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

155

 

' 'iL-icfifestsr 78:71 ‘f 37:7”-§;:zf7?¥35:f‘-’i?7¢¢11.1lies/rise7ii7iti‘;7; 3:131???

 

 

Us“:
' ’ @xp.¢-lxi7 .,

f,_ft‘ran’s'.f‘tly

Rf

 

serum

mouse 3T3 fibroblasts

22,23,
44,45,
146,170

 

mouse C127 fibroblasts

74

 

HUVEC

168

 

R82

45

 

rat mesangial

++++

195,196

 

rat tracheal

26

 

phorbol
esters

mouse 3T3 fibroblasts

22,28,
146

 

human fibroblasts (HUVEC,
ECV304)

29,64,
168

 

rat tracheal epithel. (EGV6)

26

 

rat mesangial cells

195

 

U937

49

 

IL-l

Human fibroblast (HUVEC,
ECV304, lung)

29,168

 

LPS

HUVEC

29

 

Macrophages (rat, human)

34,36,60

 

HUVEC

29

 

PDGF

mouse 3T3

28,33

 

EGF

mouse 3T3

22

 

rat tracheal epithelium

26

 

A23187

mouse fibroblasts (C127)

74

 

cAMP

mouse 3T3 fibroblasts

28

 

POE,

mouse 3T3 fibroblasts

33

 

forskolin

rat granulosa cells

171,172

 

mouse 3T3 fibroblasts

22

 

LH

rat granulosa cells

171,172

 

v-src
transform.

mouse 3T3 fibroblasts

197

 

CEF-l47

75

 

LA-90

53

 

 

v-fos, v-fes
transform.

 

mouse 3T3 fibroblasts

 

 

 

 

 

45

 

 

16

Table 3. Corticosteroids which inhibit PGHS-2 stimulation in various cell lines and
tissues. (-, stimulated PGHS-2 is negatively regulated by factor; NE, factor did not
demonstrate any effect on stimulated PGHS-2.)

 

 

 

 

l7

 

. _.‘._. L9Xp. 7. _

4 » = Senator; 75:555.:3f¢¢11°1indiissu¢g:‘:}ft :gwg, gins-NA»; iusé; :fi’raas.§;f Ref

 

aldosterone, mouse 3T3 fibroblasts 46

cortisol TPA'

 

mouse fibroblasts (3T3, C127) - 44,46,74
TPA",seI'f

 

RS-2 - N E 45
serf serf

 

dexamethasone - 26

rat tracheal epithelia (EGV6) -
TPAe TPAc

 

U937 - 49

TPA' TPA“

 

 

 

 

 

 

 

 

 

'Protein level changes “espouse t07actor, demonstrated 3y western or immunoprecipitatron.

.’mRNA level changes in response to factor, demonstrated by northern analysis or quantitative PCR analysis.
”Transcription rate changes in response to factor, demonstrated by run-on analysis.

*l‘ransient expression assays utilizing regions of the PGHS-2 gene in reporter plasmids demonstrate regulation
response to factor.

°Stimulation by TPA is inhibited in response to factor.

rStimulation by serum is inhibited in response to factor.

_0

l1

 

 

 

   
   

balance of \

of platelet a,
heart attacks
theahtithmh
aspirin or ot
stomach. col
mechanisms
or indirect.
through the
the co-oxt-
phenols, n
some of 1‘
carcinogc
cells Ihro

the inhih

18

balance of vascular prostaglandin production in favor of prostacyclin, an inhibitor of
platelet aggregation, and away from the thromboxane (produced by platelets) stimulation
of platelet aggregation [80, 81]. Studies have shown that aspirin can reduce the risk of
heart attacks, strokes, and pre-eclampsia [2, 82-86]. Presumably, these all result from
the antithrombotic effect of aspirin. Evidence from epidemiological studies indicate that
aspirin or other NSAIDs may also be effective in reducing the risk of esophogeal,
stomach, colon, and rectal cancer [87-91], although the mechanism is uncertain. Several
mechanisms have been postulated for this protection, including the inhibition of directly
or indirectly produced mutagens or carcinogen products, such as those synthesized
through the reduction of malondialdehyde, aside product of PGH2 production, or through
the co-oxygenation of various xenobiotic compounds, such as polycyclic hydrocarbons,
phenols, nitrofurans, and aromatic amines (Figure 4) [92, 93]. Blocking production of
some of these compounds by aspirin is therefore postulated to prevent the initiation of
carcinogenesis. Aspirin may also influence the establishment or growth of cancerous
cells through its inhibition of PGHS prostaglandin production. It has been proposed that
the inhibition of prostaglandins involved in such processes as the regulation of immune
cell maturation and/or function, or mitogenesis may play a role in aspirin’s anti-cancer

effect [87].

19

Figure 4. Mechanism of PGHS-mediated co-oxidation of xenobiotics. PGHS
possesses two distinct but interdependent activities: a cyclooxygenase and a peroxidase.
The cyclooxygenase thought to be initiated by a tyrosyl radical, originating from the
PGHS peroxide—mediated oxidation of the heme. In order to restore the oxidized heme,
two single-electron reductions occur, the electrons of which are donated by a co-oxidized
substrate (AH), which may include certain xenobiotics as phenols and aromatic amines.
This results in the potential production of highly reactive radical species (A0), which,
dependent on the surrounding mileu, may mediate the oxidation of other xenobiotic
compounds (XH), including aromatic amines. Therefore, the peroxidase activity of
PGHS provides the basis for two mechanisms of xenobiotic metabolism- one enzyme-
mediated, one substrate-mediated. A third pathway for xenobiotic metabolism may result
from the cyclooxygenase activity of PGHS, and therefore is the only one which
conceivably could be inhibited by non-steroidal anti-inflammatory drugs, such as aspirin:
the cyclooxygenase substrate, intermediates, and products are shown in shaded boxes.
The tyrosyl radical initiates the removal of the 13-pro-S hydrogen from arachidonic acid
(AA), forming the arachidonyl radical (AAO), followed by the addition of, first one 02
molecule to form one peroxyl radical (AAOOO), then a second molecule of oxygen to
form another peroxyl radical (OZAAOOO). Because peroxyl radicals are very highly
reactive oxidizing agents, both are capable of substrate-mediated oxidation of xenobiotic
compounds, such as aromatic hydrocarbons. Reduction of the peroxyl radical OZAAOOO
by the cyclooxygenase yields the product hydroperoxide PGGZ, which, in turn, can be
reduced by the addition of an electron, perhaps from heme, also to a highly reactive
peroxyl radical.

 

 

 

 

CYCLOOXYGENASE

 

 

20

3382253522.:
0X ii

\Ifl 2x

.ig 6..

0x1 :x
20¢ 1'. 00¢ 4..

I0< .00<

.
.
.
.
- o
.
. .
.
.
.

1‘

 

 

‘ ens £45.:
\‘(E \
\

__ \ _-\: /
. E 8»: 93...... III

 

 

 

 

 

8"; £43... j; _ 3.8%.
AA ( :2 :8: K
A J\
; mm<9xomma
E 8- .. £45... AIAo E .0. 8.5%.. IAo E 8.4.. mafia...
. K

 

mm<zm0>x0040>0

 

 

 

3.
large gYOU
hydrocarbo
polychlorol
larger poly
number of
(Figure 5)
similar tor
Each of tl
hydrocarbt
nucleus 21
elements(
Sensitive
IIfit‘tscript
endogenc
affinity f.
98]. B.
metaboh

[OXICittg

the LI);

SEHSthe

2 l
TCDD

2,3,7,8—Tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical member of a
large group of common environmental contaminants, the halogenated aromatic
hydrocarbons (I-IAHs). In addition to TCDD, the HAHs include compounds such as
polychlorobiphenyls (PCBs), and polychlorodibenzofurans. HAHs are a subgroup of the
larger polycyclic aromatic hydrocarbon (PAHs) family of compounds, which include a
number of planar, aromatic compounds such as benzo-a-pyrene, and B—naphthoflavone.
(Figure 5) [94, 95]. Despite the heterogeneity of structure, these compounds have
similar toxic effects which is presumed to result from a shared mechanism of action.
Each of these xenobiotics can bind to and activate a presumably cytosolic aromatic
hydrocarbon receptor (AhR). Once activated, the liganded receptor translocates to the
nucleus and binds to specific DNA cis-acting sequences, termed dioxin responsive
elements (DREs) or xenobiotic responsive elements (XREs) and transcriptionally activates
sensitive genes (Figure 6) [94, 96]. The ability of these xenobiotics to regulate
transcription of these genes is dependent upon their affinity for the Ah receptor. An
endogenous ligand for the Ah receptor has not been identified. The AhR has a high
affinity for TCDD compared to other polycyclic aromatic hydrocarbons (PAHs) [95, 97,
98]. Because of this high affinity of the Ah receptor for TCDD, and the slow
metabolism and elimination from the body, TCDD is commonly used in studies of PAH
toxicity.

The physiological effects of exposure to HAHs vary between species and tissues;
the LDSOS of TCDD, for example, range over 5000-fold. Guinea pigs are highly

sensitive to TCDD exposure (LD50=1 pig/kg), but hamsters are relatively insensitive

22

Figure 5. Structures of examples of polycyclic aromatic hydrocarbons. Compounds
which are substrates for the Ah receptor include halogenated aromatic hydrocarbons
(HAHs), such as polychlorodibenzo—dioxins, including 2,3,7,8-Tetrachlorodibenzo-p-
dioxin (TCDD), polychlorobiphenyls (PCBs), and polychlorodibenxofurans, and a variety
of planar aromatic PAHs, such as benzo-a-pyrene (BP) (or oxidized products of BP), and
B-Naphtholflavone (B-NF).

 

23
2,3,7,8-TatrachIorodlbanzo-p-dloxln (TCDD)

C):IO I CI
c 0 CI
Polychloroblphenyls (PCB's)
c CI . c CI
CI CI
Polychlorodlbonzoturans
c I I CI
CI 0 CI

Aromatic Hydrocarbons

 

Benzo(a)pyrene p-Naphtholflavone

24

Figure 6. Mechanism of TCDD/AhR activation. Planar aromatic hydrocarbon
xenobiotics, such as TCDD, are thought to activate transcription first by diffusion into
a cell, and subsequently by binding to a cytosolic multimeric aromatic hydrocarbon
receptor (AhR). Next, the liganded receptor undergoes a transformation, which is
thought to involve dissociation of at least one AhR subunit, hsp90. Once activated, the
liganded receptor translocates to the nucleus, associates with at least one additional
subunit, AhR nuclear translocator (amt), and binds to specific DNA cis-acting sequences,
termed dioxin responsive elements (DREs). The interaction of the liganded AhR/amt
complex with the DRE promotes transcription of TCDD-sensitive genes, including those
involved in xenobiotic metabolism, such as the genes encoding cytochromes P4SOIA1 and
-IA2, glutathione-S-transferase, and NAD(P)H oxidoquinone reductase, as well as those
encoding factors in inflammation or growth regulation, such as IL-lB, TGF-a, and PAI-
2.

 

 

25

®

Aromatic Hydrocarbon
(TCDD)

   
 
    

   
 

\~‘\\‘\:\\\\‘:§\\s‘:\\\\\

$(TCDD Rogulatod Gone)

W
W

 
  
    

   

  
   
   
 
 

    

 

  

 

 

 

 

 

 

 

 

 

 

MAM
MAM
MAM
- , glncraaaodwProtoinf, ~ ,,
‘ ,. inflammation/Simian
“ " ‘ " " ' Enzymes: rm:
CYPIA1I2 (cytochrome P450) _ V Interleukin-1 B
GIutathione-S-Transferase Transforming Growth
NAD(P)H Quinone Oxidoreductase I * A Factora
' Plasminogen Activator
Inhlbltor-2
J

 

26
(LD50=SOOO pg/kg) [99-101]. Toxicity from exposure to TCDD can include wasting

(weight loss); thymic atrophy; hepatotoxicity; chloracne (skin lesions caused by
hyperplasia and hyperkeratosis of the epidermis and squamous metaplasia of sebaceous
glands); and carcinogenesis in susceptible mice and rats [94, 95]. Although there is
controversy over human TCDD toxicity, chloracne is a commonly observed effect of
TCDD exposure in humans [95, 96, 102-104].

The etiology for the pathology of TCDD exposure is not fully understood; but it
seems clear that toxicity is mediated through the expression of TCDD sensitive genes.
Although many of these genes may not have been identified, those dioxin-responsive
genes that have been identified to date can be categorized into two groups: those
involved in the metabolism of xenobiotics, and those involved in inflammation or growth
regulation. The xenobiotic metabolizing enzymes include the cytochrome P4501A1,
glutathione-S-transferase, and NAD(P)H:quinone oxidoreductase [105-109]. Induction
of these enzymes is thought to facilitate elimination of xenobiotics from the body, but
these enzymes can also activate some xenobiotics into more mutagenic compounds. A
second set of genes that can be induced by TCDD include a set the products of which
are involved in inflammation or growth regulation including interleukin-16, plasminogen
activator inhibitor-2, and transforming growth factor-0: [110-112].

Speculation about a possible role for arachidonic acid products in the mechanism
of TCDD-induced toxicity originally arose from studies on chick cardiac edema resulting
from PAH exposure by Rifkind and Muschick. They demonstrated that the non-steroidal
anti-inflammatory drug benoxaprofen decreased PCB induced toxicity in chick embryos.

Interestingly, another NSAID, indomethacin, was inactive, leading the authors to propose

 

that lipOX)
crucial tha
cell systen
production
TCDD, P
prostaglani
such as ed
examined |
toAhR lig;
and these i:
Knutson ai
demonstra!
keritinizaii.

j“domethac

27

that lipoxygenase product production, which also is inhibited by benoxaprofen, is more
crucial than prostaglandin production in the mechanism of PAH-induced toxicity in this
cell system [116]. However, Quilley and Rifldnd later demonstrated that prostaglandin
production was induced within 24 hours in vivo in chick embryo hearts in response to
TCDD, PCBs, or the PAH, B-napthoflavone [117]. To date, the role (if any) of
prostaglandins, or other possible arachidonate metabolites in TCDD-induced responses
such as edema remains unclear, but it may be dependent upon the species or tissue(s)
examined [113-115]. In cultured cells, increases in prostaglandin production in response
to AhR ligands were also observed in vitro in Madin-Darby canine kidney (MDCK) cells,
and these increases were sensitive to indomethacin treatment [118, 119]. However, when
Knutson and Poland examined the XB/3T3 culture for initiation of keritinization, they
demonstrated that exogenously added arachidonate, PGE, or PGFZG failed to induce
keritinization. In addition, the authors claimed that a number of NSAIDs, among them,
indomethacin and benoxaprofen, did not inhibit TCDD-induced keratinization of these
cells [115]. The effects of increased prostaglandin production resulting from xenobiotic
treatment and the mechanism by which xenobiotics induce prostaglandin production have
not been elucidated.

The most intensively studied dioxin-responsive gene is the cytochrome P4501A1
gene, CypIAI. Much of what is known about the mechanism of TCDD action was
derived from the study of this gene. In the 5’-flanking region of CypIAI is a dioxin
responsive domain containing four functional DREs to which liganded Ah receptor binds
in what is thought to be a c00perative manner [120-125]. Activation of transcription of

CypIAI occurs within minutes of TCDD exposure, and does not require protein

 

 

st-nthesis.

 

hydroxyla ‘
Unli

and NAD(I
DRE [108.
DRE sequt'
element (A
The cons.
GTGACA
protein n
transcript
Tl
inflammz
are e‘ievz
elevated
fOllowin
21148 M

TCDD

28
synthesis. The induction of the P4501A1 mRNA and the resultant aryl hydrocarbon

hydroxylase (AHH) activity can be sustained for over 48 hours [105 , 106, 126].

Unlike the cytochrome P4SOIA1 gene, the glutathione-S-transferase Ya subunit
and NAD(P)H quinone oxidoreductase genes contain only one copy of the prototypical
DRE [108, 109, 127]. In these genes, xenobiotics not only induce transcription through
DRE sequences, but also through a DNA sequence termed the antioxidant responsive
element (ARE) (also the electrophilic responsive element (EpRE)) [107-109, 127, 128].
The consensus sequence for this responsive element has been determined as
GTGACAAAGC [129]. It has been proposed that an additional transcriptional activator
protein may be involved in the binding of the ARE; however, activation of this
transcription factor may depend on initial AhR activation [130].

The mechanism by which TCDD stimulates expression of genes involved in
inflammation and growth regulation is less well understood. Message levels for PAI-2
are elevated within and peak within 6 hours after TCDD (10 n_I\_/I) exposure, and remain
elevated even after 48 hours of exposure [110]. IL—lB mRNA is induced within 1 hour
following TCDD (10 nI_VI_) exposure, is maximal at 24 hours, and remains elevated even
at 48 hours [110]. Elevated TGF-a mRNA levels reach a maximum by 24 hours after
TCDD treatment [112], and TGF-a levels in the medium of cells treated with TCDD
(lOnM) have been observed 2 days afterwards [111]. However, because coincident
increases in TGF-a transcription are not observed, this gene now appears to be induced
through a post-transcriptional mechanism [112]. However, it is still not clear whether
the other genes in this category of TCDD-regulated genes also follow a similar

mechanism.

29
Analysis of the CypIAI , glutathione—S—transferase Ya subunit, and the human and

rat N AD(P)H quinone oxidoreductase 5’-flanking and promoter sequences has led to the
identification of an extended consensus DRE sequence,
(C/G)TNGCGTG(AlC)N(A/T)N(G/C)N(G/C) (Figure 7) [108, 109, 121, 123, 127 , 131].
Most critical in this sequence for AhR binding are the core GCGTG nucleotides. Unlike
other enhancers, such as the steroid responsive elements, DREs do not have dyad
symmetry. Thus, the analogy with the steroid receptor system is only superficial.
Further, the dissimilarity with the steroid system was emphasized with the recent cloning
of two subunits of the Ah receptor, which indicate the AhR belongs to the class of basic
helix-loop—helix transcriptional activators, and not the steroid receptor class of zinc-finger
motif activators [132-135].

The basic helix-loop-helix motif found in AhR is similar to that found in two
Drosophila proteins, Per, a circadian rhythm protein, and Sim, a neurogenic protein.
This domain was identified in the Amt subunit of the AhR receptor complex [132]. This
basic helix-loop-helix homologous region of approximately 250 amino acids is termed the
PAS domain (Ber Amt Sim). The Amt subunit of the AhR complex, which was
originally thought to be only involved in nuclear translocation or retention, has since
been shown to be required for AhR DNA binding activity [133, 135]. It has been
proposed that the PAS homology region is involved in the dimerization between the AhR
(ligand binding) and Amt subunits of the AhR complex, and by inference, dimerization
must be a critical step required for AhR DNA binding and transcriptional induction. Due

to the homology between the protein structures of the AhR subunits, and Sim and Per,

30

Figure 7. Alignments of DREs from the cytochrome P4501A1 and glutathione-S-
transferase genes. Sequence alignments of DREs from the mouse CypIAI [123], the
human glutathione-S-transferase Ya subunit [127], and the rat [108] and human
NAD(P)H quinone oxidoreductase [109] genes reveal a consensus sequence of
N(C/G)TNGCGTG(A/C)N(A/T)N(C/G)N(C/G). The core TNGCGTG nucleotides
appear to be the most critical in determining liganded AhR binding affinity (boxed). The
location of each of these DREs (number of nucleotides relative to the transcription start
site) are indicated on the left and right of each DRE.

 

 

 

 

31

can-

can-

Now-

hc__-

wmo-

nno_-

wco-

00

0000900

ZODZS

0<<E~000

<12000000

<0<E-‘0<<

[-‘<<E-'E—<0

0000<<0

0<<[-‘00<

 

E-

0

0

 

£on82

 

 

 

0000000

{-‘E—E-ti-‘E-tE—E—

0000000

0000000

vvn-

mum-

boa-

mmm-

0 0 0 0 0 0 0
< <1 5" 0 E" F‘ 0
t— E— [— t—~ t— E-t <|
0_ 0 0 <3 0 u o
o 0 < 0 H 0 <

 

 

 

mam-

 

 

nw__-

cho_-

mDmmeZOO

mo maca<z a:

mo £592 3.

er -emc
Emma/v.35
Mme—35%”.
83-233

$5.233

5:

Sflh

SSH

 

 

 

 

 

and the Sill
they were :

The
transcriptit
system. ‘
fragmentf
only to a l
digestion :
this mesh;
AhR- de
aceessi'oil
by TCDI
positionir

GINA] .

32

and the similarity of the gene structures of AhR and sim, it has been hypothesized that
they were all derived from a common ancestral gene [132-135].

The mechanism by which the liganded AhR interacts with the DRE to enhance
transcription is still being determined, primarily utilizing the CypIAI gene as a model
system. Watson and Hankinson demonstrated that TCDD addition protects a DNA
fragment from nuclease digestion in the cypIAI gene, whose sequence corresponds not
only to a DRE, but also to a G-rich region adjacent to it. This protection from nuclease
digestion required the AhR, suggesting AhR is binding to the DNA is a component of
this mechanism [136]. Whitlock and co-workers have also demonstrated a TCDD- and
AhR- dependent DNA bending, chromatin disruption, and increased promoter
accessibility in the CypIAI gene, which may participate in the activation of transcription
by TCDD [137-139]. However, it is not known how the AhR influences nucleosomal
positioning, or whether this mechanism applies to the enhancement of genes other than
CypIAI.

Therefore, while many details have been elucidated about the mechanism by
which TCDD effects the transcription of the CypIAI gene, relatively little is known
about the mechanisms by'which other genes, especially those involved in inflammation
and growth regulation, are induced by TCDD. In addition, although measurements of
the effect of PAHs on prostaglandin production have been made in some cell systems,
direct measurements of PGHS protein or mRNA levels in response to PAH exposure

have not been previously examined prior to the studies described in this dissertation.

CHAPTERZ

GENE STRUCTURE, TRANSCRIPTIONAL START SITE ANALYSIS,

AND ANALYSIS OF 5’-FLANKING SEQUENCES OF THE PGHS-1 GENE

Introduction

When these experiments began, there was evidence that regulation of
prostaglandin synthesis was dependent on transcriptional regulation of PGH synthase
expression. At that time, it was believed that only a single PGH synthase gene existed.
Therefore, our initial studies focused of the regulation of the PGHS-1 isozyme. In this
chapter, I will describe the isolation of the mouse PGHS-1 gene derived from genomic
clones isolated from a mouse NIH 3T3 fibroblast library. The PGHS-l gene structure
and its restriction map will also be presented. Identification of the transcriptional start
site of this gene will be discussed, as well as an initial analysis of its 5 ’-flanking region
for putative cis-regulatory elements, and an examination of regulation of this gene using
transient transfection experiments with reporter gene constructs containing the 5’-

regulatory elements.

33

34
Methods

Characterization of Genomic PGHS-l Clones. Two distinct PGHS-1 clones,
X5 and MZA were isolated by screening a mouse NIH 3T3 genomic XFIXII library
(Stratagene) using the 32P radiolabelled EcoRI restriction fragments from mouse PGH
synthase-1 cDNA (Figure 8)[39]. After plaque purification, DNA was isolated from
these phage [141] and was mapped using restriction endonuclease digestion and Southern
blot analysis. Initially, these clones were characterized using XbaI restriction
endonuclease digestion (XbaI cleaves mouse DNA relatively infrequently (average
fragment size: 6-7 kb) followed by Southern blot analysis using hybridization with
probes derived from the mouse PGHS-1 cDNA sequence. Probes were radiolabelled
with a-32P-dCTP (800 Ci/mmol, New England Nuclear), using the random primer
method [142]. For Southern blotting, restriction fragments obtained upon digestion were
separated by electrophoresis on 0.4 %-0.8% agarose gels in 1 x TBE. DNA in these gels
was denatured by treatment with 0.25 M HCl for 15 minutes, followed by 1.0 M
NaOH/l.5 M NaCl for 30 minutes. Gels were then neutralized with 0.5 M Tris-Cl, pH
7.0, 1.5 M NaCl for 30 minutes, and DNA was then transferred to nitrocellulose
membranes by capillary action in 20 x SSC (3 M NaCl, 0.3 M Na citrate, pH 7.0). The
nitrocellulose membranes were prehybridized for 1 hour at 42°C in 50% formamide
containing 5 x SSPE, 5 x Denhardt’s, 0.1% SDS and 2.5 mg/ ml denatured herring sperm
DNA. Hybridizations were for 18 hours at 42°C in 50% formamide containing 5 x
SSPE (0.75 M NaCl, 0.05 M NaH2P04, 5 mM EDTA, pH 7.4), 5 x Denhardt’s (0.02%

Ficoll, type 40, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.1% SDS

35

Figure 8. EcoRI restriction map of mouse PGHS-l and PGHS-2 cDNAs.

 

 

 

36

«.mzmvn. 950.2

 

e. and a... .332 9.68
a u q o - oi
E E E e. no . E e. F p E e. m: E
mewn 3:22
go no. a: o
as 3. pa. _ .00 an.

 

1 q q l.
PC PC PC a: as r PC PC

37

and 1 mg/ml herring sperm DNA, to which 1x106 dpm/ml radiolabelled probe was
added.

Initial mapping indicated that clones X5 and MZA contained common Xbal
fragments, and thus were probably overlapping. These clones also hybridized to only
the 5’-most sequences of the mouse PGHS-1 cDNA. Representative XbaI restriction
fragments were next subcloned into pUCl9 for additional restriction mapping with the
restriction endonucleases, EcoRI, SacI, and BamHI. Southern blots of these digests were
prepared as before. However, 17-mer Oligonucleotides prepared from the sequence of
specific regions of the PGHS-l cDNA (Figure 9) [39] and end-labelled with 7-32P-ATP
(6000 Ci/mmol, New England Nuclear) and T4 polynucleotide kinase were used as
probes in addition to the restriction fragments of these same cDNA. Hybridizations with
the cDNAs and Oligonucleotides of the digested genomic clone Southern blots allowed
us to construct a restriction map for the mouse PGHS-1 gene and 5’-flanking region.
Restriction fragments from these clones were further subcloned into M13mp19 and
sequenced with the Oligonucleotides derived from the first two exons and the first intron
(Figure 9). Mapping of the rest of the gene was determined in parallel with Elizabeth
Meade [143]. Together, the genomic map of the entire PGH synthase-1 gene was
constructed.

Characterization of the 5’ End of the PGH Synthase-l mRNA. Cytoplasmic
RNA was isolated from serum-stimulated quiescent Swiss 3T3 cells 2 hours after the
addition of fresh DME medium containing 10% fetal calf serum. RNA was isolated by
the NP-40 lysis and proteinase K digestion method [141]. The RNA was then poly A+

selected using oligo (dT) cellulose chromatography. [141].

38

Figure 9. Sequence of the mouse PGHS-l cDNA, showing the location of the
Oligonucleotides used for probes in Southern blots and primer extension analyses. 5’-
flanking region of PGHS-l from nucleotide -2489 through -1 numbered as such, while
nucleotides +1 through +2784 are from the cDNA (and are numbered as such). In
order to designate the cDNA derived from the different exons, they alternate between
lowercase and uppercase lettering. Primers are shown above and below the sequences:
primers ABOVE the sequence indicate primers that are actually derived from the reverse-
complement of the sequence indicated, while primers BELOW the sequence are identical
to the sequence underlined.

-2689
-2‘00
-2300
-2200
-2100
-2000
-1900
-1800
-1700
-1600
-1500
-1400
-1300
-1200
-1100
-1000
-900
-800
-700
-600
-500
-400
-300
-200
-100

101
201
301
£01
501

1001
1101
1201
1301
1601
1501
1601
1701
1001
1901
2001
2101
2201
2301
2001
2501
2601
2701

CAGACAIJCT
TG‘GCT11CT

AGTIGCGTGT

CAICTQCTOG
ABTIGAACAA

TTICASCCCA

 

ATCTIGCACC

AAAASTGCCT

 

CTTTCCTICC
GCAGIOGGCG

39

TIJTCAICTG

10HTCCTTTC
TOCTGGGAAA

AGAACTTCTG TRGGABAGAA GAAAAAACGA CTGTCAAIGA

CTGAGCCCAB
ACTGAGGGAG

$0
TcccccAc1fl'iltl11553d'ttllfluhccc

TCTIAGTTIG GGCAACTTTO AAAAAGGHTC

TCATTIAITT 1.3111010? AAGGNIRAAA

1:3?CCATIC ATISTTTTTG ASISCTICTT

CTCACTGCCC CTTCCTGOCC ACCCCCTCCC ACAASCCTTC

!
intramu- cream MTMA‘I‘A
mama rim-rm mm W

TITIJCTATC
CCCCACTTCC
CACCGAGGAC
AACGGAGAAG
ACCTCCTTGC
CTCAGAGAGC

TTCTGICACT GTIAAAAGAT CTTGCACCTC AAGGASCGTB AGTEICGGTC TGIOCAGTCA CCAJUCAGTI

WWW-Wuhan W W WWW
W cm ”MEWW Maya-WW meet-ice
mmmamm Wow
utoecctttg uneasy-can ctaccagcgc ondEIEIEEE';gifiii55EE’ocoaguceee llBtltIBCC cccCTGAGAr CTGGACCTGG
exuberance WWW masculine mac-rm MN» new new

III‘Itflfil‘
CTTCGGANTT
TICTCAJBCB

:ltl.l¢tac

AGACCTCTGG
Ieuuctcctc
GGTGTCCCGC
GCGTGTGCGA
G.ECI.".'
gcoatculat
scoot-tun:
TCTGCATOTB

lttfliflllct

mac-m mm sweetigccm
excommun-
W W A‘fl'CC’MCCC
WWWCH
amm-

GTGAAAGGCA
CCTGCTGAAG
tobacco-ac
eta-cents:
litflitcttt
GCTOTOGAflG
TCACAG'IflI
canctocatc
ace-cotton
ItltIOOIli
tootlltflul
ttfl‘lttttt

GASGGCTGflG
GAGGAGCATC
Icttflifltfll
abatenctu'
liiifitlili
TCATCAAOGA
IDIIII03§I
tttflilllll
ltlItIIcit
belccctaun
glonnatoct
coo-ctttcg

CGICGTGCTG
GGCCAGGAGG
CCACGTGOGA
Btltttcctfl
9.068.336.

3.3000tli.

GTCCCGAGAG

IIB‘CBOOC.
CCCAGGGCAC
AOGCCACOGG
GACGGAGAGG
TUTTTGGGTT
TGASGGGCAG
c.'¢t¢..lt
baboon-ate
tuccttctct
ASGCGCCTAC

D")

TCCTCAECCT
CTCAGTTTGC
ASTIASRAAAW

AIICCCTIGC

GAIGGCAIGA

GCAGACCGGG
CTCCCAGATC

TCTTCIABCA
TlTIICCCAA

AGGGCTGTIB

GGCTGAGCTA

TCAITfiflTTT
TCTCAGTNZA

GGGCCCTTCC

GGCCASTCAG

GATCTGTITA
AACTCCCCTC

BOIIOOtCB. IfltGIIO'OO tIIBtIBOtG IIBtI"." 00000066..
cocoontuug IOCOOIIUGO AAGAAACAGT Tle5fl9AICT TCAGCTTCTO

CRACAGCCTU TTTUCCTTCT TTGCACAACA CTTCACCCAC CAGTTCTTCA

't...‘¢tt"IES=E=EE:=TEIIEI=E’.t 30"..B'IO .‘t.t°.flct
TOTACCCACC TTCCGHOGAA CAGGCGTCCG TGTTGAJGCG CTACCCACCA

GCTTCCGGGG CTG.TGCTCT TCTCCACGfiSZCTGCCTTCCT GAACATIACC
-5 av
Mex-rm. sumo. econ-too:
ecouccogun gceuctgtcc gynaecoagt coo-acute: aaaccggocc
“summon:- outcome: ttactcam

AGCCCTTCAA

OEIIBCIIRT
TGAAJICCGA

l¢:l¢‘l|l‘ tll'l"l°t Itlcli‘IIc O‘CUISIBCC

“about”. new: «occur-.3. We:
h

I'DB‘CG..O EEEgtchaon

IISIIBIIIS
tl.tit9!lt
'Itlttlllt

WW»...
W eon-mm mum

00....9... 8......IB. ..¢...B... O..¢“CB.I

tectlfllltt BIHIIIBOCG ttclfliltflt C'Ictltct'

0°..Btt'.. 8.06%....0 Ct'fl..tt" $00.00CCBQ
...¢CC.¢" Bt.fl'¢¢... antennayoa 't.."tfltt
Iltcttflltt guinaccacc .c.'ttctlt ccccnbccaq

ctfltcttllt
tttc'tgflct
Ecttt'ttll
tlfilflifltt.
Iflctltttli
lltltllilt
Calcit'lct
tattcnctgl
ottgyulucc

”Itctttlstt

GICQOECOBt
llllnactcc
BI'GOBtIt'
CtCI'III't
Illflttttt.
oat-cobact
cccctaaaaa
3.0.3.0t90
annoyance.
acacccaanc

0‘90300339

0.30.3000.
accaaacccc
nonoccoucg
til-Intuit
ccacauugta
concoct-an
aaaaaaocco
cgttcctuvt
lilo-IOOIi
cccctccaaa

tttctcacun

mm mane-mm neurons?
D-l
mm WAC

tlflllttctl
09‘3lIIIIt
'tct'cCtoa
0.003.090.
accountttg
tttgtctgcc
OII‘Itlltt
sconce-egg
cttIIll'tt
cancgatcca
acocaacgtc
I‘Eitllili

.flctllt...

0000000000
occltctgtt
I¢I¢°|30|¢
I'IIIOIIBC
Illctttgyj
cocoa-cane
acacacacot
Itflflltlltfl
notcuustcc
tllcntgiuc
0.00.8931!
0IIGIII¢00
ottc

county's.-
°¢°°I'.lt.
ocgcocccac
tctl'.CI'I
coccacoctt
:touctocec
tantcaaiia
concoctcec
ccaatcccot
canaaoatbo
cacctcoccc

actcotctot

4O

Primer Extension Analysis. Two 30-mer Oligonucleotides were constructed from
sequences near the 5’-end of the mouse PGHS-1 cDNA. These oligonucleotides were
DD-68 5’TGTCGGCGGCAGCAGCAGGAGCAGCAGCAG-3’ (Figure 9), which is
complementary to base pairs 70-99 of the murine PGHS-l cDNA and is located in the
second exon, and DD-69, 5’CACCTGG'I'I‘CTGGCACGGATAGTAACAAC-3’ (Figure
9), which is complementary to base pairs 149-178 of the murine PGHS-1 cDNA, and is
located in the third PGHS-1 exon [143]. The Oligonucleotides were end-labelled with T4
polynucleotide kinase, and 7-32P ATP to a specific activity of approximately 2.5 x 108
dpm/ug [141]. The labelled primers (2 x 10° dpm) were mixed with 10 pg 3T3 poly A+
RNA in 30 ul of 40 mM Pipes, pH 6.4, containing 1 mM EDTA (pH 8.0), 0.4 M NaCl,
and 80% formamide. These samples were denatured for 10 minutes at 85°C, then
allowed to hybridize for 12 hours at 30°C. The resultant hybrids were precipitated with
ethanol and resuspended in 20 pl of 25 mM Tris-Cl, pH 8.3, 10 mM DTl‘, 37.5 mM
KCl, 1.5 mM MgC12, 40 units RNasin, and 1 mM each of dATP, dCTP, dGTP, and
dTl‘P. M-MLV reverse transcriptase (200 U, BRL) was then added, and the reaction
allowed to procede for 10 minutes at 22°C, then for 50 minutes at 42°C. After
phenol:chloroform:isoamyl alcohol (25:24:1) extraction, the radiolabelled cDNA was
ethanol precipitated, and resuspended in a 80% formamide loading buffer. The samples
were denatured by heating to 90°C, then loaded on to a 5% polyacrylamide/7M urea
sequencing gel [141]. In adjacent lanes, sequencing reactions using a PGHS-1 genomic
DNA clone template primered with an unrelated 30-mer oligonucleotide
(GTCCCCTCCCACCCATCTCACGGCTCCACC) primer were electrophoresed to allow

size determination of the extended product [144].

41

RNAse Protection Analysis: A BamHI/EcoRI restriction fragment containing the
first 107 bp of the PGH synthase gene (ending in the first intron) and approximately 2.3
kb of the 5’-flanking region was subcloned from clone 15 into the plasmid vector pSP65
(Figure 10). This plasmid was linearized with anI and was then used as a template to
synthesize a labeled 536 bp antisense RNA probe with SP6 polymerase (United States
Biochemical) and [a-32P]CTP (800 Ci/mmol) (NEN) [141]. The labeled probe (5 x 105
dpm) was combined with 10 pg of poly A+ RNA (or 20 pg of tRNA as a negative
control) in 20 pl of 40 mM Pipes, pH 6.4, containing 1 mM EDTA (pH 8.0), 0.4 M
NaCl, and 80% formamide. The mixture was heated to 90°C for 10 minutes then
allowed to hybridize for 12 hr at 60°C. Single-stranded RNA was removed by digestion
with RNAse A (18 pg/ml) and RNAse T1 (1 pg/ml) for 1 hr at 30°C. Proteinase K was
added (0.28 mg/ml), and the mixture was incubated a further 30 minutes at 37°C.
Following extraction with phenolzchloroform:isoamyl alcohol (25 :24:l), the protected
products were precipitated with ethanol. The products were then redissolved in 5 pl of
80% formamide sample buffer, denatured at 90°C, and loaded onto an 8%
polyacrylamide/7 M urea sequencing gel [141]. Sequencing reactions with a known
template and primer (30-mer) were run in adjacent lanes for determination of the size of
the protected products [144].

Characterization of the 5’-Flanking Region of the PGH Synthase—l Gene. An
4.5 kb Xbal restriction fragment isolated from the )6 clone, which contains the first 157
bp of the PGH synthase-l gene and approximately 4.4 kb of 5’ flanking region, was
subcloned into Ml3mp19. DNA approximately 2.5 kb upstream from the transcriptional

start site was sequenced in both orientations using the Sanger dideoxy chain termination

42

Figure 10. Construction of the plasmid used to produce an antisense mouse genomic
PGHS-1 probe used for RNase protection analysis. (A) A 5.5 kb PGHS-l genomic
EcoRI restriction fragment was isolated from the PGHS-1 AFIXII clone X5, the 3’ end
of which corresponds to an EcoRI site 41 bp into the first intron. This restriction
fragment was subcloned into the EcoRI site of the expression vector pSP65, in an
orientation such that the antisense transcript would be produced upon in vitro
transcription. (B) A second plasmid, containing a 2.3 kb PGHS-l genomic sequence was
derived from the 5 .5 kb EcoRI restriction fragment, above. A 2.3 kb BamHI/EcoRI
PGHS-1 genomic restriction fragment, extending from nucleotide -2243 to nucleotide
+107, relative to the transcription start site, was isolated and ligated into the BamHI
restriction sites of pSP65, in an orientation such that the antisense transcript would be
produced upon in vitro transcription. A 536 base radiolabelled antisense RNA probe was
synthesized by linearizing plasmid B, above, with anI restriction endonuclease and
transcribing the resultant linearized plasmid using SP6 polymerase and a-32P-CTP.

A.

til-Sm +l O 7
Xbal EcoRI Xbal EcoRl

PGHS-l CLONE as: I | I

 

-5400 ‘22“ 424 107
cEcoRl BamHI anl IicoRl
Irumnmumumun-rumourrnunmrnnurrlmmummnmmmuruumnnummmlmmurnunmrhJ

Exon A
Hindi" ‘
PSI
y
m

 

 

anl

44

 

B .54“) 4243 424
° ECORI , BamHI anl
LMIIIIIIIIllImmMinimum!Ill-Immuunmllmumuflllllmufllumm
2243 424
BamHI

 

 

4243
WI

Pats-1

EmnA

Linearize with an I 96;“ +107
424

Xnut I SP6 promotcrr. an I

aim"

. . . . ExonA
m vrrro transcription using SP6

polymerase

ammmnj Antiscnsc probe used in
$4 RNAse protection

ExonA

+107

ExonA

[

I.
3

EcoRl

45
method [144] with Sequenase-2.0 (U SB), using synthetic oligonucleotides (Michigan State

University Macromolecular Structure Facility) as primers. Sequence alignment, and
identification of putative responsive elements were performed using the Wisconsin
Genetics Computer Group (GCG) programs [145] implemented on the VAX 860
computer at the Michigan State University computer facility.

Construction of PGHS-1 5’-flanking region luciferase Vector. A 2330 bp
BamHI/HIM] restriction fragment, encompassing nucleotides -2243 through +28 of the
mouse PGHS-l gene was inserted into the BglII restriction site in the polylinker of
pGL2-Basic (Promega), which contains the luciferase cDNA, but not promoter or
enhancers as follows: from a pSP65 plasmid containing a 5.5 kb EcoRI restriction
fragment encompasing the PGHS-1 sequences -5 .5 kb through +109 bp (relative to the
transcription start site (Figure 10A), two restriction fragments were isolated: (1) a
HindIII/Nsil fragment containing sequence from approximately -3.4 kb to -201 bp of the
PGHS-1 5’-flanking region, and (2) an NsiI/EcoRI restriction fragment corresponding to
nucleotides -201 to +107. The Nsi/EcoRI fragment (2) was initially subcloned into
pSL1180, then linearized using 171M] restriction endonuclease, and the ends were blunted
using T4 DNA polymerase (Boehringer-Mannheim), and dNTPs [141]. A 10-mer BamHI
linker (New England Biolabs) was then ligated to the blunted 171M] ends of the plasmid,
subsequently digested with BamHI, then religated. This resultant plasmid was next
digested with HindIII and Nsil, and the 3.5 kb HindIII/Nsil restriction fragment (1,
above) was inserted, and the plasmid was re-circularized. A 2.3 kb BamHI restriction

fragment corresponding to nucleotides -2243 through +28 of the PGHS-l 5’-flanking

46

region was isolated and ligated into the BglII-linearized pGL2-Basic, and the orientation
of the insert was confirmed using NsiI/EcoRI digestion (Figure 11).

PGHS-2mm, which encompases nucleotides -975 to +88 of the mouse PGHS-2
gene was used in these studies (Figure 12). The insert of PGHS-2,hon was obtained by
polymerase chain reaction amplification of the XFIXII clone isolated from a NIH3T3
genomic DNA library, using primers derived from the sequence of Fletcher, et al. [146].
This PCR fragment was cloned into the HindIII/BamHI restriction sites of pBlueScript-
KS +. The resultant plasmid was digested with the restriction enzymes KpnI and BamHI,
and the PGHS-2 5’-flanking sequence fragment was isolated, and subcloned into the
KpnI/BglII sites in the polylinker region of pGL2-Basic (Figure 12). The amplified
restriction fragment was also subcloned into Ml3mp18 and Ml3mpl9 for sequencing
using the Sanger dideoxy chain termination method (Figure 13) [144]. Our sequence
contained many single base pair differences when compared with the original reported
sequence [146], but confirmed the identity of the PCR amplified product.

Transient expression of luciferase vectors in NIH3T3 cells. NIH3T3 mouse
fibroblasts were transiently transfected with the luciferase expression plasmids using the
CaClz-precipitation method [141]. Cells were grown on lOO-mm plates to approximately
70% confluency in 10 ml DME medium supplemented with 10% fetal calf serum.
CaClz-precipitated DNA ( 1.1 ml) was added to each plate and incubated for 5 hours at
37°C in an incubator containin 5% C02. The medium was next removed, the cells were
shocked by treatment with 1.5 ml of 15% glycerol/HEPES-buffered saline (140 mM
NaCl, 5 mM KCl, 0.75 mM NazHPO4 2HZO, 6 mM dextrose, 25 mM PIPES (N-2-

hydroxyethylpiperazine-N’-2-ethanesulfonic acid) solution for 1 minute, and the cells

47

Figure 1 1. Construction of the luciferase reporter plasmid containing the 5 ’-t'lanking
region of the mouse PGHS-1 gene. (A) From a plasmid containing the 5 .5 kb EcoRI
restriction fragment corresponding to sequences -5.5 kb through +107 of the PGHS-1
5’-flanking region (which was also used to produce the antisense mouse genomic PGHS-1
probe for RNase protection analysis, see Figure 10A), two restriction fragments were
isolated: a HindIII/Nsil fragment containing sequence from approximately -3.4 kb to -
201 bp of the PGHS-l 5’-flanking region, and an NsiI/EcoRI restriction fragment
corresponding to nucleotides -201 to +107. The Nsi/EcoRI fragment was initially
subcloned into pSLll80, then linearized using 171M] restriction endonuclease, and the
ends were blunted using T4 DNA polymerase (Boehringer-Mannheim), and dNTPs [141].
A 10-mer BamHI (New England Biolabs) was then ligated into the blunted HIM] site,
digested with BamHI, and religated. This resultant plasmid was next digested with
Hindi]! and N517, and the 3.5 kb HindIII/Nsil restriction fragment was inserted, and the
plasmid was re-circularized. (B) A 2.3 kb BamHI restriction fragment corresponding to
nucleotides -2243 through +28 of the PGHS-1 5’-flanking region was isolated and ligated
into the BgllI-linearized pGL2-Basic, and the orientation of the insert was confirmed
using NsiI/EcoRI digestion.

48

A o “:5“ ca. 3443 -2243 424 -201 +3 I 107
Ecol! Will yum Xul Nril mm alt!

L.-..........i........t......................t.....t...fl_.t
\/

HMflfll
.thflI/Ahflfinynun

 

 

 

 

W
SI
Bail
+1 07
Econ!
bull +3!
”I"
PGH-t

Digest with EflM!
Blunt ends. Add BamHI
linkers. Religate.

 

 

Digest with Hind!!! / Nsi I
Ligatc Hind!!! l Nsi I fragment

47

Figure 11. Construction of the luciferase reporter plasmid containing the 5’-flanking
region of the mouse PGHS-1 gene. (A) From a plasmid containing the 5.5 kb EcoRI
restriction fragment corresponding to sequences -5.5 kb through +107 of the PGHS-l
5 ’-flanking region (which was also used to produce the antisense mouse genomic PGHS-1
probe for RNase protection analysis, see Figure 10A), two restriction fragments were
isolated: a HindlII/Nsil fragment containing sequence from approximately -3.4 kb to -
201 bp of the PGHS-l 5’-flanking region, and an NsiI/EcoRI restriction fragment
corresponding to nucleotides -201 to +107. The Nsi/EcoRI fragment was initially
subcloned into pSL1180, then linearized using 171M! restriction endonuclease, and the
ends were blunted using T4 DNA polymerase (Boehringer-Mannheim), and dNTPs [141].
A lO-mer BamHI (New England Biolabs) was then ligated into the blunted 171M! site,
digested with BamHI, and religated. This resultant plasmid was next digested with
Hind!!! and NsiI, and the 3.5 kb HindIII/Nsil restriction fragment was inserted, and the
plasmid was re-circularized. (B) A 2.3 kb BamHI restriction fragment corresponding to
nucleotides -2243 through +28 of the PGHS-1 5’-flanking region was isolated and ligated
into the BglII-linearized pGL2-Basic, and the orientation of the insert was confirmed
using NsiI/EcoR! digestion.

48

A o «-5400 ca. 3443 424 -201 +3 1 +107
Ecol! rum 3...”, X1"! Nrtl mu] Eco!"

l........................l....... .................................... Lamar
\‘Lm/V

W"

 
   
  
  

Hind!!! [Milli-am

 

 

Digest with RflM!
Blunt ends. Add BamHI
linkers. Religate.

 

 

Digest with Hind!!! / Nsi I
Ligate Hind!!! l Nsi I fragment

 

Mi Hind"
m (on. 4443)

Digest with Barn!!!

 

-2 243 +28
at H! Barn HI
Illlllllllllflllll W.m—_:
Exon A

  
     

I Digest pGLZ-Basic
with Bglll

 

 

 

 

 

 

 

50

Figure 12. Construction of the luciferase reporter plasmid, PGHS-2mm, containing
the 5’-flanking region of the mouse PGHS-2 gene. (A) A 1079 nucleotide PCR
fragment, was generated from a KFIXII mouse NIH3T3 PGHS-2 genomic clone, using
the following primers: 5’-primer:CCAAGCTI‘CCAACACAAACACAGTAGGAAGATA,
3’-primer: CCGGATCCGGTGGAGCTGGCAGGATGCAGTGCT. The primers were
derived from mouse PGHS-2 5’—flanking region sequences reported by Fletcher, et al.
[146]. Hind!!! or BamHI restriction sites were added on the 5’-primer and 3’-primer,
respectively. (B) The PCR fragment was digested with Hind!!! and BamHI restriction
endonucleases, and ligated into the Hind!!! and BamHI restriction sites of pBluescript-
KS +. The resultant plasmid was subsequently digested with KpnI and BamHI restriction
endonucleases, to excise the PGHS-2 5’-flanking region PCR fragment insert, plus an
additional 5’-36 nucleotides, derived from the polylinker region of pBluescript-KS+.
(C) This KpnI/BamHI restriction fragment was ligated into the Kpnl/Bglll sites of pGL2—
Basic, immediately upstream from the luciferase coding region. Therefore, the insert in
PGHS-2m“, contains sequences corresponding to -975 through +88 (relative to the
transcriptional start site) of the PGHS-2, plus an additional 42 non-PGHS-Z-derived
nucleotides on its 5’-end, and 6 nucleotides on its 3’-end.

51

f-PCR primer
( adbd HinDIIl cite)

 

ccu.ctthACACAAACACAGl'AmAAGATATCIIAACA A CAAMACATCACIICI’CI'ACIKAAATAATTT‘ITTAT
QAACACI'GTTTCI‘GAATGAAGTGAATTTTTTTTTAAGGTTAGIGAGAATAAWAG'I'TATOOAAAAATAACI’ATTAG
TTTTOATWTAACATACTTCI‘TGTAAACATGGACAGAAAAAG‘I‘I‘TTI'AAAACXII’ACATTTGAAAACAAATTCI’
rrcrnaranrncnm GTGAAAAGAGmATCAa AATGATM AAGCI'ATGTAACAGCAGmg GAAAI TAG) TAAA
(KAATGO GTOGACACI'TAGCATTGIIATGA AGTWCK'I'CAGCATCTGTCC Cl'mg AA AG; (ISAGTGO: A0003 CTCCI‘
GTGACI’mAGAAc WWAGTTWTGA AAGACI'I‘CAAGTI'AATTCIIACIIAGTACAGATGTGGACGTI‘GACAGAOG
ACACI’CATTTCATTAAAAATAGAAGAAATTAAATTAAGIIGTIMOTGTGWTGCTCTGAGCAGGAGCACIITCAGACI
000m AGTCXK‘GAGAGGTGAGJGGATTGI'TTAMTAGGACLTTAGATGIGGGAGCG; AAGCTGTGACACI'CTTGAG
CITTI'AWACI‘GATWCKIGGA oouacxnononmuonomocnmcnoococ AGACI’CACKIBAA
(ZACAWCTOGAMGATGGAGAGWWAGCTUWOGCACCAMGGGGCAGCCAAGCKIIAGCI‘TCW
"WWWGUCAWTMGTWAAAAWWWCTTGCOCA ACI'CACI'GA AGCAGAGAGOGGGAAAA
GTKXTTWTHXIGGAACKIJAACXDGAAAGACAGAGTCACIIACTA(BTCACIITGGAGTMCTTACAGACTTAAAAO
caaoorrcrmrrnocaoocnoncrca AACTWAMAA. IMTCAWG‘I’CAGTCAWCTCTMCAWA;
ancrcrocncrocntrroocnmcrg juice].

 

3‘-PCR [rim
(added Ban!!! cite)
PCR
Digestion with Hind!!! and BamHI
.975
mam emu

ExonA

Digest pBS-KS-t- with

<—
BamHI and Hind!!!
<—

 

 

 

m =

m1

NM

Digest with KPH/DUI”! W '
DI .
42 hp derived from pas-x3+ '
polylinker +Hind!!! sic oIPCRprimer ' '

\ -915 8

+8
Kan! find!!! you”)
I l

 

52

42 bp derived from pBS-KS+
polylinker +Hind!!! rite of PCRprirner

-975 +8 8
K9" lid!!! BamHI

l
ExonA

 

‘—
Digest pGL2-Basic
with Kpn! I Bglll

 

 

 

POI! (A) sifllll
(It! background reduction)

 

 

 

 

53

Figure 13. Sequence of PGHS-2,1,“, insert. A 2.3 kb HindIII/BamHI restriction
fragment derived from PCR amplification of the 5’-flanking region of PGHS-2 was
ligated into the HindIII/BamHI sites of M13mp18 and Ml3mpl9, and sequenced by the
method of Sanger, et al. [144] from both ends using the M13 (-40) sequencing primer.
In addition, one internal primer, DD-9l (CCTGTGG'ITCGCTGAGT) was used to bridge
the sequences obtained from both ends of the insert. Sequence from which the two PCR
primers were derived are underlined. The PGHS-2 transcription start site is bolded and
indicated above with (V). A number of deviations between this sequence and that
previously reported for the mouse PGHS-2 5’-flanking region by Fletcher, et al. [146]
were observed. Any omission (by Fletcher, et al. as compared to our sequence) is
indicated above the nucleotide (.); insertions are indicated above in small, lowercase
notation. Subsequent sequencing of the 5’ 378 bp of this sequence, which was isolated
directly by restriction endonuclease digestion from the original XFIXII PGHS-2 NIH3T3
genomic clone, confirmed that at least 14 of these discrepancies (12 single-bp omissions,
2 single-bp insertions) were not the result of PCR amplification errors. The source of
the discrepancies between our sequence and that and that of Fletcher, et al. has not been
resolved. Sequence which is inserted into PGHS-2mm, but which does not correspond
to authentic PGHS-2 5’-flanking region sequence is indicated by lowercase bolded
lenefing.

-1017
-1000
-950
-900
-850
-800
-750
-700
-650
-600
-550
-500
-450
-400
-350
-300
-250
-200
-150
-100

-50

51

cgaggtcgac
TCCAACACTA
ACACTGTTTC
GCTAGTTATG
CTTCTTGTAA
AAATTCTTTC
TAAAAGCTAT
CACTTAGCAT
GAGTGCCAGG
AGACTTCAAC
CTCATTTCAT
TGCTCTGAGC
GGGATTCCCT
TCTTGAGCTT
CCCCAAAGAG
GGAGGGATGG
AAGGGCAGCT
TGCCGCTGCG
GGTGGGGGTT
CGTGGAGTCC

v
AGTTGTCAAA

I
CACGAAGGAA

54

ggtaccg

ggccccccct

ggtatcgata agcttCCAAC ACAAACACAG TAGGAAGATA
CACCTCTCTA GGCAAATAAT TTTTTATCAA

TCAAAAACAT
TGAATGAAGT
GAAAAATAAC
ACATGGACAG
TAGTGATACA
GTAACAGCAG
TCCGATGAAG
GGCTCCTGTG
CTAATTCCAC
TAAAAATAGA
AGCGAGCACG
TAGTTAGGAC
TTAGGCCCCC
CGCCAGACTA
AGAGGGCGGT
TCCCGGCTTC
GTTCTTGCGC
GGGGAAGCCT
GCTTACAGAC
CTGCGAGCTA

CTCAGCACTG

GAATTTTTTT
TATTACCTTT
AAAAAGTTTT
TCCGGTGAAA
GGGGAAAATA
TGGAGCTCAG
ACTGCAGAAG
CAGTACAGAT
AGAAATTAAA
TCAGACTGCG
CTTAGATCCC
ACTGATGCGC
GGCGGAGACT
GCAGCTCTCT
CTTCGTCTCT
AACTCACTGA
AAGCGGAAAG
TTAAAAGCAA
AGAGCTTCAG

CATCCTGCCA

TTAAGGTTAG
TGATCCGTTG
TAAAAGGTAC
AGAGTTGCTG
CCTTAAAGCA
CATCTGTCGC
CCGGAGGGTA
GTGGACCCTG
TTAACCGGTA
CCCGAGTGGG
GGGAGGGGAA
GACTGGGAGG
CAGCGAACCA
TGGCACCACC
CATTTGCGTG
AGCAGAGAGG
ACAGAGTCAC
GGTTCTCCCC
GAGTCAGTCA

GCTCCACqu

GGAGAATAAG
CCATAACATA
ATTTGAAAAC
ATCAAAATGA
ATGCTGTGGA
TGGGAAAGGC
GTTCCATGAA
ACAGAGGACA
GCTGTGTGCG
GAGAGGTGAG
GCTGTGACAC
AAACCGGAGA
CAGGGCGCCT
TGGGGCAGCC
GGTAAAAGCC
GGGAAAAGTT
CACTACGTCA
ATTAGCAGCC
GGACTCTGCT

gtcc 94

-1001
-951
-901
-851
-801
-751
-701
-651
-601
-551
-501
-451
~401
-351
-301
-251
-201
-151
-101

-51

50

55

were next washed twice with PBS. DME medium (4.5 ml) containing 0.2% fetal calf
serum was added and the cells were incubated for 24 hours. Next, the addition of 0.5
ml of fetal calf serum, or DME containing 0.2% fetal calf serum was added and the
incubation was continued for 8 hours. The medium was removed and the plates were
washed twice with 5 ml PBS. The cells were harvested in 0.3 ml of cell lysis buffer
(Promega) into microcentrifuge tubes, and the cell debris was pelleted by centrifugation
at 12,000 x g for 5 minutes at 4°C. The supernatant was decanted, and assayed for
luciferase activity using a Luciferase Assay Kit (Promega) and a Turner TD-20E
luminometer, or an LKB scintillation counter. Luciferase values were normalized to
protein quantity as measured by the method of Bradford [147], and statistical significance

was assessed using a paired Student’s t test at the 95% confidence level.

56
Results

Characterization of the Mouse PGH Synthase—l Gene, Two different, overlapping
AFIXII NIH3T3 genomic clones, termed X5 and M2A were mapped using restriction
endonuclease digestion. The approximate location of the first two exons of the PGHS-1
gene in these genomic fragments were initially determined by Southern analysis of the
restriction endonuclease digests of the genomic clones using 32P-labelled oligonucleotide
probes derived from the cDNA sequence. A more precise mapping of the first two exons
within these clones, their intron/exon borders, and the sequence of the first intron was
obtained by sequencing these clones using the same oligonucleotide primers.

A complete restriction map of the PGH synthase-l gene was obtained by
combining my results for the 5’ end of the gene with those of Dr. Elizabeth Meade, who
characterized two additional clones, x25 and A66, derived from the 3’ end of the gene
(Figure 14) [143]. The PGH synthase gene extends over 22 kb, and is composed of 11
exons ranging in size from 70 to 1268 bp, separated by introns of 93 bp to 6.2 kb in
length. Each splice site conforms to the consensus donor/acceptor splice site sequence
(GT/AG) (Table 4). The nucleotide sequence of each exon and the flanking intronic
sequences is shown in Figure 15. Exon A (70 bp in length) and Exon B (96 bp in
length) are separated by only 98 bp, one of the smallest introns. The final exon of the
PGHS-1 gene, K, is the largest (21268 bp), and encodes the final 120 amino acids,
including the active site serine (Ser 532) and the polyadenylation signal sequence. The
other exons (C-J) have more moderate lengths (84-287 bp). The intron/exon borders,

as well as the relative arrangement of the exons of the mouse PGHS-1 gene are

57

Figure 14. Restriction endonuclease map of the mouse PGHS-l gene, and the
)tFIXII clones from which it was derived. Exons are indicated by closed rectangles
above and below the line. The restriction sites are X, XbaI; P, Pstl; H, HindIII; B,
BamHI; R, EcoRI; S, Sac]. Complete XbaI, BamHI, EcoRI, and Sac! restriction maps
of the individual phage clones were determined for the regions included in the gene map
compilation, but additional Hind!!! or PstI sites may exist.

 

58

250 8.2.2.? :0.—

 

 

 

 

 

 

 

. . . .
z _ _ : ..._ .2. o = < .23...
u .
Jill-41...“. = 1 .. :i._1L3_ .d.
x x 5...» a : mm :5. m a a: can a an x m as}... a a x
2.; 2. am 2.
2.. n“. <2...
on. a. ..
.... d I o-H-IOQ-U—Fouu =8—
1 _ _ ._. _ _ BEBE I
x x x x
a";
= _ _ .
x x x
<2.“
_ _ _ _
x x
a...
. _ _ _

59

Table 4. Exon/Intron borders of mouse PGHS-1.

 

 

60

no.
8.
8.
ex
an
ax
an
an
8.
on

m.H
m.N
m.~
N.H
mm

b.N
mmH
own
N.m
mm

3:.
.353

4040
0994
4040
0090
0490
4400
0009
4090
4409
0440

0400
0049
9099
0400
0900
0044
0090
0090
0400

0090

.9004
.0044
.0040
.0904
.9040
.0044
.9000
.0040
.9004

.9040

0404
0000
0494
0400
0000
0444
0404
0094
0400

0904

meHA
wvH
hwm
hem
em
NmH
¢¢H
H¢H
bHH

4009010403:th6

61

Figure 15. Sequence of the mouse PGHS-l gene. Exon sequences are indicated in
uppercase, while intron sequences are in lowercase. Approximate distances between
intron sequences are given. The sequences from which the primer extension
oligonucleotides DD68 and DD69 were derived are underlined.

 

 

gtgacaactg
GGAGCCAGCC
tcggtggcct
CTGCTCCTGC
aaaattccaa
999t9tt989
cactaaaggg
ttt9999399
tcatctgcaa
gctcacaacc
cactaggctc
actctttctc
98999t9999
cagTCAATCC
CGGGCTACTC
cctgccttct
ctgtggtagt
actcctgttt
ctcaggtctt
CCCCTCGTTC
GGTACTCACA
gataaatcaa
GTCCAACCTT
CAITCTGCCC
ctcttcaggc
aggcagaaaa
tctgtctgct
cccaaccagG
CCAACAICCT
TAGGCCACGG
ttctctgacc
AGTACCAth
actaccagag
gtggcctcct
atccatctta
caacaaatca
ttgtgtaaac
tgagatggat
gatccctggg
acataaataa
cacaaaccca
gcagagaacc
gtggccatac
ggtatactga
aggtagtaaa
atcccacagG
AGGCAGATGG
TGCGACCIGC
cgagaggcag
tcaggcccag
attggtcacc
ttaccctacc
tatttttgtg
ctccttgtct
cactggggat
ggtaaagcag
agtcctatgt
tgttcatttc
TGCAGCACTT
TGGAATITAA
TTAACACTTC

62

9339939939 t99999t993 gccgtgagat 999t999399
GTTGGCATTG CACATCCATC CACTCCCAGA GTCATGAGTC
gcaggcggag ccttgaacgc taggctcaac {CtCtCttCC
TGCTGCCGCC GACACCCTCG GICCTGCTCG CAGATCCTGG

gtcctgcgcc
gttatggaat
agtcgactcg
89999t9989
gtgggcataa
atggtgctca
tgccaaggca
tgactatttt
tttccgtcta
CIGTTGTTAC
AGGCCCCAAC
ctgaccatgg
tcttcatgct
ggtccctatc
tccagaggga
ACCCATITCC
59t999t9t9
QQQCtQQCBt
ATCCCCAGCC
TCTGTACCCA
agagcaggca
ggcaactaag
tcgtcctgat
GAAGAAACAG
GTTTGCCTTC
thgagtatg
tcagGTAGAC
agtgctggca
tgaaggaaac
ggggatattt
gccatgggcc
tgaacctgac
cagagctgtc
tttaatagca
agagatctga
atgtaataca
gtagctgaaa
tggccttccg
tcacactgga
ttgctgtgta
gtcggaacac
TGCTGGACGG
CTGTGGGCCA
TGAAGGAGGA
9888968999
gtctccaggc
tcaaacccca
ctcaggaacc
tctcctacgg
gagtcactac
aagagtagcc
cccagcaggc
caa...~1.6
ccatgcattg
GAGTGGCTAT
CCATCTCTAT
TATGCTGGTG

...ttcagg tatcttttct

tatgactttt
actcttctgg
gagtcttcat
ggaaagtgaa
gcagcatcac
TGTTCTGCAI
GCCTTACACC
acaagcaaag
gagagacacc

tatgggctcc
agctgctcac
tttcatgggg
gtgctgcaca
gtacactggg
GTGGCTGTGG
TCTTTCCAGG
agactgggat
actttccagg

9t9996tt99
9999‘CCCt9
atccccctcc
aggaagtgaa
tgaagcctga
tctggtagaa
99889t9999
catggtgcag
tggtgtatgg
IATCCGTGCC
IGTACCAICC
cttcttttcc
cggtgtccct
ctctcaccgt
tggactcagt
TGCTGACACA
QQQCtQQCCC
999899399C
CTCCGACCTA
AAGACTGCCC
ggaaagtcat
cgtcatgcct

tccatctccc ttccaggcgg
gagaatggct agggcctacc
tggtctcttg tattttatcc
gacatggaca gttggcctga
tgtccctccc ttctgcagaa
accttcccat accctggcct
cccataacac attgtcttat
ggagggattg agaactttcc
9t9999C899 9C899t6899
AQAACCAGGG TGTCTGTGTC
gtgagcaaga ctccagtccc
ctccttgtct ggctttgatc
tgtcttattc tgggtcctgg
gggttgctca ggtctttcct
gactcccact ttccctgcag
TGGATACTGG CTCTGGGAAT
ctctaatctg gagaacctgg
3999889t99 ggggtaactg
CAACTCAGCG CATGACTACA
CACACCCATG GGGACCAAAG
ttctgaccca ttttttagat
atggtgacaa agaaagccag

gaa.....-2.4 kb-..... gggatgtagg

TTACCAGATG
TTTGCACAAC
t999868999
CTTGGCCACA
t999989999
caggaggaca
tcatttcctt
caaccatcaa
tgtctggata
caacagtagt
tactctactt
ttcctgcaag
aattaaaaag
catcagcttt
gcatccacat
ggcatactac
cccaagtggt
aggagccggc
AGAGGTGTAC
GGAGGTGTTT
GCATCCCACG
ttaggaccct
tgtggcttca
gtttgtttct
cccaatttct
gggtgagtcc
ctctgggtac
8989988998
tgcaatgtga
kb-.......
agctctgctg
TTCCTGCAGC
CACTGGCATC

TTCAGCITCT GGCCCAACAG
ACTTCACCCA CCAGTTCTTC
agggagtctc aggtgccaca
TTTATGGAGA TAATCTGGAA

acagtgtaa gggtctccct
gagagtaaag 96t9998998
agctgtttca ctccctggaa
gaaaccataa tctatcaggg
tgggtgaacc acagtttata
gtgagcctca tatgtaattc
gggctggtaa gtggctcagc
ttatcctctg acctccacag
gttctcattg gagatggctc
aaaaatgtaa aactataggg
agaggctctc aacctctgga
tactact...-100 bp-...
aaggcaagag ttggcttagg
tgtggagagc tccaagacgg
CCACCTTCCG TGGAACAGGC
GGGTTGCTTC CGGGGCTGAT
TGGGATGATG AGCAGCTCTT
ggtgttactg aacctgggga
gttcctacac gcgctgactt
cccttgtgct gtacccagtt
ctctctctgt ctatcagcct
ataacctaaa atgtcagatg
aagtgggcat ggctggttcc
caggctgatt cttcggaagt
agacactgtt aatcagactt
gtgaccaagg gagtaaatga
accttgtttc ttatcttccc
TCAAGTITGA CCCGGAGCTG
CACTCATGCC CAACTCCTTC

ggacctgcca
gtgagtccga
ttctgcagGA
GGTGCCCTCA
actccctgat
tgg......

ttggttctgc
tatgaccatt
atggaaggct
cctctctgaa
cagctgtttc
cttgggcccc
aggctacttg
CGCTTTGGCC
caactctcct
catgttccca
ctctttcatg
atagactcct
CTGAGATCTG
ITGTGAATGC
catttgataa
atgtgggaac
ICAGCTGGGA
9t38998899
gggtaaagca
8996898996
tacaagcaag
CTGCTGCTGA
AAGACCTCTG
tgcaggctat
CGACAGTATC
tgtctctctt
gtaacagggc
tgggcaccat
gtcaaataca
catacatgac
aaatttccta
tggtaaaggc
ctttgctttg
tctctagatt
ctggagaatt
actccagctc
tctccctctc
gcaggcactt
atcttggagg
GTCCGTGTTG
GCTCTTCTCC
CCAGACCACT
gtcacagcct
gctatgagcc
tggccttaaa
ttgtagaatt
gtttcctgcc
aattatagag
ctgtatagcc
aggacttcag
ctccagcatc
cttgtagGAG
CTGTTCCGAG
CAAGTGGGCI

9t96t99996
ccccagtggt
AGGAGTCTCT
CCAthaggt
tcccctttct
-S.4kb-...
aaagcctctt
tcctggtgac
aggcagatcc
gcccacaaac
ctagcttcag
atgcatggat
agggtatctc
TCGACAACTA
gctccctgag
cgtgagctct
tgttatcact
gtttggtccc
GACCTGGCTT
CACCTTCATC
889689t999
agcctgtcat
GTCCTTCTCC
99Ct9t99C3
atgcaagaca
tgactatggt
agctaggcat
GAAGGGAGTI
GAAAGATGGG
catcagaatc
ACCTGCGGCT
ataaagatgg
aaaagcaaat
gatggaactt
ggattaccag
tgtcaggggt
gtggcttcag
acttgccacc
ataccaggat
tcataaactt
989tC89699
taaatgatct
ctcctcctcc
agggagcagc
89996C8989
ATGCGCTACC
ACGATCIGGC
CGCCTCATCC
gagcccgagc
tagaaggaag
gtcctcagca
tggtagattc
tggaccctgc
gctaattcac
cagaccttta
ccgacccatg
8968998989
AAACCATCAA
CCCAGTTCCA
CACAAGAGTA

AGTGCTGGAG
gcccccacgc
CGCTCTGGTT
gcccccgccg
QCtCQCQQtQ
cggccgccgt
tcctggaatt
cttttctggc
tgtgtttcta
atggcgtttg
ctccaccagc
t999Ct9989
taatacttgt
CCAGTGTGAT
atcatctcta
ttacttagtc
cctaagggta
tatcctctca
CGGAATTCTC
CGAGAAGTAC
tggagaaact
tatttttgtt
AATGTGAGCT
aggaactagc
gcatgtctag
tatcatggta
cttctgccct
CATTCCTGCC
TCCTGGCTTT
catgtgatct
CTTCAAGGAT
Ct999t8990
aaagaacggg
gtacacatga
ggtacaggca
acaggctgta
taacaaaagt
aagcctgaca
gctcttgcct
tgtaattgaa
ttgagagcac
gatgccctct
tcccccactc
aggcagatgg
tcaggttgcc
CACCAGGTGT
TTCGTGAACA
TTATAthga
cagggtttgg
gaaggctctg
caccagcatc
taggtgactc
acctctgtcc
cggcttctag
ccagagaggc
ttgacacggc
gaggagcact
AATTGTCATT
ATATCGAAAC
CAGCTACGAG

TTGCACCCGA
gtcccggaat
TCCCQIQQIQ
gatgcccgtg
ccggggaata
caattaacct
gattgctgag
cttattttcc
atatctgggt
tggtgtttgc
ttggctctgg
gtccagggca
ttctcaccca
TGTACTCGCA
tacccccaca
ccctcccacc
cttcctatag
ccgtgggttg
TGCGGCCCAG
TCATGCGCCT
aagatttcca
ctgcagTGCG
ACTATACTCG
ttgggttttc
gatcaaggaa
ccccagggtg
ttcctacctc
CCCCAGGGCA
ACCAAGGCCT
gaatggaccg
GGGAAACTTA
aaattgaaga
gtagatttct
ctgagtcagt
aaccacagtc
gcaagatttc
aaaaaacaga
gccagtgttt
ctgcacacag
taagtagatt
ctgctgactc
tctggcctct
cgtctctcag
9CC8999Ct9
aggcggcctc
CCCGCCTGAA
TAACCGCGIG
gcacatgccc
ctacccatac
agtacatttg
aaacctcagc
agggacaaga
agaccaggct
aagagttggt
caggcctgtg
tgaggttgcc
tttcacaaga
GAGGAGTATG
CGCATCGCCA
CAGTTTTTAT

GACTATGGGG TIGAGGCACT GGTGGATGCC TTCTCTCGCC AGAGGGCTGG CCGthaagc tt..-3.0 kb°
ttcctgctca attatacatg attatttcag

tctgtgtgtg
Cttgtttctt
actttatttt
cttcagtgtt
ctacaccaga
ATGTCATCAA
AGCTCACAGg
93t9C999t9
‘9999t9399

tgtgtgtgtg tgtgtgtgtg
acttggcctg tagcttacct
tctttctttc aataaagatg
gaggaagcag acacattgtg
ttctg...-100 bp-.....
GGAGTCCCGA GAGATGCGCC
taggttgctg catcctgggc
gccagcattg tggctgagca
gtaataaata aaaaatttcc

gagaaataga
tgtgagagag
agtgagctag
acactacctg
ggcctctaag
tctcttacca
TACAGCCCTI
atagtctctg
ttgtcaggaa
aggtataaaa

acttccttaa
agagagagag
gtaggctgtc
ttggtgtgac
tgagacgcag
gATTGGIGGA
CAATGAATAC
cccttgaggc
gacagtgaat
t89899399t

aactgatgct
agaggtcaga
caacaggccc
tccaaggaac
99t3999Ct8
GGTAGGAACT
CGAAAGAGGT
atgtccagaa
gtgacagtgg
gcacttcagg

ccttagtttt
tgtgtgtgtc
cggggatctg
tcaagttact
gagccagact
ITGACTATCA
ITGGCTTGAA
agtacagatc
cctactgatt
atatggcaaa

gggaaagcaa
agatgctgaa
tcagtggttg
ttaagctcaa
attcaggatc
taaacaagtc
tatcctccta
ttcgctttgt
tattggacct
gcggatccaa
taaaagatct
actggtgtga
catcatctgt
tttctcatga
gttaaatttt
catccccagg
GTACGGTGAC
GAIGGGGGCT
GGGCTTCAAC
TIACCCTGGA
TCCTGATGAA
CCTAGAATAT
TGGCTCTTTG
CACACACCTT
CAGCCATGGA
AAAAAATCCC
AGAAGGTCCT
GTTCAGTGAA
TAAGGTGTTC
CAGTICTATC
tctctaagta
tgcttcaacc

aaggatagag
999tt999tt
agaatttgcc
cgactgccta
tttatcatca
ctggatattg
cggtgagcgt
ctgttggtgc
gcggatcttt
tgttcttgtc
ctactctctc
cagaccctgt
ggacatgcaa

gtaagaaaag
gtatcctgtc
cagcatacaa
tatggcagaa
gctacaggcg
gcgcgcaaac
99t9t69999
899t993339
ctgcgtcgtt
tgccacaggc
ttctccccca
acccatagga
ctgtcagagt

aaagcg...-100 bp-....

tacgatgttt
ctgatcttaa
ATCGAIGCTT
CCCTTTICCC
CTTGTCAACA
GATGACGGGT
GACAGTCCTT
TGTGATTTTG
TTGGCAACTC
AATCCCAGCA
TGCATAGTGA
TTGAAAGTTA
GTTCCTGGTC
ATGGACACAG
TTGGGAGCCA
CCCATCCAGA
tcctctgtaa
ttccctttgg

cctctagttg
gaaaatcatg
TAGAGTTCTA
TCAAGGGCCT
CAGCCTCACT
CTGTCTTAGI
GATGTGGGTT
CCACTTTCGG
AGAAAGTCAG
CITGGAAGGC
GACTCITICT
ATGGGGTCCT
AATGATCTAT
AACAAAAGAA
CACTIAGACT
TCTTTGCTTG
gtgacacttt

tagatgcttg
tagtggttcc
gaggtcattg
attgccgcca
gtagcgccct
tctactgatc
ccagtgtggc
taaaacggca
gggctacccg
acatgactta
ccccccttcc
998t999ttt
ggaacagagt
atgtccaggt
ctcaaattgt
agtgactgcg
CCCGGGGTTG
CCTAGGGAAT
GAAGAAACTG
GAGACGCTCC
TTCGIGGCTT
ATGTTGAATT
ATTTCTGGTT
AGAGGCAGTT
CAAAACAAAC
CAATITTCTT
AACATGGGCC
CCCAGTGTCC
CITTCCAAAG
TGGCAGCTGT
cctcctaccc

63

tctgggagat
taagtactga
gcttaacccc
cttctggtta
atgccagcca
ccgatggtcg
BCQQQCtSCQ
ctggataacc
gataacgccg
cgtgtacatg
cccaaatttc
gaggaaataa
ctgggatcag
tgaagggcct
tcttctgtag
tcctcttgct
CTGCTGGAGA
CCCATCTGTT
GTCTGCCTCA
ACTGAGCTCT
GGCATTGTGA
CTTTGTIAAC
GATITGGAAT
GGAICICTGG
AAACAAACAA
CCTAGAATTT
AAAACATTCC
AGCAAGAATT
ATGTGGAGGG
TICTCATGAA

aC39t99939

gccataatct gtcccttcag tctg

gctaggtaac
caaagatgat

aaagggagat
aggagtatct

ttgat...-100 bp- .....

tgactggttg
agcccgtgat
atactgccga
cgctggggac
tggacgaaat
ggcacccgga
gccacacaga
cccagacgtt
atggagagag
tagatgagct
atttgttctt
tctctgtcac
ccacagGAGA
AGTGCCAGCC
CCCCAGAGTA
ACACCAAGAC
GAGGGAGCTG
GAGCTGATGC
TAAGAAAGTT
ATAGGCITAA
GAGTITGAGG
ACAAACAAAC
GGAGGCCTCT
CAACTTGAAT
GCCTTGCCCA
AACAGATGGA
GCTAATAAAA
acttttccag

ctgattgacg
ccgtccggtg
caggcacgtc
scattgagaa
cctcgacgtc
agtgcagcga
gatgtatata
998689t998
cccatgtctg
cccacggtcc
tctttcctga
ccccatataa
GAAGGAGATG
CAACTCCATC
CTGGAAACCC
CTGCCCCTAT
GAAAGCAGCC
ICACATTTGA
AGAAGTGGTT
AACTTTATAT
CCAGTTTGGC
AACTTTTAGA
TCAGAATGTT
GTCTAGAATG
AACCTACGTC
CTCATCIATG
TTctttctcc
gagcttaccc

tgacagtgca
99tt99tt99
aagtgaaatt
9996968696
gaaggcagta
aggtggtgtc
tacatggcgc
9889998tt9
attattgaaa
attaaaaata
gttagagtcc
tctcttttgt
tggaacaatc
ctattctctg
aaactagatc
GCTGCTGAGT
TTTGGAGAAA
AGCACGTTCG
GTTTCCTTCC
ICTGGAGGGA
AACTTTGGGT
TTGTCTGCCI
TATAGGGTAG
CIATATAGTG
ATGTAGAATT
GACTATCIGA
IGGAATTGGT
IACGCCAAAG
ATCTTGGTTG
aaagttgctt
tgtagaagcc

ctagcatctc
ggagttgtgg
99tct9t9t9
gccaaacacc
aaccgctgat
tgccacgcgc
aagttaacga
atggcgtgtt
ccagtattcg
aaataggtct
tcttgtctcc
ctcatgtgtc
atcttgatga
atgactgcag
caggagcttc
TGGAGGAGCT
GTATGATAGA
GTGGTGACGT
GTGTGCCAGA
GGAGTTTTGT
CTTACCCTTG
CCTCAGAACT
GGTGTGGTTG
AGTTCTAGGC
CCGTAAAAAA
CAGGTGACTC
ICATTTTCCT
GTCAAGGCAG
GAAACCACCA
gctatgtttc
ttatctttga

64
essentially identical to those reported by Yokoyama and Tanabe for the human PGH

synthase-1 gene [61].

Characterization of me 5’ end of me PGH Synthase-l Gene. The position of the
transcriptional start site of the PGHS-1 gene was mapped using primer extension and
RNase protection analyses. Primer extension analyses were performed using two 30-mer
oligonucleotide primers complementary to the coding sequence near the 5’ end of the
PGHS-l cDNA (Figure 9). A 126 nucleotide extension product obtained from the primer
DD-68, and a 205 nucleotide extension product obtained from the primer DD-69, indicate
that the transcriptional start site is located at an adenine residue 64 base pairs upstream
of the ATG translational start site [39] (Figure 16). Both primers produced extended
products with "ragged ends" (1'. e. they produced a single large extension product, and
two smaller extension products, each a single nucleotide shorter). It is not known
whether this is a result of variable transcription initiation or capping, or incomplete
primer extension.

Results of the RNase protection analysis were used to confirm the location of the
transcriptional start site, as determined by primer extension anaylsis. cRNA,
complementary to the 5’ end of the cDNA produced by in vitro transcription of a
linearized pSP65 plasmid in which the 5’ end of the PGH synthase-l gene had been
subcloned, protected RNA fragments of 68-73 nucleotides in length. The sizes of the
protected fragments correlate well with a 70 nucleotide protected fragment, which was
predicted from the results of the primer extension analysis (Figure 17).

Approximately 2400 bp upstream from the transcriptional start site was

sequenced, and examined for putative promoter or enhancer elements (Figure 18). No

65

Figure 16. Mapping of the transcriptional start site of the mouse PGHS-1 gene
using primer extension analysis. (A) Schematic of the primer extension experiments
showing the size of the extended products obtained with oligonucleotides DD-68 and DD-
69. Poly A+ mRNA (10 pg) was annealed to (B) the 30-mer oligonucleotide DD-68,
TGTCGGCGGCAGCAGCAGGAGCAGCAGCAG, complementary to base pairs 70-99
of the PGHS-1 cDNA or (C) the 30-mer oligonucleotide DD-69,
CACCCTGGT'I‘CTGGCACGGATAGTAACAAC, complementary to base pairs 149-178
of the PGHS-1 cDNA, and was extended with reverse transcriptase. The extended
products were analyzed on a 5% polyacrylamide denaturing gel and their sizes
determined by comparison with the dideoxy-termination products from sequencing
reactions run in adjacent lanes of a known template-primered with a 30-mer
oligonucleotide. The autoradiogram of the sequencing lanes resulted from a 12 hour
exposure, while those of the primer extension were from a 72 hour exposure.

 

66

2% :2m Ecozatomcmfi ommfigm 10a .4

mean

0.90004004404000900094904590:05.08944090400409000090000900940400090090090
m m o o _

 

00.500494 ._. 8E8909000909090400<<0000904090

o»o<crack?o<oooB<ooEooEo<oot<ooctoooo<ooo <oo<oooo<ooto<oohoo§

e

00000.50.901000900400004000.900094040900004.0090000090484005009044040.90

 

4444444! I

 

(ZEE

mean .HHIIITIII an mom
mmméim 10a

Soc H“ an ear

 

gut

67

 

:126
12p25

ll

 

 

 

 

68

 

69

Figure 17. Mapping of the transcriptional start site of the mouse PGHS-1 gene
using RNase protection analysis. A labelled 536 nucleotide antisense cRNA probe
corresponding to the region surrounding the translation start site was synthesized as
depicted in Figure 10. The labelled probe (5 x 105 dpm) was combined with 10 pg poly
A+ mRNA or 20 pg tRNA for a negative control and hybridized for 12 h at 60°C.
Single-stranded RNA was removed by digestion with RNase A (18pg/m1) and RNase T1
(lpg/ml) for 1 hour at 30°C. The size of the protected product was determined by
electrophoresis on an 8 % polyacrylamide/7 M urea sequencing gel. Sequencing reactions
with a known template and primer (30-mer) were run in adjacent lanes for determination
of the size of the protected products. No bands were observed in the tRNA control lanes
(not shown). The autoradiogram of the sequencing lanes resulted from a 12 hour
exposure, while those of the protected products were from a 72 hour exposure.

 

 

70

 

71

Figure 18. Sequence of the S’-flanking region of the mouse PGHS-l gene, showing
location of putative responsive elements. 5’-upstream sequence, exons A and B and
intron a are shown. Intron a sequence is in lowercase letters. Numbering begins with
the transcriptional initiation site (+1) and the translational start site is in boldface.

-2489
-2450
-2380
-2310
-2240
-2170
-2100
-2030
-1960
-1890
-1820
—l7SO
-1680
-1610
-1540
-1470
~1400
-l330
-1260
-1190
-1120
-1050
-980
-910
-840
-770
-700
-630
-S60
-490
-420
-350
-280
-210
-140

-70

71

141

AGAACTTCTG
AGGGTTGACT
CCCTGGGTTG
CCTTTCTTTG

AGCAACAACC

AP-l
MW

GCCTGTATTA
ACACTCCCAT
TAGCAAGGGC
GTTCAAATGG
TTCTTGATTC
CAGGACAGGT
GAGTGGAGGA
GGAACAGAAC
CCCAGCTGGC
CCAGGGCTCC
CTGTAGAGCA
TGTTCCTTTC
GCCTCTGGGT
CGAGGACACT
CCAATGACCC
AGCCCTGGAA
GATCTGTATA
TATACAAATG
TATGCCATAC
TTAATTCACT
CCCCTACACT
GTCCATTTTA
TAGGAATGTA
TGTTGGCGGG
ATGGCTTCTC
TTTGTTCTGC
ATGGATGCAT
CCATGCAGTA
GTGACAACTG

AGTGCTGGAG
xr-r

gtgagtccga

taggctcaac

TGCTGCCGCC

TAGGAGAGAA
GCCCTCTACA
TGAGCTTTCT
GATGGACCAA
AGCATCTAAA
cccrrrcccr
ATACCCTAGC
TGCAGCCCTC
TCATGCTCTC
GAAGCAGGCC
CCAGGCTAAG
CTGGGAGTGA
TGAGAATCAT
crcaccrrrc
TGGTTACCAG
Acrrcrrccc
TGGGAACAGC
CTGAGCCCAG
GCGGGCGTGG;
GTGGCTTTGC'
AAAGGCTTAG
TCTAAGTTAG
TTTTTTTCTA
TTATAATTTT
ATATTTTTTG
TTACATCCCA
TCTCCTCTGA
TAAATAAATA
AATTACAGCT
CAGGAGTGGC
ACCTCCTTGC
CTACAAGCCT
TTCTGACACT
ATTAATAAAA
GAAGGAGGAG
TTGCACCCGA
ccccagtggt
tctctcttcc

GACACCCTCG

GAAAAAACGA
AGGTCTACAT
AGTAGAACAA
GTGTTAACTC
TGTTCTCTAC
TGTCTACTTC
TGCTCTCTTG
CTTCACTGCA
TACTGGCATG
CTAAGGGCTT
TCTCCTCCCT
GCTACAGGTG
GCCAGCTCTT
CTCCCAGATC
AGGGGTCTTA
CTGGTCTGTC
TCTGGTTTGG

AGATGCCCGA

‘72

ACAACTTCT
CTGTCAATGA
TCAGGACGTT
GGATGCTCCC
TGTGTTTTCC
CTCTCTATCC
TTCCTTGCTC
GCCTCTTGCT
TCTGTCTTCT
GGTCCTGCCT
GTCTATGGTC
CCAGGGTTAG
TGACTCCTTG
CTTGTATTTC
TCATTATTTT
AATCTGGGGA
CTGGCTTGCT
AAGCAAAAGG

GAGCCAGCAT

ATCTAGCACC
CTGGAGCAGA
GGCTCTGGAC
CCAACTTGGG
AGCCACTGCC
ATACTCCAGG
CACCCCCTTG
TCTCCACTGT
GATGGCATGA
TTGCTACCCA
AAAGAGAAGA
GGCTGAGCTA
GGTCCAGGCC
TTGGTCACCA
GCTGACAATC
TGTGGAGAAA
CAGTTTCCCT
GGCACCCCAT

AAGGATGAAT

ccA;Apcqccfgccrccdaax ACTGAGGGAG
TcdrflflrdrcfirchCCrrcr'AGGCTCAGTT

... r ,I t i z r .
crccrhrzhc‘ocrcccnher*bcccarrcac

telr
cccaaérrrc

TGGTGTTTTT
CTTTTAAAAC
ATATCTACTT
CTCACTGCCC
GTAGGTGGGG
CACCGAGGAC
CCTCATGTCT
DR!
AGTAGCGTGT
TCCTCATCCT
TGTCCTCCTT
GTAAAAAGAT
CGTCTTGGCT
Sp-X
TGGGGGTGGA

GGAGCCAGCC

.fi' f,’ -

:AAAAAGGTTC

.‘9 fl

TTTAAATGTC
AACTCCCCTC
TATATGTATC
CTTCCTGGCC
GCCCCCTGGG
CAGCAAGGAC
GACCTGGCCT
TTTCCATGCA
TCTTCAAGCA
CGTGCCCATC
CTTGCACCTC
AAGGCTGGAG
GCCGTGAGAT

GTTGGCATTG

AI-Z
gcccccacgc gtcccggaat

ttctgcagGA

GTCCTGCTCG

AGGAGTCTCT

CAGATCCTGG

TéTGACCACA
TCATTAATTT
ACCTTTAACT
TTTAAAAAAT
ACCCCCTCCC
TATCCCTCAC
TAAAGGTGAG
CTGCCTGCTC
GGACCTCGTG
AGCTATGGTT
CTCAGAGAGC
AAGGATCGTG
CAGAGGAAGT
ggéiégeacc
CACATCCATC
tcggtggcct
CGCTCTGGTT

GGTGCCCTCA

ACTTGAGGGG
CAGACAATCT
ACAAGAGCAA
TATTCATCTG
TGTCCCTGAG
TCAACTGGTA
GTTAGGCTTG
ACCCATCCTT
AGGGCTGTAG
CTTGGTCCTA
AAGGAGACTT
GTTAGCATTT
CAGAGTGAGG
GAAGGTCCCC
ACTTGACCCA
CTGAAGCTGG
AGAGCAGTTG
CAGGCCCCAC
GGGCCCTTCC
AGAGCTGGCA
TCCCCCACTG
CTGGAGGGTG
GAGTAGCCAC
TATTTTGTGT
ATCAGTAAAC
TTTGTTTTTT
ACAATCCTTC
TGCCTAGGGT
CCAACTGGCA
AACGGAGAAG
TGCAGGCTGG
CAAACCAGCC
CTCAGTTTGC
AGTTACGGTC
TGGCGGTCAG

GGACCTGCCA

AGGAAGGCAC
CAACTACTGG
GGAAAGGCGG
CATGTTGGAT
CATTGTCCCT
AAGAGCTGGC
GTCCTTGGAG
CATCAACTGC
AGCATGGTTA
TTATCCTGGT
GAACTCAGTT
GTTTTCACTA
GCAGACCGGG
TGTCCTCTGG
CAGCCCCTTG
TCTCAGTATA
GCTTTTGTCC
CTTTCCTACC
ACCAAGCCAT
AGGAAAAGGA
TACAAAGGAG
GCATTTGGCT
TGCCACATGG
AAGGATAAAA
AAAATTGCCT
AATTTTTTAC
CCCCACTTCC
GGTTTTGCAT
GGCTGTGTAT
GAAGCTCTTC
ACTGGACTGC
TCTTCCACCA
TATATCCCAA
TGAGCAGTCA
CCTGTCAGAG
GTGCTGGGGC

t
GTCAIGAGTC

AF
CACTCCCAGA

QCGQQCQQag
TCCCCTGCTG

CCAG

ccttgaacgc

CTGCTCCTGC

 

menus
region [l3
at position
sequences.
been shut?

piOmO'tEt'S.

 

promoters [‘

Othe
resembled tl
to +72, +'
Interestingl
anegatit'e:
sees nee it
torn regu
tese m b J
ltT/ClCtG
addition, :
dioxin res

heated at

1123mm

73

consensus TATAA- or CCAAT— box promoter element sequences were located in this
region [148-151]. However, two putative Spl binding sites (GGGTGG) were identified
at positions -47 to -41, and -30 to -24 relative to the transcriptional start site. These
sequences, which are similar to the canonical Spl binding site sequence, GGGCGG, have
been shown not only to bind Spl and enhance transcription in several papillomavims
promoters, but have a proposed role in directing transcriptional initiation in TATAA-less
promoters [152-155].

Other putative regulatory elements were also identified. Three sequences which
resembled the consensus Ap—l binding site ('I’(G/C)A(G/C)TCA(G/C)) are present at + 65
to +72, +72 to +79, and -2097 to -2090 relative to the transcriptional start site [156].
Interestingly, one putative Ap-l binding site (at -2097 to -2090) was located adjacent to
a negative glucocorticoid regulatory element (nGRE) (-2123 to -2109). This regulatory
sequence in other genes, such as proliferin, has been proposed to mediate glucocorticoid
down regulation of phorbol ester-induced transcription [157, 15 8]. A sequence which
resembles the Ap-2 binding site consensus sequence
((T/C)C(G/C)CC(C/A)(G/C)(G/C)(G/C)) was located at +93 to +101 [159]. In
addition, a sequence (5’-GGCAGTAGCGTGTITI‘CCA-3’) resembling the consensus
dioxin responsive element (DRE) ((C/GTNGCGTG(A/C)N(A/T)N(G/C)N(G/C)) was
located at -403 to -385. This sequence has been shown to mediate the modulation of
transcription caused by xenobiotics such as TCDD in other genes [121, 123, 131].

To determine whether these transcriptional regulatory elements were active, we
constructed reporter gene constructs containing the 5’-flanking region (Figure l 1). These

were transfected into NIH3T3 cells and tested for their response to serum. After

74

transfection of luciferase reporter gene constructs containing 2.3 kb of upstream
regulatory regions of PGHS-l into 3T3 fibroblasts, we failed to detect significant
induction of reporter enzyme activity in response to serum stimulation, although in cells
transfected with a PGHS-2 reporter plasmid, serum stimulated luciferase expression was

observed (Figure 19).

75

Figure 19. Luciferase activities observed upon serum induction in transfected 3T3
cells. NIH 3T3 cells were transfected with: (A) pGLZ-Basic, the parent luciferase
expression plasmid (which does .not contain a promoter or enhancers for luciferase
expression); (B) pGLZ-Control, the parent luciferase expression plasmid containing the
SV40 promoter and enhancer sequences; (C) the'pGL2-Basic plasmid containing 2.3 kb
of PGHS-1 5’-flanking region, described in Figtrpe 11, (PGHS-1); or (D) the pGL2-Basic
plasmid containing 1 kb of PGHS-2.5’-flankir‘ig region, described in Figure 12 (PGHS-
2,,,,,,,). The transfected cells were then stimulated:with 10% fetal calf serum, or treated
with carrier, alone. Luciferase values are expressed as the fold induction observed in
serum-treated cells over those treated with carrier alone. Error bars indicate standard
deviation. ('P< 0.05 vs. Basic; n=4 in each group)

76

 

 

 

- 1 u
5 4 3 2 «I

ccoEEo... :53» can: 5:25.: Eon:
>=>=o< 820:2...

 

Control PGHS-1 PGHS-2

388“:

(short)

77
Discussion

Genomic clones containing the mouse PGH synthase—1 gene were isolated and
characterized. This work, together with work by Dr. E. Meade on the 3’-end of the gene
provide a complete structure of the PGHS-1 gene. The restriction map of the mouse
PGHS-l gene is remarkably similar to that observed for the human gene [61]. Such
conservation of structure and sequence suggests that gene structure, itself, may, in some
manner help regulate of PGH synthase-1 expression.

The first two exons of the mouse PGH synthase-1 gene appear to be associated
with clearly defined functions in the mature protein (Figure 20). Exon A, for instance,
is the transcription and translation initiation domain and contains the entire 5’-
untranslated region and translational initiator. The signal peptide, the signal peptide
cleavage site and the amino terminus of the mature protein are entirely located in exon
B. The next exon, C, contains an epidermal growth factor homology domain, that
appears to allow dimerization of the PGH synthase-1 monomer [56, 66]. All other exons
of PGH synthase-1 do not appear to encode defined separate functional domains but
instead appear to collectively comprise the catalytic domain of the enzyme. Interestingly,
in addition to the 60% similarity between the amino acid sequences of PGHS-1 and
PGHS-2, the genes encoding these two isozymes also show a similar intron/exon
structure [146]. The main differences between the coding regions of two enzymes are
found on the amino-end, where significant differences also exist between the two genes.
Mouse PGHS-l contains 9 amino acids more hydrophobic leader sequence than PGHS-2.
Differences are also seen at the carboxyl-end of PGHS-2 which contains an additional 18

amino acid inserted region, but these differences are not reflected in the relative gene

78

Figure 20. Correlation of the mouse PGHS-1 exons with specific functions of the
PGHS enzyme. Nucleotide sequences encoding residues associated with proper
functioning of PGHS are distributed throughout the PGHS-l gene structure. These
include a putative signal peptide in exon B; an EGF-homologous domain proposed to be
involved with dimerization of PGHS-l [56] in exon C; N-linked glycosylation sites in
exons C, E, and I [57]; heme-ligand histidines in exons F (proximal heme ligand, His”),
and I (distal heme ligand, His“) [56, 67]; the cyclooxygenase active site tyrosine (387)
[68] in exon I, the aspirin acetylation site (Serm) [39, 160] in exon K.

552.5 use gamma—«who amnion—Ecum— mbmafinmm 00m

 

 

 

79

4
_._ __ a _e_.__a_a_e_a
a; a a am a
e. read ovvo $0 dear 00 «a... 9.
99/ % «lure %0 é/f V 4 00%
Q .P 9% 00 02m 6.? (Q.
990.. 9% 940.» 2 and Q 90 . ea
.9 a... a
%%9 $0.0 0% 8). 0&6 3..va
6 0 (J r50 0.0
a . 4 re.

80

structure. While the gene structure of mouse PGHS-1 and PGHS-2 are similar, they are
quite dissimilar in size (22 kb vs. 8 kb for PGHS-l and PGHS-2, respectively), and are
located on different chromosomes (chromosome 1 for PGHS-2 and chromosome 2 for
PGHS-l in mice) [24, 29]. Despite the fact that PGHS-2 has an almost identical
exon/intron structure to that of mouse PGHS-1 [146], the major structural difference
between the two genes is an additional exon in PGHS-l, formed from the apparent
splitting of exon A in PGHS-2.

Both PGH synthase-1 and PGH synthase-2 catalyze similar activities although the
regulation of the two forms differ greatly [69, 70]. PGH synthase-l is constitutively
expressed [22, 29, 46, 64], and thus believed to produce prostaglandins that regulate
"house-keeping" functions in cells. PGHS-2, on the other hand, is highly inducible, and
believed to be involved in production of prostaglandins that regulate inflammation, cell
growth, and ovulation [32, 42, 75, 161, 162]. Despite the similarities in the coding
regions of the two PGHS genes, and their similar genomic structure, the 5’ and 3’
untranslated regions have no significant homology, suggesting these regions are
potentially responsible for the differential expression of the two isozymes.

PGHS expression may be regulated at the transcriptional or post-transcriptional
levels, or both. While PGHS-l is constitutively expressed in many tissues, increases
in PGHS-l mRNA levels have been observed in response to factors such as serum in
NIH 3T3 fibroblasts [44], or TPA in rat trachea epithelial cells and in the monocytic cell
lines THP-l or U937 [26, 49, 72]. Co-ordinate elevations in PGHS-l protein levels
were only observed in the monocytic cell lines THP-l and U937. The increase in PGHS-

1 expression does not appear to be a direct effect of TPA stimulation, but rather an

81

indirect effect of differentiation of these cells into macrophages initiated by TPA
treatment [49, 72]. Therefore, although regulation of PGHS-1 expression may not occur
acutely, it may be regulated developmentally in many cells. This hypothesis was
strengthened by the recent report in which elevations in PGHS-1 mRNA and protein
levels were observed in ovine pulmonary artery samples during the last trimester of
gestation through the first four weeks of life [163]. In contrast, PGHS-2 appears to be
acutely regulated in a number of cells in response to stimuli, but is not detected at
significant levels in unstimulated cells, fully differentiated cells or tissues [22]. PGHS-2
mRNA and/or protein levels have been shown to be positively regulated by v-src
transformation in chicken embryo fibroblasts [53, 75]; serum in mouse fibroblasts [22,
28, 44, 47]; phorbol esters in mouse fibroblasts [22, 28], human monocytes [30, 49], rat
tracheal [26] and human endothelial cells [64]; hCG in rat granulosa cells [32]; PDGF,
EGF, and forskolin, calcium ionophore, and cAMP in mouse fibroblasts [22, 28, 74];
interleukin-1 in human endothelial cells [29]; and lipopolysaccharide in human monocytes
[30], and endothelial cells [29, 64].

PGHS-1 gene expression may be mediated through one of the putative regulatory
elements identified in the 5’ flanldng region of the PGHS-1 gene. The PGHS-1 gene
contains, among other putative responsive elements, three putative Ap-l sites, which in
other genes have been shown be involved in protein kinase C-mediated transcriptional
induction, as well as one putative Ap-2 binding site, which may mediate CAMP-induced
transcription [156, 159, 164]. Using transient expression assays in 3T3 fibroblasts
employing 2.3 kb of 5’-flanking sequence and the promoter of PGHS-1 driving a

heterologous luciferase reporter gene, we failed to detect a significant increase in

82

luciferase activity upon serum induction in cells transfected with this construct, even
though a small induction in PGHS-l transcription and mRNA levels has previously been
observed in this cell system [44]. Several explanations for this result exist: First, the
putative responsive elements identified in the 5’-flanking region of PGHS-l may not be
actual cis-acting factors, and transcription induction may be controlled by regions not
included in the reporter construct. Second, the reporter construct may not accurately
reflect the context of the PGHS-1 promoter and regulatory regions, and transcriptional
induction may involve modulation of structural features of the whole PGHS-1 gene.
Third, because PGHS-1 transcription was enhanced only a small degree by serum in 3T3
fibroblasts (approximately 3-fold induction of mRNA was observed) [44], it is
conceivable that the luciferase assay system is not suffiently sensitive to detect this level
of induction.

While PGHS-1 mRNA levels have been shown to increase in a number of cell
lines in response to stimulators such as serum, PDGF and IL-1, no examples of a
coordinate increase in PGHS-l enzyme levels have been reported. This lack of co-
ordination between the mRNA and protein levels suggest that post-transcriptional
regulatory mechanisms may regulate expression of PGHS-1. Regulation of mRNA
stability clearly seems to be involved in the regulation of PGHS-2 expression. Analysis
of the 3’-untranslated regions of the PGHS-2 cDNA revealed the presence of 11 copies
of the sequence AUUUA, which has been implicated in the mRNA destabilization of
other short-lived mRNAs [165-167]. These sequences are not found in the PGHS-l
mRNA [22, 39, 75]. The PGHS-2 mRN A has a relatively short half-life (estimated 0.5-2

hours), while the half-life of PGHS-l mRNA is comparatively stable (t1,2 > 3 hours) [26,

83
44, 45, 168]. It has been reported that the anti-inflammatory glucocorticoid,

dexamethasone, inhibits stimulation of PGHS-2 expression. Dexamethasone not only
inhibits PGHS-2 transcription, but also reduces PGHS-2 mRNA stability, and may also
affect translation of the PGHS-2 message [44, 45 , 168]. Although there is evidence for
regulation of PGHS-2 expression through post-transcriptional mechanisms, no such
evidence exists for post-transcriptional regulation of PGHS-1.

Two different, alternatively spliced products of both human PGHS-1 and chicken
PGHS-2 have been reported. For PGHS-1, Diaz and co-workers describe one product,
which maintains the same splice sites as reported earlier for the mouse PGHS-1 gene,
and a second, 111 bp shorter product, which arises from an incorrectly spliced exon
9/exon 10 junction, which does not disrupt the reading frame. In their system, the larger
product is preferentially produced under stimulated conditions [169]. On the other hand,
Xie and co-workers isolated a chicken PGHS-2 cDNA clone, in which the first intron
was not spliced out, which resulted in an altered translational reading frame. They
further illustrated that this anomalous intron l-containing PGHS-2 mRNA was produced
only in nondividing cells [75]. As yet, these alternatively spliced forms of PGHS have
not been confirmed in other systems, and the role of alternative splicing in PGHS
expression remains largely unexplored.

Transient expression assays using reporter constructs containing the 5’-flanking
region of PGHS-2 have proven useful to a number of researchers in identifying regions
in the gene responsible for induced transcription. Fletcher, et al. demonstrated that a
luciferase expression vector containing a restriction fragment corresponding to

nucleotides -963 to +70 of the PGHS-2 gene (analogous to our PGHS—2shon vector)

84

conferred an apparent 8-fold induction in luciferase expression upon serum treatment in
NIH3T3 cells. In comparison, the magnitude of induction we observe in cells transfected
with PGHS-23hort is only an approximate 4-fold. The reason for this discrepancy is not
readily apparent. Fletcher, et al. further demonstrated that a restriction fragment
containing sequences corresponding to nucleotides -371 to +70 of the mouse PGHS-2
gene in luciferase reporter construct was sufficient to confer both serum and TPA
inducibility in mouse 3T3 fibroblasts [146]. Related studies have been performed using
PGHS-2 from other species. Xie and co-workers were able to determine that a region
between nucleotides -158 and -3 was necessary to detect significantly elevated CAT
activity upon serum treatment over background levels in reporter construct-transfected
3T3 fibroblasts [170]. Unfortunately, this research group did not attempt to differentiate
between serum-stimulated CAT expression and basal CAT expression.

The most detailed studies of responsive regions of the PGHS-2 gene have been
performed using rat PGHS-2 sequences, and rat granulosa cells. Using CAT reporter
plasmids, Sirois and co-workers demonstrated the importance of the region between
nucleotides -192 and -53 of PGHS-2 in forskolin-, leutinizing hormone-, and follicle-
stimulating hormone— (but not gonadotropin-releasing hormone-, or interleukin IB-)
induced expression in transfected rat granulosa cells [171]. Through the use of
electrophoretic mobility shift assays, using extracts from hCG—stimulated rat granulosa
cells and labelled DNA fragments derived from the rat PGHS-2 upstream region, in
combination with further CAT transient expression assays, they were able to demonstrate

that a C/EBPE responsive element, located between nucleotides -142 and -l20 was, in

85
part, responsible for the activation of PGHS-2 expression by forskolin, LH, and FSH

[172].

Interestingly, each of these research groups intimated the possible presence of an
inhibitory domain in the 5’-flanking region of PGHS-2, which depresses both basal and
stimulated expression of the reporter enzyme. Inclusion of PGHS-2 gene sequences
upstream from nucleotide -371 in mouse, -192 in rat , and -755 in chicken in reporter
constructs appeared to reduce both basal and induced expression of reporter enzyme in
transfected cells [146, 170, 171].

Comparison of the 5’ flanking region of PGHS-2 genes from diverse species
reveal a number of common putative responsive elements are present at approximately
similar relative locations in the gene [146, 171]. In addition to the previously discussed
C/EBPB element, these elements may potentially be involved in the induction of PGHS-2
transcription. Included among these are examples of sequences, such as an NFKB site,
and two additional C/EBP/NF-IL6 sites, which are found in the regulatory regions of
other genes whose products are involved in the inflammatory response [173, 174]. A
putative Ap-2 binding site, which may further mediate CAMP induction of transcription
of this gene [159, 170], as well as consensus sequences for Ets responsive elements,
were thus located [175]. It may be hypothesized that the action of activated trans-acting
factors, binding to these or other, as yet undiscovered responsive sites in the 5’-flanking
region of PGHS-2 may serve to activate or derepress transcription.

Anti-inflammatory glucocorticoids have been shown to reduce prostaglandin
synthesis by inhibiting stimulated synthesis of PGH synthase protein; therefore, it was

encouraging to identify a negative glucocorticoid responsive element in the 5’ flanking

86

region of PGHS—l, which might be involved in the anti-inflammatory action of
glucocorticoids [157, 158]. Subsequent reports have demonstrated that the PGHS-2 gene
is a major site of action for glucocorticoid regulation of prostaglandin production after
inflammatory stimulation [41-43]. As mentioned before, glucocorticoids inhibit PGHS-2
transcription, reduce PGHS-2 mRNA stability, and may also affect PGHS-2 translation
[44, 45]. However, the inhibitory effect of glucocorticoid on stimulated expression could
not be simulated using reporter plasmids containing the 5’-flanking region of the PGHS-2
gene [170], signifying a complexity in the mechanism of glucocorticoid inhibition of
PGHS-2. Dexamethasone has shown at least to depress moderately the elevation of
PGHS-1 protein observed upon TPA treatment of U937 monocytic cells [49], as well as
elevations in PGHS-1 transcription in 3T3 fibroblasts upon semm stimulation [44], and
PGHS-1 mRNA in TPA-treated rat tracheal epithelial cells [26]. So, while the anti-
inflammatory effect of glucocorticoids may not be mediated through the putative nGRE
in the 5’-flanking region of the PGHS-1, it potentially could be involved in the down-
modulation of PGHS-1 levels in these cells.

One unexpected outcome in the analysis of the 5’-flanking region of PGHS-1 gene
was the identification of a putative dioxin responsive element (DRE). DREs mediate the
transcriptional modulation resulting from exposure to a number of xenobiotics of the
polycyclic aromatic hydrocarbon (PAH) family, of which the halogenated aromatic
hydrocarbons (HAHs), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), seem to
be the most effective. Experiments examining PGHS regulation by HAHs will be

described in Chapter 3.

87

Through the mapping and analysis of the mouse PGHS-1 gene and its 5’ flanking
regions, it was hoped that more information about the regulation of prostaglandin
synthase expression could be obtained. Although a number of putative responsive
elements were located in the 5’ flanking region of this gene, there is no indication at this
time that any are functional in regulating prostaglandin synthase in response to external
factors, although potentially they may be involved in tissue-specific regulation of PGHS-l
expression, or in the terminal differentiation of cells. Rather, the subsequent cloning and
expression studies on a new isozyme, PGHS-2, implicate its role in prostaglandin
synthesis in response to external factors, while PGHS-1 has been relegated to a role as
a "housekeeping" enzyme, responsible for immediate prostaglandin production upon
stimulated arachidonate release in response to internal factors. So, while PGHS-2 is the
key prostaglandin-producing isozyme involved in inflammation, ovulation, or
mitogenesis, it is likely that PGHS-l is critical in producing prostaglandins involved in

maintaining vascular integrity and function, kidney function, and stomach protection.

CHAPTER 3

INDUCTION OF PGHS-2 AND PGHS-1 BY 2,3,7,8-TETRACHLORODIBENZO-P-

DIOXIN (T CDD) IN MADIN DARBY CANINE KIDNEY (MDCK) CELLS

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical member of a
large group of xenobiotics, the halogenated aromatic hydrocarbons (HAHs). This group
includes, not only TCDD, but also the polychlorobiphenyls (PCBs), the
polychlorodibenzofurans, and can include metabolized forms of aromatic hydrocarbons
(AH) such as benzo-a-pyrene [94-96]. These compounds are grouped together because
they share a common mechanism of action. HAHs, including TCDD, diffuse into cells
and bind a specific cytosolic receptor, termed the Aromatic Hydrocarbon Receptor
(AhR). The possibility that HAHs might regulate PGHS expression was first suggested
by the identification of a putative dioxin responsive element (DRE) 396 bp upstream from
the transcriptional start site of the PGHS-l gene. Subsequently, when a second PGHS
gene, PGHS-2, was cloned, its promoter also contained DRE sequences, one at -l65 bp
and another at -1069 bp upstream from the transcriptional start site. The determination

of these DRE sequences in the promoter regions of PGHS-1 and PGHS-2 raised the

88

89

possibility that dioxin might regulate expression of these genes. Previous reports had
indicated that exposure of Madin Darby canine kidney (MDCK) cells to xenobiotics
resulted in increased prostaglandin synthesis by a mechanism that appeared to require
protein synthesis [118,119]. We therefore decided to determine directly whether dioxin

could effect PGHS expression in these cells.

90
Methods

Cell Culture: MDCK cells grown to approximately 60% confluence on 100 mm
tissue culture plates in DME medium supplemented with 8% calf serum and 2% fetal calf
serum (Hyclone), were serum-starved by replacing the medium with 4.5 ml fresh DME
medium containing 0.2% fetal calf serum for 48 hours. The cells were then exposed to
1 nM TCDD in DMSO, or to the DMSO (0.1%) alone. At appropriate time points,
medium was removed, the cells were washed twice with ice-cold PBS and harvested by
scraping into PBS, and collected by centrifugation. Typically, approximately 2 x 107
cells (10 10 cm plates) were used per time point for the isolation of nuclei, mRNA, or
microsomal membranes.

Western Analysis: To prepare microsomes, TCDD-treated or control MDCK
cells (2 x 107 cells) were resuspended in 0.1 M Tris-Cl, pH 7.5 and sonicated three times
for 10 seconds on a setting of 6 (Heat Systems-Ultrasonics, Model 225-R), then the
homogenate was centrifuged for 10 minutes at 4°C at 10,000 x g. The supematants were
transferred to polyallomer tubes, and centrifuged a second time at 100,000 x g for 1 hour
at 4°C. The resultant microsomal pellet was resuspended in 0.2 ml of 0.1 M Tris-Cl,
pH 7.5 using a glass homogenizer.

Protein content of the microsomal samples was quantitated using the Bradford
method [147]. MDCK microsomal proteins (100 pg) were then separated by
electrophoresis on a 10% SDS-polyacrylamide gel and transferred electrophoretically to
a 0.45 pm BA-S nitrocellulose (Schleicher & Schuell) [67]. Microsomal membranes
derived from cos-I cells transfected with mouse PGHS-2 (5 pg) were electrophoresed in

separate lanes as controls [176]. Blots were blocked in a Tris-buffered saline (TBS)

91

solution containing 3% nonfat dried milk overnight. For PGHS-2, an affinity-purified,
antisera produced against the peptide DD-2 (C-Y-S-H-S-R-L-D-D-I-N—P-T-V-L-I-K)
derived from the C-terminal insert of mouse PGHS-2 [44] was used as a primary
antibody. For a control the PGHS-2 selective antisera was preincubated for 30 minutes
with 20 nmoles of the DD-2 peptide in a total volume of 100 pl. Western blots were
hybridized with the primary antisera was for 1 hour at room temperature in TBS
containing 1% nonfat dried milk and 0.1% Tween-20. The filters were then washed
three times for 10 minutes in TBS containing 1% nonfat dried milk and 0.1% Tween-20.
The blots were next hybridized to a horseradish peroxidase-conjugated goat anti-rabbit
secondary antibody (BioRad) (1:2000), for 1 hour at room temperature and washed
again. Protein bands were visualized by photoluminescence and autoradiography using

the ECL photoluminescence kit (Amersham) according to manufacturer’s instructions.

Nuclei and Cytoplasmic RNA Isolation: For preparations of nuclei for run-on
assays, freshly isolated MDCK cells (approximately 2 x 107 cells) were first resuspended
in 2 ml nuclear isolation buffer (10 mM Tris-Cl, pH 8.6, 140 mM NaCl, 1.5 mM
MgC12, 0.5% Nonidet P-40, 1 mM DTT, and 40 U/ml RNasin (Promega)). Nuclei were
pelleted by centrifugation at 4°C for 5 minutes at 1000 x g. RNA was isolated from the
supematent, while the nuclei pellet was washed by resuspending in 2 ml nuclear isolation
buffer without DTT and RNasin and collected by centrifuging for 5 minutes at 1000 x
g at 4°C. This washed nuclei pellet was resuspended in 0.2 ml nuclei storage buffer (50
mM Tris-Cl, pH 8.3, 5 mM MgC12, 0.1 mM EDTA, 40% (v/v) glycerol), and frozen

in liquid nitrogen.

92

To isolate RNA, the supernatant was added to 2 ml of 2 x Proteinase K buffer
(0.2 M Tris-Cl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 0.2 (w/v) SDS), and the mixture
was incubated at 37°C for 45 minutes. Following phenolzchloroformzisoamyl alcohol
(25:24: 1) extraction, the cytoplasmic RNA was precipitated by adding 8 ml ethanol.

Northern Analysis: Cytoplasmic RNA was poly A+ selected using oligo-dT
cellulose (Boehringer—Mannheim) by a batch method [141]. 10 pg of poly A+ RNA of
each sample was then separated on a 1% agarose, 2.2 M formaldehyde, 1 x 3-[N-
morpholino]propanesulfonic acid (MOPS), pH 7.0 gel [141]. RNA was transferred by
capillary action to 0.2 pm BA-S nitrocellulose (Schleicher & Schuell) using 20 x SSC (3
M NaCl, 0.3 M Na citrate, pH 7.0) . Northern blots were prehybridized in 50%
formamide, 5 x SSPE (0.75 M NaCl, 0.05 M NaHzPO4, SmM EDTA, pH 7.4), 5 x
Denhardt’s solution (0.02% Ficoll, type 40, 0.02% polyvinylpyrrolidone, 0.02% bovine
serum albumin), 0.1% SDS, and 2.5 mg/ml denatured herring sperm DNA for 2 hours
at 42°C, followed by hybridization with a PGHS-2 probe. Following autoradiography,
these blots were hybridized to a glyceraldehyde—3-phosphate dehydrogenase (GAPDH)
probe for normalization. Hybridization conditions were at 42°C for 18 hours in 50%
formamide, 5 x SSPE, 1 x Denhardt’s solution, 0.1% SDS, 1 mg/ml denatured herring
sperm DNA, containing 2 x 10" dpm/ml 32P-labelled probe. cDNA probes were
radiolabelled using a-32P-dCTP (New England Nuclear, 800 Ci/mmol) by the random
primer method [142]. The PGHS-2 probe was derived from a 1.16 kb EcoRI cDNA
restriction fragment containing the 5’ end of the coding region of mouse PGHS-2 [44].
The GAPDH probe is a 1.2 kb Pstl restriction fragment from the mouse GAPDH cDNA

[177]. A second exposure to XAR—5 xray film was used to visualize the GAPDH-

93

hybridizations. This probe to the constitutively expressed GAPDH gene was used to
normalize PGHS hybridizations to correct for variations in sample size and transfer
efficiency.

Nuclear Run-on Assays: Using a minifold slot blotter (Schleicher & Schuell),
10 pg of five cDNA probes were blotted on to strips of 0.2 pm supported nitrocellulose
(BA-S, Schleicher & Schuell): (l) the entire coding region of mouse PGHS-2 in pUCl9
[176]; (2) the entire coding region of mouse PGHS-1 in pUCl9 [176]; (3) mouse 62
macroglobulin in pKC7, (a constitutively transcribed control); (4) mouse c-fos in pUC19;
(5) pUCl9, (non-specific background control) [44]. Before slot blotting, the plasmids
were first linearized with an appropriate restriction endonuclease, then were extracted
with phenol:chloroform:isoamyl alcohol (25:24:1), and ethanol precipitated. The probes
were next resuspended in TE (10 mM Tris—Cl, pH 8.0, 1 mM EDTA) and denatured by
treating with 0.1 volumes 3 M NaOH for 1 hour, at 65°C. Following neutralization with
one volume of 2 M ammonium acetate, the samples were applied to the nitrocellulose.

Transcription in the thawed isolated MDCK nuclei was re-initiated by addition of
400 pl of2 x Reaction Mix (10 mM Tris-Cl, pH 8.0, 10 mM MgC12, 300 mM KCl, 1
mM each of ATP, CTP and GTP, 200 uCi of a-32P-UTP (800 Ci/mmol), 5 mM DTT,
and 160 units of RNasin (Promega)). Transcription was allowed to procede for 30
minutes at 30°C. Next, 40 pl of RNase-free DNase (BRL) was added and the reactions
were incubated for an additional 10 minutes at 30°C. Proteinase K (Boehringer-
Mannheim) was then added (400 pg/ml) in 840 pl of 2 x SETY buffer (100 mM Tris-Cl,
pH 7.4, 50 mM EDTA, 2% SDS, 100 ug/ml yeast tRNA) and the incubation was

continued at for 45 minutes at 37°C. Following extraction with

94

phenolzchloroform:isoamyl alcohol (25 :24:1), the samples were precipitated with 1
volume ethanol and 0.75 volumes of 7.5 M ammonium acetate. The resultant RNA was
resuspended in 100 pl TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and denatured by
adding 2 pl of 3 M NaOH and incubating on ice for 10 minutes. 2.5 M HEPES free
acid ( 12 pl) was then added to neutralize the samples. The labeled RNA was precipated
using 10 pl of 3.0 M sodium acetate, and 200 pl of ethanol, then resuspended in 50 ul
TE prior its addition to the hybridization solution.

The slot-blotted nitrocellulose strips containing the cDNA probes were
prehybridized for 1 hour at 42°C in 50% formamide, 5 x SSPE, 5 x Denhardt’s solution,
0.1% SDS, and 2.5 mg/ml denatured herring sperm DNA. The strips were next
hybridized for 48 hours at 42°C in 50% formamide, 5 x SSPE, 1 x Denhardt’s solution,
0.1 % SDS, 1 mg/ ml denatured herring sperm DNA, with the radiolabelled RNA. After
hybridization, the strips were washed twice at room temperature for 10 minutes in 2 x
SSC, 0.1 % SDS; once in the same solution at 50°C, then rinsed in 2 x SSC at room
temperature. This was followed by a 15 minute treatment in 2 x SSC containing 10
pg/ml RNase, and two rinses in 2 x SSC, 0.1 % SDS at room temperature. The
resultant hybridized strips were exposed to XAR-S xray film for up to 10 days.

Construction of Luciferase Vectors: The TCDD-inducible control vector,
pGUDLUC1.1, which contains the DRE-responsive domain from the 5’-flanking region
of the cytochrome P4501A1 gene, including four dioxin-responsive elements (DREs),
joined to the mouse mammary tumor virus LTR promoter, was a generous gift of Dr.
Michael Denison. pGL2-Basic (Promega), contains the luciferase cDNA, but no

promoter or enhancers (Figure 21).

95

Figure 21. Luciferase reporter control plasmids, pGL2-Basic and pGUDLUCl.l.
The plasmid pGL2-Basic, used as a negative control, contains no promoter or enhancers
to drive luciferase expression, and is used as the parent plasmid for all other luciferase
expression plasmids. The plasmid pGUDLUC1.1, used as a TCDD-inducible control,
contains the TCDD-inducible domain of the CypIAI gene, which includes four DREs
upstream from an MMTV-LTR, which drives luciferase expression.

96

 

 

 

 

  
  
 

wrvrrw one: (4)
1829 bp

Hindlll 1876

 

97
PGHS-2,1,“, and PGHS-2long were constructed using pGL2-Basic. PGHS-2m,

contains sequences -975 to +88, relative to the transcriptional start site (Figures 11 and
12). PGHS-2long (which contains the 5’-flanking sequences corresponding to base pairs
—3l97 to +88 was constructed from PGHS-2m by the ligation with a 2.6 kb PstI
restriction fragment, containing sequences further upstream of PGHS-2,1,0“ (Figure 22).
The Pst I restriction fragment was obtained by digestion of the same AFIXII NIH3T3
mouse PGHS-2 genomic clone. This fragment was subcloned into M13mpl9, and the
sequence of both strands of this 2.6 kb was determined by the Sanger dideoxy chain
termination method (Figure 23), using oligonucleotide primers (Table 5) [144]. To
facilitate the ligation of the 2.6 kb PstI restriction fragment to the PGHS-2,hon sequence,
aKpnI site at the 5’ end of the PGHS-2M, was changed to a PM! restriction site. The
2.6 kb Pst! restriction fragment was then subcloned into the PSI! sites of the PSI] site-
inserted PGHS-2m, plasmid, and its orientation was confirmed by restriction enzyme
mapping.

Transient expression of Luciferase Vectors in MDCK cells: MDCK cells were
transiently transfected with the luciferase expresssion plasmids using the CaClz-
precipitation method [141]. MDCK cells were grown to approximately 50% confluency
in 10 ml DME medium supplemented with 8% calf serum, 2% fetal calf serum. CaClz-
precipitated DNA ( 1.1 ml) was added to each plate and incubated for 5 hours at 37°C
in an incubator containing 5% C02. The medium was next removed, the cells were
shocked
by treatment with 1.5 m1 of a 15% glycerol/HEPES-buffered saline (140 mM NaCl, 5

mM KCl, 0.75 mM NazHPO4 2HZO, 6 mM dextrose, 25 mM PIPES (N—2-

98

Figure 22. Construction of the luciferase reporter plasmid PGHS-21m, containing
the 5’-flanking region of the mouse PGHS-2 gene. PGHS-2m contains PGHS-2
genomic sequence corresponding to nucleotides -3197 to +88 in the parent plasmid
pGL2-Basic. It was assembled using the PGHS-2,hon plasmid (which contains sequence
corresponding to nucleotides -975 to +88 of the PGHS-2 gene), whose construction was
described in Figure 12, which had been modified by the addition of a PstI restriction site
in the KpnI site on the 5’ end of the insert, using an adaptor. A 2.6 kb Pst! restriction
fragment, which overlapped with the PGHS-2M, insert, and contained sequences further
upstream from the PGHS-2m, insert sequence, was initially identified by Southern
analysis, utilizing the 5’ PCR primer oligonucleotide, as a probe. This 2.6 kb PstI
restriction fragment, which corresponded to nucleotides -3197 to -574 relative to the
transcriptional start site of the PGHS-2 gene was isolated, and ligated into a Per-digested
modified PGHS-2mm. The correct orientation of the PSI! fragment insertion as confirmed
using EcoRV restriction digestion.

99

 

._. 3: u.

 

 

 

    

       
  

   

 

 

     
  

 
    

 

4.9852 8.8:
__ 59:58 .3315 4.8
:32 95
«3. 38m ._ a $2. ._. ream
9.23:9. :3
.328:
E
3.. 1.5 __ i 8
83:83
68:3: «.90.. S = .8 8+ 0. m5.
.528: 34.0..
._.um 22.262 3:8»qu 8: 5.
:5 .29. 21.8 .2
an .23 .330
0334 033.0%
229:; .2522... 689898895
-owzo 4 54 44 54444 049000004009000
+
5.4 :3 acm—
ee 28 4 e4 ._. b+
_ I. ...
~ 94$. N ”fig 045/ ‘g
3% § 4.2 m-
29?. N010;
[— — q _ .
N-m=0n_ 4 4.34 4246.90 4 =4 .QGO—U Now—.HMVA— axmrfl K
—/%\20: 4. a — m-
:37. N-m:0n_

E 8&2 2255.4 320..

units
.15.

a
my
their
lll lie
Iii <th

l
f

l Psi

0 he

Stet

. rt

1

100

Figure 23. Sequence of the PGHS-2long insert, showing locations of putative
responsive elements. A 2.6 kb Pst! restriction fragment, corresponding to nucleotides -
3197 to -574 of the PGHS-2 gene was ligated into the Ru! sites of M13mp19, and
sequenced in both orientations using the dideoxy termination method [144]. This
sequence, was compiled with that of the PGHS-2m, insert (Figure 13), with which it
overlaps 378 bp, to obtain the sequence of the PGHS-2m insert. The location of the 5’
end of PGHS-2m, is indicated with an asterisk. The transcription start site is bolded.
Putative responsive elements are underlined.

umn

-3195
-3150
—3080
-3010
~2940
~2870
-2800
—2730
-2660
-2590
-2520
-2450
-2380
-2310
-2240
-2170
-2100
-2030
-1960
~1890
-1820
-1750
-1680
-1610
-1540
-1470
-1400
-l330
-1260
-1190
-1120
-1050
-980
-910
~840
-770
-700
-630
-560
~49O
-420
-350
-280
-210
-140
-70

71

AGTGCCCATA
TACTTCTACA

'- 8“
mm

TGTCCTTTAT
ATATCTGAGT
TACCCGTGTC
GTCATTTATT
CATTATAAAA
ATTCAAGCAG
TTCCTTTTTT
ATGTACCCAA
ATCTTATTAA
TGGGACAGTT
CTTCTCAAAA
TCTTGTCCTC
ATCTGAGGTT
ATGTTTAATG
TTGAATTTGT
CTGGCTGTCC
TCAATGCTGG
TCAGAATATA
TTGAAGATTT
TTCGTATCAG
CALGAACTAT
CCGTGTAAGA

AGGATCACTG
TTTCTTTCCT
TATCTTTCTT
TAGCCAGATG
TTGAGGCCAG
GAGGAAAAGA
TCCAGTGTAT
TTAGAACGAA
CAGAAGAGGG
AGAATCTCAA
GTGTACAGAG
GGAGCCTCAT
AATATTCCCC

TTTTA
TACCTATTCT
CTAAAGAATG
TTTACTTTTA
GTGGTGATAC
CTAGCTCCAT
AAAAGAAATA
GCATGAACTT
AATCTACCTA
CAGTAAAACT
AACTCTTTCA
AACCACCAAA
TACCATTGTG
TGTAACCAGT

n;
ACAAAATACT

-.
GCAGCCAATT

CAGGTCTGAA
ACAGATTAGA
TCTTTAAATG
AATGTTTTCT
TGAAACGTGC
GATTAAGGGC
CTTCTCTACA
CACTAGAAAA
GAGCTCTAAC

TTTCTTACTG
TCAACCAACT
TCAAGTAGTC
TTTTTTTTTT
TCTGTGGACC
CAACATCACC
TAATGGGCAT
TGTTCGTACA
AAACTTAGTT
TCTCCAAAAG

Ihd
TTAAAGGGAT

GGCTGGGAGT

a
GTCACTGTGT

recaerraaar
arrccarcac
rccrrcrcar
crrrrcrrrr
ggcaenarar
aerrcaarrr
arectcaacn
TTTTATCAAA
AAAAATAACT
TAAAAGGTAC
TAAAAGCTAT
TGGAGCTCAG
GTTCCATGAA
TAAAAATAGA
ccccacrcoc
rcrrcaccrr
eccccneacr
rcccccxecc

lth
GTTCTTGCGC

TATTCTTACT
GGTCGATTGG
GAGCAGGGGT

AGTGTAATTT cacncrcrdi'

TTTAGTTTCC
CAAACACAGT
CACTGTTTCT
ATTACCTTTT
ATTTGAAAAC
GTAACAGCAG

GCCATACCTG
CTCATGTAGC
TTCTGGGAGA
Inn;
ATTCCCCCAT
CTTGCTGTGT

101

TTGTTCTGCC
TGAAGAACTG
CTCTATAAAA
ACTTTCTCTT
AAGTCTTCAA
CTATGGAGTG
ACAATTAATA
TACATTTAAT
ACAATCTACC
CAACAGAATG
TATACTAACA
ATTCCACACA
CTTTGCCATA
ATTCCAATTG
TTCCCCTTAT
AGTTCTCCTG
CAACCATACA
TGAAGGTGAT
TTCTTTTAGT
AGGGAGGCCT
ATCCACCACC
ACAATTAATA
CAGTCAAGAA
TGACACCATG
AAAACAGTCT
GACTGATTAA
ATCAAATTAG
CTTCCGCAGA
GTGGATATTC
TACCCAGCCT

1
GTCATTTTTT

TCCTTTTCTT
AGGAAGATAT
GRATGAAGTG
GATCCGTTGC
AAATTCTTTC
GGGGAAAATA

’
carcrcrc63°rcceaaaccc

AGACTTCAAC

Inna
CTAATTCCAC

AGAAATTAAA TTAACCGGTA

GAGAGGTGAG
TTAGGCCCCC
CAGCGAACCA

lhfl
GGGATTCCCT

ACTGATGCGC
CAGGGCGCCT

no
TCCCGGCTTC

n.
AAGGGCAGCT

AACTCACTGA

AGCAGAGAGG

ACAGAGTCAC CACTAEEECA CGTGGAGTCC
AGTTGTCAAA CTGCGAGCTA AGAGCTTCAG

CATCCTGCCA

GCTCCACC

arrrrccrcc
CCAACACTAT
AATTTTTTTT
CATAACATAC
TAGTGATACA
ccrraaacca
GAGTGCCAGG
CAGTACAGAT
GCTGTGTGCG
TAGTTAGGAC
GACTGGGAGG
GGAGGGATGG
crrccrcrcr
GGGAAAAGTT
GCTTACAGAC
GAGTEKETCA

CTCATGTGTA
ACTGCTATCA
ACAAATAAAA
TGAATGGGAT
GCCCAGCATG
AGTTCCAGGA
AAAAGCCAAA
ATCAACACTG
TAGCATCTTA
TTCTCAAAGG
AAATATATTT
GTTCATCACT
CAGAACAGTT
TGCCTGTACA
GTCCTTATTT
TCATTCCGTA
GATAATTTTA
TATTCTCTAT
TTTTGTGAGA
CAGAGATCTG
ACCCCCTTTG
TTAGATGAGG
TGTGGAATTT

-- m
mam-£929...

crccrrrcac
ACATGACTAG
CTTGCACATG
crcaccrarr
rccccrrrca
Gerrcaaacc
Trarccarrc
Trrrcccrac
CAAAAACATC
TAAGGTTAGG
TTTCTTGTAA
TCCCGTGAAA
arccrereea
Gccrccrcrc
GTGGACCCTG
TGCTCTGAGC
crraearccc
AAACCGGAGA
AGAGdEEGGr
camiéars
GGTGGGEEiT
TTAAAAGCAA
ccncrcrccr

TGAAGGCTTC
AATGCACTAC
CTATTCTTGC
ATTGTTTGAA
TCAGAGGCAG
TGGCCATGAC
CCCAAAACAA
CATGTTTAAT
GGAGAAAAAA
TGTTAGTACA
TCTACAACAG
ACACTTGCTA
TGAACCATCT
GTGTGCTCTG

arrsr GTAC
chrcrrrar
rrrccrcrgg
II-XL‘
TTAAGGAATA
AGGCAGGCAA
TACACAGAGA
AAACAAAAAT
rarcrxaorc
GTTGCTGAAA
rrrccrrxrr
ACACTTAAAT
TATAATAAAC
TGATTTGGTT
GCATCCCAGA

au
TCAAAACTGC

GGACTTCCTT
GTATATGGAA
CTTGCATTTC
GCCCTGTTTT
CAGGATTTTT
CTTAGACTCC
CCATAATCTC
ATGGAGTTGG
TATACAGTTC
ATTTAAAATA
TTGAAATTTC
AACAAAGGCC
AAGCAAACAG
TGCTAGAGTT
TTTTTGTTTT

CTCTCCTTAC
TTCTATGATT
TCCCCTCACT
CTGTGTATAG
TTTTTCTCCC
TTTTAAAACA
TCAAAGTCAG
CTAGCTGAAG
AAACAGAAAA
CCATAAGACT

up:
CTGGGTGACA

TTAAAAAAAA
TCCTAAGTGC
GTTTTGTTCT
CTTAACTGTC

Mrl
ATTTTCCTGA

an
ACGCCAAGAA

TCCATCCTCA
ACCTCTCTAG
GAGAATAAGG
ACATGGACAG
AGAGTTGCTG
CACTTAGCAT
ACTGCAGAAC
ACAGAGGACA

CGTACAGTTT
CACCCCCTCC
GCAAATAATT
CTAGTTATGG
AAAAAGTTTT
ATCAAAATGA
TCCGATGAAG
CCGGAGGGTA
CTCATTTCAT

can
acccacc§g§____

GGGAGGGGAA
CCCCAAAGAG
GCAGCTCTCT
GGTAAAAGCC
nun;
GGGGAAGCCT
GGTTCTCCCC
CACGAAGGAA

TCAGACTGCG
GCTGTGACAC
CGCCAGACTA
TGGCACCACC
TGCCGCTGCG
AAGCGGAAAG
ATTAGCAGCC
CTCAGCACTG

102

Table 5. Oligonucleotides used to sequence the 5’ flanking region of PGHS-2.

103

 

' " ‘ s “ i v Sequel-‘93 ’1(i;ii-giflf‘f

 
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DD-91 -257 CCTGTGGTI‘CGCTGAGT -273
DD- 105 -850 GCC'ITATI‘CTCCCTAAC -866
DD-106 -2 890 TCAGAGGCAGAGGCAGG -2 874
DD- 108 -1 158 GTAACACAGCAAGACCC -1 174
DD- 109 -255 1 GTTCTCAAAGGTGTI‘AG -25 35
DD-l 10 - 1607 CGAACTI‘CAGCTAGGAA -1623
DD-l 1 1 -2260 GTGTGCT CT GGCATCCC -2244
DD-l 13 ~1871 GCACGTTI‘CAGGACAGC - l 887
DD-l 14 - 1974 'I'I'I'I'TCCCCTCAC'I'ITG - 195 8
DD-l 15 -2221 AATTGGCTGCTI‘TTGAG ~2237
DD-l 16 - 1614 GAAG'ITCGTATCAGGAG - 1598
DD-l 17 -2568 CTGCCCTC'I'I'CTGCTGC -25 84
DD-l 18 - 1229 TGATA'ITCTCCCCTITC -1212
DD-l 19 -29 12 TATCACCACCATCTGGC -2928
DD-120 -866 GTIAGGGAGAATAAGGC -850

 

 

 

 

 

104

hydroxyethylpiperazine-N’-2-ethanesulfonic acid) solution for 1 minute, and the cells
were next washed twice with PBS. DME medium (4.5 ml) containing 0.2% fetal calf
serum was added and the cells were incubated for 24 hours. Next, 1 nM TCDD (or
0.1% DMSO vehicle control) was added and the incubation was continued for 8 hours.
The medium was removed and the plates were washed twice with 5 m1 PBS. The cells
were scraped into PBS (4 ml per plate) and were collected by centrifugation at 4°C for
5 minutes at 200 x g. The cell pellet was resuspended in 0.3 ml 10 mM Tris-Cl, pH
7.8, and the cells were lysed by sonication. The cell lysate was then transferred to
microcentrifuge tubes, the cell debris was removed by centrifugation at 12,000 x g for
5 minutes, and the supernatant was assayed for luciferase activity with a Turner TD-20E
luminometer, using a Luciferase Assay Kit from Promega. Protein was quantitated by
the method of Bradford [147]. Statistical significance was assessed using paired t tests
at the 95% confidence interval.

Quantitation of Run-on Assays, Northern and Western Blots: All quantitations
of autoradiographic bands were performed by densitometry using a Bioimage Visage 110

image analyzer.

105
Results

To determine whether TCDD regulates expression of the PGHS gene products,
we first conducted Western blot using antiserum specific for PGHS-2. A clear elevation
of two bands of approximately 69 and 72 kDa were observed within 2 hours after TCDD
stimulation in Western blots of MDCK protein samples after hybridization with a PGHS-
2 specific antibody. These bands correspond to the sizes of the PGHS-2 as expressed in
cos-1 cells (Figure 24) [57]. PGHS-2 levels peak 4 hours following TCDD treatment at
a level 25-fold higher than in unstimulated cells. The 69 and 72 kD bands were not
observed in these Western blots when the primary antibody was preincubated with an
excess of DD-2 peptide (Figure 24). In similar western blots, using antisera directed
against PGHS-1, we failed to detect PGHS-l in MDCK proteins, suggesting that PGHS-l
expression is below the level of detection in MDCK cells.

To determine the mechanism for PGHS-2 protein induction, we examined TCDD-
induced changes in PGHS-2 mRNA. Northern blot analyses were performed using poly
A+ RNA isolated from MDCK cells treated with lnM TCDD or vehicle control, with
32P-labeled probes corresponding to the coding region of mouse PGHS-2, and to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PGHS-2 mRNA levels increased
by 1 hour and peaked 2 hours following TCDD addition. PGHS-2 mRNA levels
remained elevated only 4 hours (Figure 25). Analogous transient increases in PGHS-2
mRNA levels are observed in serum-stimulated fibroblasts [44]. The time course of the
increases in PGHS-2 mRNA, followed by increases in PGHS-2 protein, suggested that
the regulation of PGHS-2 mRNA levels, possibly by transcriptional mechanisms, may

control PGHS-2 expression in response to TCDD. To examine whether regulation

106

Figure 24. Western blot analysis of TCDD induction of PGHS-2 in MDCK cells.
MDCK cells which had been serum-starved for 48 hours were stimulated with either 1
nM TCDD in 0.1% DMSO carrier (+TCDD), or in 0.1% DMSO, alone (-TCDD). At
the indicated times, cells were collected and microsomes were prepared. Microsomal
MDCK proteins (100 pg), as well as a murine PGHS-2 control, obtained from
transfected cos-I cells [176] were separated on SDS-PAGE and transferred to
nitrocellulose filters as described in Methods. Filters were incubated with (A) the PGHS-
2 peptide-specific antisera. The blot was subsequently incubated with a horseradish
peroxidase-conjugated goat anti-rabbit antibody, and protein bands were visualized by
photoluminescence using the ECL system (Amersham) on XAR-5 X-ray film. Shown
is a representative blot; induction of PGHS-2 protein was observed in four independent
experiments. (B) Relative intensities of the protein bands in A were quantitated by
densitometry. The size of the canine PGHS-2 is apparently similar to that of the mouse
PGHS-2 protein, approximately 70 kDa. This band can be competed off, by the pre-
incubation of the antisera with the peptide antigen.

107

«.mzoa
330:.

.4:

o

.4 m N
000.. oz

_.

I998...

106

Figure 24. Western blot analysis of TCDD induction of PGHS-2 in MDCK cells.
MDCK cells which had been serum-starved for 48 hours were stimulated with either 1
nM TCDD in 0.1% DMSO carrier (+TCDD), or in 0.1% DMSO, alone (-TCDD). At
the indicated times, cells were collected and microsomes were prepared. Microsomal
MDCK proteins (100 pg), as well as a murine PGHS-2 control, obtained from
transfected cos-I cells [176] were separated on SDS-PAGE and transferred to
nitrocellulose filters as described in Methods. Filters were incubated with (A) the PGHS-
2 peptide-specific antisera. The blot was subsequently incubated with a horseradish
peroxidase-conjugated goat anti-rabbit antibody, and protein bands were visualized by
photoluminescence using the ECL system (Amersham) on XAR-5 X-ray film. Shown
is a representative blot; induction of PGHS-2 protein was observed in four independent
experiments. 03) Relative intensities of the protein bands in A were quantitated by
densitometry. The size of the canine PGHS-2 is apparently similar to that of the mouse
PGHS-2 protein, approximately 70 kDa. This band can be competed off, by the pre-
incubation of the antisera with the peptide antigen.

am
i in:
fit
an
an
red by
SW
em
uh
me
en

+T DD

N TCDD

mouse
PGHS-2

hr

m7

PGHS-2-

 

w m . o. . ma . .m.
C-0a05n— NtmT-MUQ 50 940—0030:-

it...

0

U40n4

Fold Induction of PGHS-2 Protein

108

B.

TCDD INDUCES PGHS-2 PROTEIN LEVELS

 

60

 

 

 

time (hr.)

109

Figure 25. Northern blot analysis of TCDD induction of PGHS-2 in MDCK cells.
MDCK cells which had been serum-starved for 48 hours were stimulated with either 1
nM TCDD in 0.1% DMSO carrier (+TCDD), or in 0.1% DMSO alone (-TCDD). At
the indicated times, cells were collected, and cytoplasmic, poly A+ RNA was isolated
as described in Methods. 10 pg poly A+ RNA was separated on a 1%
agarose/ formaldehyde gel and transferred to nitrocellulose filters as described in
Methods. (A) Filters were hybridized with a 32P-radiolabelled mouse PGHS-2 1.16 kb
EcoRI cDNA restriction fragment. The blots were then exposed for 2 weeks to XAR-5
X-ray film. The filters were subsequently re-hybridized with a 32P-radiolabelled mouse
GAPDH 1.2 kb Pst! restriction fragment, and re-exposed to XAR-S X—ray film for 12
hours to visualize the GAPDH bands. Shown is a representative Northern blot; induction
of PGHS-2 mRNA was observed in seven independent experiments. (B) Relative PGHS-
2 mRNA expression in MDCK cells iTCDD were quantitated by densitometry on a
Bioimage Visage 110 image analyzer. Values are expressed as the integrated optical
density of the PGHS-2 bands, normalized to the integrated optical density of the GAPDH
bands for each time point.

 

 

 

110

tilt. t1 .
w _ .
mm 3 Hi .I . _rfi

 

w

 

_
’10540

111

 

+TCDD

TCDD INDUCES PGHS-2 mRNA LEVELS

 

No TCDD

 

I
100

. d . . . . . . 0

O 0 0 0
8 6 4 2 o

B fod<eaézo$ 296.. <2:

2:23 c. 8:32.. 2o".

MP1

 

trans
put.
pub.
has.
hans.
dl-hh
use
won

PGHS

 

consul
hnreas
30tnini
TCDD

”ofihen

dSdeS (

112

of PGHS-2 transcription occurred, we measured TCDD-induced changes in PGHS—2 gene
transcription by nuclear run-on assays. In these assays, radiolabelled transcripts
produced in isolated nuclei following TCDD-stimulation were hybridized to cDNA
probes immobilized on nitrocellulose. Hybridization intensity indicates the relative
transcriptional activity of the gene when the nuclei were isolated. We measured the
transcription of four genes: PGHS-2 [44], PGHS-1 [39], the constitutively transcribed
BZ-Macroglobulin, and c-fos [44]. While our cDNA probes were all mouse derived,
these cDNAs are sufficiently conserved in other species such that we surmised they
would detect transcription from the respective dog genes. The parent plasmid of the
PGHS vectors was used as a background control, B2-Macroglobulin was used as a
constitutively transcribed internal control, while c-fos was used as a positive control.
Increased transcription of the PGHS-2 gene was observed in TCDD-treated (1 nM) cells
30 minutes following treatment. The rate of PGHS-2 transcription peaked 2 hours after
TCDD stimulation, coincident with the peak in PGHS-2 message levels observed by
northern blot analysis. Increases in PGHS-1 transcription were not evident in these
assays (Figure 26).

To begin to identify TCDD responsive regulatory elements in the 5’ flanking
regions of the PGHS-2 gene, luciferase expression plasmids were constructed, using the
promoter and 5’ flanking region of the PGHS-2 gene inserted upstream of the firefly
luciferase coding region. Two constructs were made: one containing sequences
corresponding to —975 bp to +88 bp relative to the PGHS-2 transcriptional start site
(called PGHS-2mm), which contains only one of the putative DREs (Figures 11 and 21);

the other containing sequences corresponding to -3l97 bp to +88 bp relative to the

113

Figure 26. Transcription of the PGHS-2 and PGHS-1 genes as determined by
nuclear run-on assays. MDCK cells which had been serum-starved for 48 hours were
stimulated with either (A) 0.1% DMSO alone (-TCDD), or (B) 1 nM TCDD in 0.1%
DMSO carrier (+TCDD). At the indicated times, cells were collected, and nuclei
isolated as described in Methods. The nuclei were incubated with 32P—UT P for run-on
analysis as described in Methods. Labelled transcripts obtained from re-initiated
transcription in these nuclei were hybridized with nitrocellulose strips to which 10 pg of
each of the following mouse cDNA probes had been blotted: PGHS-2 in pUCl9, PGHS-
l in pUCl9, 62 macroglobulin in pKC7, c-fos in pUCl9, and the plasmid pUC19. (C)
Relative PGHS-2 transcription rates in MDCK cells iTCDD were quantitated by
densitometry of the autoradiograms on a Bioimage Visage 110 image analyzer. Values
are expressed as the integrated optical density of the PGHS-2 bands, normalized to the
integrated optical density of the constitutive B2MG bands for each time point. Shown
is a representative experiments. Induction in the relative rate of PGHS-2 transcription
was observed in five independent experiments.

114

arose
Sum

«04.0

  

.E e m N F
tamofiioz

I: II I.il.q‘di ill-Ii:

‘ M

fl'fi‘ail 35- 41*} 5.5 ‘

m “M-
"anfi 'AA

 

 

+TCDD

hr.

0.5

115

A

r- H t

PGHS-2
PGHSJ
c-ios
82M
pUC19

Fold Induction in Relative
Transcription Rates (PGHS-2/B2MG)

116

TCDD STIMULATES TRANSCRIPTION OF PGHS-2

 

 

 

 

 

Time (hr.)

Fold Induction In Relatlve
TranscrIptIon Rates (PGHS-2/82MG)

116

TCDD STIMULATES TRANSCRIPTION OF PGHS-2

 

 

 

 

 

Time (hr.)

117

transcriptional start site (called PGHS-2m), which contains both putative DREs (Figure
22). These plasmids, and the appropriate control vectors, were then transfected into
MDCK cells, and tested for their ability to confer TCDD inducibility to the luciferase
gene (Figure 27). Transfection with both the PGHS-2m, and PGHS-2long led to a
relatively high basal expression of luciferase. This high basal promoter activity was
unexpected because PGHS-2 is normally not expressed in unstimulated cells, and such
high basal activities have not been observed in other reporter construct systems [146,
170-172]. A statistically significant induction of luciferase expression was observed in
transfected cells treated with 1 nM TCDD (PGHS-2mm 1.7-fold increase and PGHS-2m,
2.0-fold increase respectively). Because the magnitudes of induction were approximately

equivalent in cells transfected with either PGHS-2m, or PGHS-2 it seems likely that

to...
the TCDD responsive element(s) is in the PGHS-2 5’-flanking region that we examined,
located within the first 975 bp upstream from the transcriptional start site. The additional
putative DRE located at nucleotide -1067 of the PGHS-2 gene, does not appear to
function to increase TCDD responsiveness in this system. Indeed, cells transfected with
PGHS-2long exhibited depressed basal luciferase expression relative to cells transfected
with PGHS-2mm, suggesting the presence of an inhibitory domain between nucleotides -
975 and -3197. Similar reductions in basal and stimulated reporter gene expression have

been observed in other CAT or luciferase reporter plasmids when longer 5’-flanking

sequences of the PGHS-2 gene are compared to shorter sequences [146, 170, 171].

118

Figure 27. Luciferase activities observed upon TCDD induction in transfected
MDCK cells. MDCK cells were transfected with (A) pGLZ-Basic, the parent luciferase
expression plasmid (which does not contain a promoter or enhancers for luciferase
expression); (B) the pGL2-Basic plasmid containing 1 kb of PGHS-2 5’-flanking region,
the construction of which is described in Figure 12 (PGHS-2m”); (C) the pGL2-Basic
plasmid containing 3.2 kb of PGHS-2 5’-flanking region, the construction of which is
described in Figure 22 (PGHS-2,0,8); or (D) pGUDLUC1.1, a TCDD-inducible control,
which contains the TCDD-inducible domain of the gene encoding mouse cytochrome
P4501Al, CypIAI (P450). The transfected cells were then stimulated with 0.1%
DMSO alone (filled bars), or 1 nM TCDD in 0.1% DMSO carrier (open bars).
Luciferase values are expressed as arbitrary luminescence units per pg cytosolic protein.
Error bars indicate standard deviation. ('P < 0.05 no TCDD vs. TCDD treated within
groups; I’P < 0.01 basalshon vs. basalm).

119

 

 

8.3x

 

I No TCDD
2.0

D TCDD
x

 

 

7x

 

1’.

1.

 

 

 

 

250

1

o-

d d u d
0 0 o
0 5 5

100-

2 1

.529... 9.43:... €93.94.
3.2.04 388.2...

Short Long P450

Basic

(x10)

Promoter

120
Discussion

These experiments were conducted to determine whether 2,3,7,8-
Tetrachlorodibenzo—p-dioxin (TCDD) affected PGH synthase expression in the Madin-
Darby canine kidney cells. We adopted MDCK cells as a model system because
previous reports by Hassid and Levine [118, 119], indicated that xenobiotics elevated
prostaglandin synthesis in these cells. Because TCDD is one of the most potent and
long-lived HAHs it was chosen as a model compound to study the effects of xenobiotics
on PGHS expression.

While we failed to detect TCDD-induced changes in PGHS-l expression, TCDD
did dramatically and rapidly increase PGHS-2 transcription, mRNA and protein levels.
TCDD appears to increase PGHS-2 expression by transcriptionally activating the PGHS-2
gene (Figure 28). Transcriptional activation was demonstrated by nuclear run-on assays,
by increases in PGHS-2 mRNA, and by using transiently-transfected luciferase reporter
plasmids containing the PGHS—2 promoter and 5’-flanking region. Two luciferase
reporter gene plasmids containing different lengths of the 5’-flanking region of the
PGHS-2 gene were constructed. Both PGHS-2 constructs led to high basal luciferase
expression even in the absence of TCDD. Because PGHS-2 expression is not observed
in unstimulated MDCK cells, this strong basal PGHS-2 promoter activity was surprising.
It indicates this transient expression system does not accurately mimic all of the in vivo
characteristics of the PGHS-2 promoter. It is likely that PGHS-2 sequences, possibly in
introns or further upstream of the PGHS-2 promoter, constitutively repress PGHS-2 in
vivo, and these sequences are not present in the luciferase constructs. PGHS-2

expression may also be dependent upon the nucleosomal structure of the PGHS-2 gene.

121

Figure 28. Sequence of events in TCDD-stimulation of PGHS-2 expression. The
time-course of events in the TCDD induction of PGHS-2. Values are expressed as the
percentage of maximal induction for each event: transcription rate induction, as
determined by run-on analysis; mRNA level elevations, as determined by Northern
analysis; and increases in PGHS-2 protein levels, as determined by Western analysis.

122

Induction of Transcription, mRNA Levels,
and Protein Levels by TCDD

 

120

     

 

C

2 mRNA Protein

“

.2 v i

3 100 r I

E

'3

(D .5.
80 " ‘0'.

a I

.§

x Transcript.

a 60 -

E

"6
4O -

0

Di

0

on.

c 20

Ci

0

h

0

a

 

 

 

j

0
N4

4
Time (hr.)

123

Nevertheless, an approximate two—fold stimulation of luciferase activity was observed in
PGHS-2M and PGHS-2m transfected MDCK cells upon TCDD treatment. We would
have predicted that TCDD-induced transcription measured with the luciferase reporter
gene would have been more pronounced, considering the high level of expression of the
endogenous gene in response to TCDD. However, this induction is probably masked in
the reporter assay by the high constitutive expression driven by the isolated promoter.
Constructs containing the four DREs derived from the cytochrome P4SOIA1 gene
upstream from the MMTV-LTR, demonstrated an 8.5 fold induction in luciferase
expression upon TCDD treatment. The four DREs probably work synergistically [178,
179], so the level of luciferase expression driven by a single DRE, such as might exist
in our constructs, is within the range of what would be expected. However, in
comparing the TCDD-stimulated luciferase activity resulting from transfection with
PGHS-2M, or PGHSW, it was observed that the longer 5’-flanking region, which
contains two putative DREs did not confer TCDD-responsiveness any better than the
shorter 5’-fianking region, which has only one DRE. These results suggest that all of
the functional TCDD—responsive elements of this gene are found within the first 975 bp
of the transcriptional start site. The one DRE consensus sequence
(CCTGCGTGCCGCTGC) located within this region is centered around nucleotide ~165.
The ”ideal" AhR binding site, GTI‘GCGTGAGAAGAG, as has been previously defined
using in vitro using gel retardation assays [131, 179]. Point mutations of this
oligonucleotide identified essential nucleotides within this consensus necessary for AhR
binding. Paradoxically, the DRE at nucleotide -lO67 would be predicted to be a "better"

DRE (in terms of AhR binding) than that at -165 (Table 6). We have not yet determined

124

Table 6. Assessment of the relative AhR binding affinity for the putative PGHS-2
DREs. The sequences of the PGHS-2 DREs (one at nucleotides -1068 to -1078
nucleotides and the second at -168 to -154 nucleotides, relative to the transcriptional start
site) are compared to a consensus DRE sequence derived from the mouse CypIAI gene.
The core sequence TNGCGTG are shaded. Relative AhR binding affinity for the PGHS-
2 DREs was assessed based on two previous reports quantitating the effect mutations of
each nucleotide in this consensus DRE had on AhR binding [131, 179]. The fold
reduction in the binding affinity caused by the point mutation at each position examined,
which differed from one of the PGHS-2 DREs is displayed above (in the case of the 5’-
most PGHS-2 DRE) or below (for the 3’-most PGHS-2 DRE). In some cases, the two
research groups reported different values; in those cases, a range of values are displayed.
In order to obtain a quantitative idea of the relative ”quality” of the PGHS-2 DREs, we
assumed these values were multiplicative to obtain a numerical "total fold reduction" in
the relative binding affinity.

125

 

E2332 28

 

   

mmo N-m:Om
mun ..memeZOU..
mum N-m:Om

v2.33.

 

     
  

2.0—.38..

   

9mm
-vd.

 

E2832 28

 

5.33.2
28 .53

 

126
whether the TCDD effect is, in fact, mediated by TCDD-liganded Ah receptor binding

to the DRE at position ~165. However, the rapid time course of TCDD induction of
PGHS-2 in MDCK cells suggests a direct effect of TCDD on PGHS-2 transcription.

While we observed no changes in PGHS-l expression, it must be noted that in
each assay, the probes used for the Northerns, Westerns, and run-ons were derived from
murine PGHS-l and MDCK cells are from a canine source. Canine PGHS-1 and PGHS-
2 have not been cloned to date; however, the high degree of sequence identity of PGHS-l
[24, 39] between all other species cloned to date, and the cross-reactivity of the antisera
with all other species suggests that we could have detected PGHS-1 protein, had it been
present in these cells.

The effects resultant from PGHS-2 induction in TCDD-induced toxicity are not
known. TCDD—regulated gene products can be categorized into one of two general
categories: the xenobiotic metabolizing enzymes, and proteins involved in the
inflammation or cell growth regulation (Figure 6). The xenobiotic metabolizing enzymes
include cytochrome P4SOIA1 [105, 106] and cytochrome P4SOIA2 [180], glutathione-8-
transferase Ya subunit [107], and NAD(P)H quinone oxidoreductase [108, 109].
Stimulation of the expression of these genes serves to bioactivate or detoxify xenobiotics,
and limit their toxic effects. PGHS has the ability to oxidize various substrates including
some xenobiotics, such as polycyclic hydrocarbons, phenols, nitrofurans, and aromatic
amines [92, 93]. Many of these reactions are analogous to the reactions catalyzed by
cytochrome P4508 [92, 93]. Thus, PGHS could be classified among this group of

TCDD-regulated genes which are involved in metabolism of xenobiotics and induction

127

of PGHS could serve to enhance the detoxification and/or bioactivation of the xenobiotics
that induce its expression.

TCDD also regulates genes encoding factors involved in the inflammatory
response, and may be involved in cell growth regulation. Examples of gene products that
fall into this category are interleukin-13, platelet activator inhibitor-2, and transforming
growth factor oz [110-112] (Figure 6). PGHS-2 is also implicated in the inflammatory
response [41-43]. It is also an immediate early gene possibly involved in producing
prostaglandins involved in growth regulation [22, 28, 181]. Therefore, like the other
genes in this category, PGHS-2 may be involved in the mechanism by which some
xenobiotics, like TCDD, exert their toxic effects.

Because of the complexity and diversity of the toxic effects manifested upon
TCDD exposure, it is difficult to decisively identify the role of PGHS-2 in TCDD
toxicity; however, we can speculate (Figure 29). Carcinogenesis, for instance, is one
TCDD-linked toxic effect, in which PGHS-2 potentially may be involved. While it has
long been known that tumor promoters, such as phorbol esters, which mediate their
action through the stimulation of protein kinase C, induce PGHS-2, the role of PGHS-2
or the prostaglandins produced by PGHS-2 in the etiology of tumor promotion has not
been fully elucidated. TCDD, although also capable of inducing tumor production, is
not thought to mediate its action through protein kinase C. Induction of PGHS-2
expression and prostaglandin synthesis by TCDD may be a direct mechanism for tumor
promotion by HAHs. PGHS-2 has a speculative role in two stages of carcinogenesis:
mutagenesis and mitogenesis. Stimulation of PGHS-2 expression by TCDD or phorbol

esters potentially causes an increase in the bioactivation of xenobiotics into more

128

Figure 29. Speculative roles for PGHS-2 in TCDD-induced toxic effects. Potentially,
PGHS-2 can mediate a number of the diverse physiological effects, which have been
observed in different cell types and/or species, such as: carcinogenesis, through either

elevation of cellular mutagenesis or mitogenesis; chloracne; cardiac edema; or thymic
atrophy.

129

3323
3:55

«Save a m
0532:". 0.2—:3 «32.0 oz... «Bozo 2:030

mecca oEmmoEEmo
no 22095.: 22: 2
mozofiocox 3 532685

 

 

 

3.2.3 B :o=m>=om

 

 

 

. * CO:wU_XOnOO
Ag .DQV OZOEOCQX
m.X.._. .w m.mun. *
m=mo Dczcomoa w=00 _w‘/EQU_QW .Qm m=00 065560 .00 w=00 _0 .33

59.5 2&2... do \

A/DDU...

130

mutagenic forms [92, 93]. Additionally, xenobiotic tumor promoters, such as TCDD or
phorbol esters, may uncouple the regulation of expression of PGHS-2 from its
conventional physiological inducers, and thereby inappropriately stimulate the
prostaglandin production involved in cell growth and/or maturation. Further
understanding in this area may not only be of interest on a mechanistic level, but also
may be clinically important. Recent studies [87-91] regarding the use of aspirin in the
prevention of certain cancers, further supports the conjecture that active PGHS, or
prostaglandins, such as those produced by PGHS-2, may be fundamental in the pathway
of tumor establishment and/or growth. Interestingly, PAI-2 and IL-13 are also induced
by the tumor-promoting phorbol esters as well as TCDD [110, 182-184], leading one to
wonder whether all of these genes are common to the pathways of tumor establishment
or growth induced by phorbol esters and TCDD.

In addition to carcinogenesis, PGHS-2 and prostaglandins produced by PGHS-2
may be involved in the etiology of other toxic responses observed upon TCDD exposure.
Prostaglandins produced by PGHS-2 may be involved in the alterations in cell growth
seen in chloracne. However, PGHS-2 does not appear to play a role in the keratinization
aspect of chloracne [115]. Because prostaglandins participate in the inflammation
response, many researchers were led to postulate that prostaglandins may be involved in
xenobiotic-induced edema. In fact, Riflcind and co-workers demonstrated in a chick
embryo model system that arachidonic acid metabolism may be important in the toxic
effects observed after xenobiotic exposure, including cardiac edema [116, 117]. Lastly,
induction of prostaglandin production upon TCDD treatment may function to promote

thymic atrophy.

131

Recently it has been demonstrated that thymus has a high level of expression of
a thromboxane A2 (TxAz) and PGE2 (EP2) receptors, which are particularly high in CD4'
8' and CD4“8+ immature thymocytes [185]. It has been proposed that, under normal
conditions, TxA2 may play a role in negative selection of self-recognizing immature
thymocytes. Immature thymocytes were examined after treatment with a TxA2 receptor
agonist, STA2 (9,11-epithio-11,12-methano-thromboxane A2). The agonist induced dose-
dependent DNA fragmentation, and apoptosis in CD4+8" immature thymocytes, which
was inhibited with a TxA2 antagonist [185]. It has been hypothesized that TxAz,
produced by antigen-presenting cells (APCs) in the thymus in response to the binding of
a self-recognizing immature thymocyte during negative selection may signal the immature
thymocyte to apoptose, resulting in its negative selection (Figure 30). Because PGHS
is central to thromboxane production, it thus follows that abnormally high levels of
PGHS—2 in thymic APCs, which have not been recognized by an immature thymocyte
may impact the normal negative selection process of immature thymocytes.

Interestingly, it has been shown that TCDD appears to cause a selective deletion
of the population of thymocytes which undergo negative selection (CD48 and CD4+8+),
which appears to be mediated by cells other than the immature thymocytes themselves
[186-190]. Therefore, a possible mechanism by which TCDD-induced thymic atrophy
occurs can be postulated: TCDD causes increased expression of PGHS-2 in thymic
APCs, resulting in inappropriately high levels of PGHz, subsequent TxA2 overproduction
by the APCs, and ultimately to abnormal deletion of immature thymocytes, and thymic
atrophy. Although the exact mechanism of TCDD-induced PGHS-2 expression is not yet

discerned, it seems likely that uncoupling the regulation of expression of PGHS—2 from

132

Figure 30. Models for thromboxane Az-mediated thymocyte negative selection under
normal conditions, and after exposure with TCDD. (A) Under normal conditions, a
self-recognizing immature CD48 or CD4+8+ thymocyte (*) binds to a thymic antigen-
presenting cell (APC), stimulating the production and release of thromboxane A2 (TXAZ).
This TXAZ, in turn binds to this thymocyte, triggering apoptosis. On the other hand, an
immature CD48 or CD4"8+ thymocyte, which does not recognize a self-antigen will not
trigger TXA2 production, and will not be negatively selected. (B) Under TCDD-exposed
conditions, apoptosis is triggered in all immature CD48 or CD4+8” thymocytes, due to
the production of TXA2 in all thymic APCs, generated from the elevated PGH2 precursor
levels, resultant from the high TCDD-induced PGHS-2 activity.

133

€23.62 Echo: 8. so:
map—hon: m.moha0n< «<5. 3 5:93.35 33:00:: ES 5 5...; 60:26:.

«mica 833 000... .NSC. 00:35 «2.3255

an» @5538? -.=om .5: new 9.3.682. $8 5.0%
m.mo._.._0a< zo_._.<¢=._.<2 an? so 5025 3.02.3.3 .o

+ coEcuooo. o. 3:032 5 ~59 30:35

   

 

 

mzoEozoo omoanzwnaom

 

WNFKUOEIP EDP<§¢E
2308»... 9.3.5002. .:8 >420. Dm\~<x.P/'

swim

Iggy: Ear—HE:-

 
    

 

4/
.92 0.2%! _mzoEozoo ._Sfioz—

< ‘

 

134

its customary physiological inducers (hormones, cytokines, growth factors) due its
stimulated expression by non-physiological compounds such as TCDD or TPA, may be

one avenue by which aberrant cell growth or maturation arises.

CONCLUSION

An interesting dichotomy of functions has arisen from the study of the regulation
of the prostaglandin G/H synthases. Evidence from this laboratory and others, supports
the hypothesis that PGHS-1 is constitutively expressed in a wide variety of cells.
Although levels of PGHS-1 appear to remain constant within a given cell type,
expression varies among cell types, indicating that although PGHS-1 expression is not
acutely regulated in response to external stimuli, it is regulated developmentally [22, 29,
46, 64]. This is underscored by the fact that the only cell types which exhibit stimulus-
induced regulation of PGHS-1 expression in vitro appear to be cells undergoing terminal
differentiation, such as that observed in monocytes as they differentiate into macrophages
[49, 72]. Recently, one example of developmental regulation of PGHS-l in vivo was
reported. PGHS-l mRNA and protein levels were shown to become elevated in
pulmonary artery segments of fetal lambs as they develop through the third trimester of
gestation and during the first four weeks of life [163].

PGHS-2, on the other hand, is rapidly and highly expressed in cells as a result
of stimulation by such factors as hormones, growth factors, cytokines, tumor promoters,
or endotoxin (lipopolysaccharide), but PGHS-2 does not appear to be expressed in

unstimulated cells [22, 26, 28-30, 32, 44, 47, 49, 64, 74].

135

136

The differences in the regulation of expression between the two PGHS isozymes
may underscore their physiological function. Prostaglandins are involved in the
maintenance of routine body functions (water resorption in the kidney,
vasodilation/vasoconstriction, platelet aggregation, bone resorption, and stomach
function), which require immediate, on demand, synthesis. These "housekeeping"
functions may be maintained by the constitutive level of PGHS-1 expression. In contrast,
prostaglandins also participate in responses to external challenges (inflammation or
mitogenesis), and other episodic functions, such as ovulation. Routinely high levels of
prostaglandin production in these cells would be inconsistent with their purpose.
Therefore a highly inducible, but short-lived pool of prostaglandin synthetic capacity is
desired under these conditions. This is achieved through the tight regulation of PGHS-2
expression.

The mechanism by which expression of these genes are controlled appears to
entail both transcriptional and post-transcriptional aspects. Upon appropriate stimulus,
transcription of PGHS-2 is quickly and drastically elevated. Transcriptional induction
is often transitory, as are the resultant increases in mRNA levels, indicating both tight
transcriptional control and an unstable PGHS-2 mRNA [26, 44, 45, 168]. Stimuli induce
PGHS-2 by either the protein kinase C or cAMP second messenger pathways, usually.
However, only one signal pathway has been demonstrated unequivocally to date: a
C/EBP responsive element located in the 5’-flanking region has been demonstrated to
mediate forskolin-, LH-, or FSH-induced PGHS-2 transcription [172]. Further dissection
of the 5’-flanking region of the PGHS-2 will undoubtedly reveal other cis-acting

responsive elements responsible for induction of PGHS-2 expression by other factors.

137

Modulation of PGHS-1 transcription may only be in response to differention
signals in undifferentiated cells, or as a result of cell-specific signals. As yet, the
mechanism by which these stimuli may alter PGHS-1 expression is not clear. A role for
post-transcriptional regulation of PGHS-1 expression is also speculative. Some cells,
such as 3T3 fibroblasts, and rat tracheal epithelial cells, respond to certain external
stimuli with increases in transcription and mRNA levels of PGHS-l [26, 44, 45].
However, co—ordinate increases in PGHS-1 protein levels do not correlate with increases
in mRNA, suggesting post-transcriptional regulation may occur.

While PGHS-2 levels have been shown to be tightly controlled by the actions of
a number of stimuli, the demonstration of its transcriptional induction by the prototypical
xenobiotic 2,3,7,8-tetrachlorodibenzo—p—dioxin (TCDD) in any cell type, is new.
Assuming this result can be extended to cells and tissues in addition to MDCK cells,
induction of PGHS-2 activity may provide an explanation for a variety of TCDD-induced
toxic effects including carcinogenesis, chloracne, edema and thymic atrophy. The
putative mechanism underlying the role that PGHS—2 may play in each of these TCDD-
induced toxic effects is based on the notion that because PGHS-2 is critical in a number
of systems, it must remain tightly regulated by its physiological signals (cytokines,
growth factors, hormones) for normal function. Should this regulation go awry due to
aberrant induction by non-physiological factors such as tumor promoters and other
xenobiotics, it could have far-reaching pathophysiological repercussions.

In addition to explaining how TCDD-induced changes may arise, TCDD may also
be used as a tool to help explain how certain presumably spontaneous pathophysiological

conditions arise. For example, there has been much recent interest in various

138

epidemiological studies in which routine aspirin intake negatively correlates with the
incidence of certain types of cancer [87-90]. In addition, some tumors have been
demonstrated to show altered prostaglandin production profiles [91]. This may indicate
that PGHS-2 may not only be involved in TCDD-induced tumor promotion, but may also
be a more general component in the establishment or growth of tumors.

While a virtual explosion of data regarding the regulation of prostaglandin
synthesis has materialized in recent years, it appears that a plethora of questions remain
to be explored. In regards to PGHS-l: by what mechanism and which factors are
involved in the developmental and cell-specific regulation of PGHS-l? What role does
this regulation play in normal development, and might abberations in this regulation
result in pathophysiological conditions? In regards to PGHS-2: What other promoter
or enhancer elements in addition to the C/EBP responsive element may be involved in
the regulation of PGHS-2 expression? How is PGHS-2 post-transcriptionally regulated?
In addition, specific questions regarding the recent evidence of the induction of PGHS-2
expression in MDCK cells by TCDD leads one to ask: how general is this induction?
Can it be observed in other cell types and tissues and from different species? In
addition, can the in viva TCDD-induced toxic effects be diminished by aspirin treatment?
What might this tell us about other pathophysiological states— those not induced by
TCDD? Because prostaglandins are important in a wide variety of physiological
functions, and appear to be involved in some of the most common pathophysiological
conditions, this field will require, and will most likely receive, a great deal more

attention in the future.

BIBLIOGRAPHY

BIBLIOGRAPHY

Smith, W.L. (1989). The eicosanoids and their biochemical mechanisms of
action. Biochem. J. 259, 315-324.

Oates, J.A., FitzGerald, G.A., Branch, R.A., Jackson, E.K., Knapp, H.R., and
Roberts, L]. (1988). Clinical implications of prostaglandin and thromboxane A2
formation. N. Eng. J. Med. 319, 689-698.

Oates, J.A., FitzGerald, G.A., Branch, R.A., Jackson, E.K., Knapp, H.R., and
Roberts, L.J. ( 1988). Clinical implications of prostaglandin and thromboxane A2
formation: second of two parts. N. Eng. J. Med. 319, 761-767.

Hedin, L., Gaddy-Kurten, D., Kurten, R., DeWitt, D.L., Smith, W.L., and
Richards, LS. (1987). Prostaglandin endoperoxide synthetase in rat ovarian
follicles: content, cellular distribution and evidence for hormonal induction
preceding ovulation. Endrocn'nology 121, 722-731.

Noort, W.A., deZwart, F.A., and Keirse, M.J.N.C. (1988). Changes in urinary
6-keto—prostaglandin F,“ excretion during pregnancy and labor. Prostaglandins 35,
573-582.

Huslig, R.L., Fogwell, R.L., and Smith, W.L. (1979). The prostaglandin
forming cyclooxygenase of ovine uterus: relationshp to luteal function. Biol.
Reprod. 21, 589-600.

Moonen, P., Klok, G., and Keirse, M.J.N.C. (1984). Increase in concentrations
of prostaglandin endoperoxide synthase and prostacyclin synthase in human
myometrium in late pregnancy. Prostaglandins 28, 309-321.

Weksler, BB. (1987). Regulation of cyclooxygenase activity in human vascular
tissue. In " Advances in Prostaglandins, Thromboxane, and Leukotriene Research"
(B. Samuelsson, R. Paoletti, and P.W. Ramwell, Eds), Vol. 17, pp. 238-243.
Raven Press, New York.

Kuehl, F.A., and Egan, R.W. (1980). Prostaglandins, Arachidonic Acid, and
Inflammation. Science 210, 978-984.

139

10.

ll.

l2.

13.

14.

15.

l6.

17.

18.

19.

20.

21.

140

Needleman, P., Key, S.L., Denny, S.E., Isakson, RC, and Marshall, GR.
(1975). The mechanism and modificatio of bradykinin-induce coronary
vasodilation. Proc. Natl. Acad. Sci. USA 72, 2060-2063.

Weksler, B.B., Ley, C.W., and Jaffe, EA. (1978). Stimulation of endothelial
cell prostacyclin production by thrombin, trypsin, and the ionophore A23187. J.
Clin. Invest. 62, 923-930.

Grenier, F.C., Rollins, T.E., and Smith, W.L. (1981). Kinin-induced
prostaglandin synthesis by renal papillary collecting tubule cells. Amer. J Physiol.
241, F94-F104.

Kirschenbaum, A.G., Lowe, W., Trizna, W., and Fine, LG. (1982). Regulation
of vasopressin action by prostaglandins: evidence for prostaglandin synthesis in
the rabbit collecting tubule. J. Clin. Invest. 70, 1193-1204.

Gimbrone, M.A., and Alexander, R.W. (1975). Angiotensin II stimulation of

prostaglandin production in cultured human vascular endothelium. Science Wash.
DC 189, 219-220.

Albrightson, C.R., Baeziger, N.L., and Needleman, P. (1985). Exaggerated
human vascular cell prostaglandin biosynthesis mediated by monocytes: role of
monokine and interleukin-1. J. Immunol. 135, 1872-1877.

Smith, W.L., and Marnett, L]. (1991). Prostaglandin endoperoxide
synthase:structure and catalysis. Biochim. Biophys. Acta 1083, 1-17.

Egan, R.W., Paxton, J ., and Kuehl, F.A. (1976). Mechansim for irreversable
self-deactivation of prostaglandin synthetase. J. Biol. Chem. 251, 7325-7335.

Lapetina, E.G. , and Cuatrecasas, P. (1979). Rapid inactivation of cyclooxygenase
after stimulation of intact platelets. Proc. Natl. Acad. Sci. USA 76, 121-125.

Kent, R.S., Diedrich, S.L., and Whorton, A.R. (1983). Regulation of vascular
prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit
aorta. J. Clin. Invest. 72, 455-465.

Smith, W.L., and Lands, W.E.M. (1972). Oxygenation of polyunsaturated fatty
acids during prostaglandin biosynthesis by sheep vesicular glands. Biochemistry
11, 3276-3282.

DeWitt, D. (1991). Prostaglandin endoperoxide synthase: regulation of enzyme
expression. Biochim. Biophys. Acta 1083, 121-134.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

141

Kujubu, D.A., Fletcher, B.S., Varnum, B.C., Lim, R.W., and Herschman, HR.
(1991). TISlO, a phorbol ester tumor promoter inducible mRNA from Swiss 3T3
cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J. Biol.
Chem. 266, 12866-12872.

DeWitt, D.L., Day, 1.8., Sonnenburg, W.K., and Smith, W.L. (1983).
Concentrations of prostaglandin endoperoxide synthase and prostaglandin 12
synthase in the endothelium and smooth muscle of bovine aorta. J. Clin. Invest.
72, 1882-1888.

Funk, C.D., Funk, L.B., Kennedy, M.E., Pong, A.S., and Fitzgerald, G.A.
( 1991). Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDN A
cloning, expression, and gene chromosomal assignment. Faseb J. 5, 2304-2312.

Hoff, T., DeWitt, D., Kaever, V., Resch, K., and Goppelt-Struebe, M. (1993).
Differentiation-associated expression of prostaglandin G/H synthase in monocytic
cells. FEBS Letters 320, 38-42.

Hamasaki, Y., Kitzler, J., Hardman, R., Nettesheim, P., and Eling, TE. (1993).
Phorbol ester and epidermal growth factor enhance the expression of two
inducible prostaglandin H synthase genes in rat tracheal epithelial cells. Arch.
Biochem. Biophys. 304, 226-234.

Dewitt, D.L., Rollins, T.E., Day, J.S., Gauger, J.A., and Smith, W.L. (1981).
Orientation of the active site and antigenic determinants of prostaglandin
endoperoxide (PGH) synthase in the endoplasmic reticulum. J. Biol. Chem. 256,
10375-10382.

Ryseck, R., Raynoschek, C., Macdonald-Bravo, H., Dorfman, K., and Bravo,
R. (1992). Identification of an immediate early gene, pghs-b, whose protein
product has prostaglandin synthase/cylooxygenase activity. Cell GrOWth & Difler.
3, 443-450.

Jones, D.A., Carlton, D.P., McIntyre, T.M., Zimmerman, G.A., and Prescott,
S.M. (1993). Molecular cloning of human prostglandin endoperoxide synthase
type II and demonstration of expression in response to cytokines. J. Biol. Chem.
268, 9049-9054.

Nusing, R., and Ullrich, V. (1992). Regulation of cyclooxygenase and
thromboxane synthase in human monocytes. Eur. J. Biochem. 206, 131-136.

Maier, J.A.M., Hla, T., and Maciag, T. (1990). Cyclooxygenase is an
immediate-early gene induced by interleukin-1 in human endothelial cells. J. Biol.
Chem. 265, 10805-10808.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

142

Sirois, J. , and Richards, J .S. (1992). Purification and characterization of a novel,
distinct isoform of prostaglandin endoperoxide synthase induced by human
chorionic gonadotropin in granulosa cells of rat preovulatory follicles. J. Biol.
Chem. 267, 6382-6388.

Lin, A.H., Bienkowshki, M.J., and Gorman, R.R. (1989). Regulation of
prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-
derived growth factor. J. Biol. Chem. 264, 17379-17383.

Lee, S.H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W., Zhong, H., Liou,
S., Simmons, D., and Hwang, D. (1992). Selective expression of mitogen-

inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J.
Biol. Chem. 267, 25934-25938.

O’Sullivan, M.G., Huggins, E.M., Meade, E.A., DeWitt, D.L., and McCall,
CE. (1992). Lipopolysaccharide induces prostaglandin H synthase-2 in aveolar
macrophages. Biochem. Biophys. Res. Commun. 187, 1123-1127.

O’Sullivan, G.M., Chilton, F.H., Huggins Jr., E.M., and McCall, CE. (1992).
Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of

prostanoids involves induction of a novel prostaglandin H synthase. J. Biol.
Chem. 267, 14547-14550.

DeWitt, D.L., and Smith, W.L. (1988). Primary structure of prostaglandin G/H
synthase from sheep vesicular gland determined from the complementary DNA
sequence. Proc. Natl. Acad. Sci. USA 85, 1212-1416.

Merlie, J.P., Fagan, D., Mudd, J., and Needleman, P. (1988). Isolation and
Characterization of the complementary DNA for sheep seminal vesicle
prstaglandin endoperoxide synthase (cyclooxygenase). J. Biol. Chem. 263, 3550-
3553.

DeWitt, D.L., El-Harith, E.A., Kraemer, S.A., Andrews, M.J., Yao, E.F.,
Armstrong, R.L., and Smith, W.L. (1990). The apirin and heme-binding site for
the ovine and murine prostaglandin endoperoxide synthases. J. Biol. Chem. 265,
5192-5198.

Roth, G.J., Machuga, E.T., and Ozols, J. (1983). Isolation and covalent structure
of the aspirin-modified, active-site region of prostaglandin synthase. Biochemistry
22, 4672-4675.

Masferrer, J.L., Seibert, K., Zweifel, B., and Needleman, P. (1992).
Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. Proc.
Natl. Acad. Sci. USA 89, 3917-3921.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

143

Sano, H., Hla, T., Maier, J.A.M., Crofford, L.J., Case, J.P., Maciag, T., and
Wilder, KL. (1992). In Vivo cyclooxygenase expression in synovial tissue of

patients with rheumatoid arthritis and osteoarthritis and rats with adjuvant and
streptococcal cell wall arthritis. J. Clin. Invest. 89, 97—108.

Crofford, L.J., Wilder, R.L., Ristimaki, A.P., Sano, H., Remmers, E.F., Epps,
H., and Hla, T. (1994). Cyclooxygenase-l and -2 expression in rheumatoid
synovial tissues: effect of interleukin-16, phorbol ester, and corticosteroids. J.
Clin. Invest. 93, 1095-1101.

DeWitt, D.L., and Meade, E. (1993). Serum and glucocorticoid regulation of
gene transcription and expression of the prostaglandin H synthase-1 and
prostaglandin H synthase-2 isozymes. Arch. Biochem. Biophys. 306, 94-102.

Evett, G.E., Xie, W., Chipman, J.G., Robertson, D.L., and Simmons, D.L.
( 1993). Prostaglandin G/H isoenzyme 2 expression in fibroblasts: regulation by

dexamethasone, mitogens, and oncogenes. Arch. Biochem. Biophys. 306, 169-
177.

Kujubu, D.A., and Herschman, HR. (1992). Dexamethasone inhibits mitogen
induction of the T1810 prostaglandin synthase/cyclooxygenase gene. J. Biol.
Chem. 267, 7991-7994.

Kujubu, D.A., Reddy, S.T., Fletcher, B.S., and Herschman, HR. (1993).
Expression of the protein product of the prostaglandin synthase-2/TISlO gene in
mitogen-stimulated Swiss 3T3 cells. J. Biol. Chem. 268, 5425-5430.

Koehler, L., Hass, R., DeWitt, D.L., Resch, K., and Goppelt—Struebe, M.
(1990). Glucocorticoid-induced reduction of prostanoid synthesis in TPA-
differentiated U936 cells is mainly due to a reduced cyclooxygenase activity.
Bioch. Pharm. 40, 1307-1316.

Hoff, T., DeWitt, D., Kaever, V., Resch, K., and Goppelt-Struebe, M. (1993).
Differentiation-associated expression of prostaglandin G/ H synthase in monocytic
cells. FEBS. Lett. 320, 38-42.

Fu, 1., Masferrer, J.L., Seibert, K., Raz, A., and Needleman, P. (1990). The
induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in
human monocytes. J. Biol. Chem. 265, 16737-16740.

Raz, A., Wyche, A., and Needleman, P. (1989). Temporal and pharmacological
division of fibroblast cyclooxygenase expression into transcriptional and
translational phases. Proc. Natl. Acad. Sci. USA 86, 1657-1661.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

144

Bailey, J .M., Makheja, A.N., Pash, J., and Verma, M. (1988). Corticosteroids
suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in
Cultured vascular cells. Biochem. Biophys. Res. Commun. 157, 1159-1163.

Han, J., Sadowski, H., Young, D.A., and Macara, LG. (1990). Persistant
induction of cylcooxygenase in p60v-src-transformed 3T3 fibroblasts. Proc. Natl.
Acad. Sci. USA 87, 3373-3377.

Regier, M.K., DeWitt, D.L., Schindler, M.S., and Smith, W.L. (1993).
Subcellular localization of prostaglandin endoperoxide synthase-2 in murine 3T3
cells. Arch. Biochem. Biophys. 301, 439-444.

Otto, J .C., DeWitt, D.L., and Smith, W.L. (1994). unpublished observations.

Picot, D., Loll, P.J., and Garavito, RM. (1994). The X-ray Crystal Structure
of the membrane protein prostaglandin H2 synthase. Nature 367, 243-249.

Otto, J.C., DeWitt, D.L., and Smith, W.L. (1993). N-glycosylation of
prostaglandin endoperoxide synthases-1 and -2 and their orientations in the
endoplasmic reticulum. J. Biol. Chem. 268, 18234-18242.

Roth, G.J., Siok, C.J., and Ozols, J. (1980). Structural characteristics of
prostaglandin synthetase from sheep vesicular gland. J. Biol. Chem. 255, 1301-
1304.

Yokoyama, C., Takai, T., and Tanabe, T. (1988). Primary structure of sheep
prostaglandin endoperoxide synthase deduced from cDN A sequence. FEBS Letters
231, 347-351.

Feng, L., Sun, W., and Xia, Y. (1993). Cloning two isoforms of rat
cyclooxygenase: differential regulation of their expression. Arch. Biochem.
Biophys. 307, 361-368.

Yokoyama, C., and Tanabe, T. (1989). Cloning of the human gene encoding
prostaglandin endoperoxide synthase and primary structure of the enzyme.
Biochem. Biophys. Res. Commun. 165, 888-894.

Yokoyama, C., Toh, H., Miyata, A., and Tanabe, T. (1990). Cloning and
characterization of human cyclooxygenase gene and Primary Structure of the
Enzyme. In "Advances in Prostaglandins, Thromboxane, and Leukotriene
Research" (B. Sameulsson, Ed.), Vol. 21, pp. 61-64. Raven Press, New York.

Simmons, D.L., Levy, D.B., Yannoni, Y., and Erikson, R.L. (1989).
Identification of a phorbol ester-repressible v-src-inducible gene. Proc. Natl.
Acad. Sci. USA 86, 1178-1182.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

145

Hla, T., and Neilson, K. (1992). Human cyclooxygenase-2 cDNA. Proc. Natl.
Acad. Sci. USA 89, 7384-7388.

Wen, P.Z., Warden, C., and Fletcher, BS. (1993). Chromosomal organization
of the inducible and constitutive prostaglandin synthase/cyclooxygenase genes in
mouse. Genomics 15, 458-460.

Toh, H. (1989). Prostaglandin endoperoxide synthase contains an EGF-like
domain. FEBS Letters 258, 317-319.

Shimokawa, T., and Smith, W. (1991). Essential histidines of prostaglandin
endoperoxide synthase. J. Biol. Chem. 266, 6168-6173.

Shimokawa, T., Kulmacz, R.J., DeWitt, D.L., and Smith, W.L. (1990).
Tyrosine 385 of prostaglandin endoperoxide synthase is required for
cyclooxygenase catalysis. J. Biol. Chem. 265, 20073-20076.

Meade, E.A., Smith, W.L., and DeWitt, D.L. (1993). Differential inhibition of
prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and
other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 268, 6610-6614.

Holtzman, M.J., Turk, J., and Shornick, L.P. (1992). Identification of a
pharmacologically distinct prostaglandin H synthase in cultured epithelial cells.
J. Biol. Chem. 267, 21438-21445.

DeWitt, D.L., Kraemer, S.A., and Meade, EA. (1991). Serum induction and
superinduction of PGH synthase mRNA levels in 3T3 fibroblasts. In
"Prostaglandins and related compounds 11" (B. Samuelsson et al. , Eds), Vol. 21 ,
pp. 75-90. Raven Press, Ltd. , New York.

Smith, C.J., Morrow, J.D., Roberts, LL, and Marnett, L]. (1993).
Differentiation of moncytoid THP—1 cells with phorbol ester induces expression
of prostaglandin endoperoxide synthase-1 (Cox-l). Biochem. Biophys. Res.
Commun. 192, 787-793.

Wong, W.Y.L., and Richards, LS. (1991). Immuno-detection of two
antigenically distinct molecular weight variants of prostaglandin H synthase in the
rat ovary. J. Biol. Chem.

O’Banion, M.K., Sadowski, H.B., Winn, V., and Young, DA. (1991). A serum-
and glucocorticoid-regulated 4 kb mRNA encodes a cyclooxygenase-related
protein. J. Biol. Chem. 266, 23261-23267.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

146

Xie, W., Chipman, J.G., Robertson, D.L., Erikson, R.L., and Simmons, D.L.
(1991). Expression of a mitogen-responsive gene encoding prostaglandin synthase
is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA 88, 2692-2696.

Moncada, S., and Vane, J .R. (1979). Pharmacology and endogenous roles of
prostaglandin endoperoxides, thromboxane A2 and prostacyclin. Pharmacol. Rev.
30, 293-331.

Brooks, P.M., and Day, RD. (1991). Nonsteroidal antiinflammatory drugs-
differences and similarities. N. Eng. J. Med. 324, 1716-1725.

Smith, W.L., and DeWitt, D.L. (1994). Differential interaction of prostaglandin
endoperoxide synthases with nonsteroidal anti-inflammatory drugs. Current
Opinions in Investigatory Drugs 3, 1-20.

Futaki, N., Yoshikawa, K., Hamasaka, Y., Arai, 1., Higuchi, S., Iizuka, H., and
Otomo, S. (1993). NS-398, a novel non-steroidal anti-inflammatory drug with
potent analgesic and antipyretic effects, which causes minimal stomach lesions.
Gen. Pharmacol. 24, 105-110.

Hamberg, M., Svensson, J., and Samuelsson, B. (1975). Thromboxanes: a new
group of biologically active compounds derived from prostaglandin
endoperoxides. Proc. Natl. Acad. Sci. USA 72, 2994-2998.

Moncada, S., Gryglewski, R., and Vane, J .R. (1976). An enzyme isolated from
arteries transforms prostaglandin endoperoxides to an unstable substance that
inhibtis platelet aggregation. Nature 263, 663-665.

Willard, J., Lange, R.A., and Hillis, L.D. (1992). The use of aspirin in ischemic
heart disease. N. Eng. J. Med. 327, 175-181.

Lewis, H.D., Davis, J.W., Archibald, D.G., Steinke, W.E., Smitherman, T.C.,
Doherty, J.E., Schnaper, H.W., LeWinter, M.M., Linares, E., Pouget, J.M.,
Sabharwal, S.C., Chelsler, E., and DeMots, H. (1983). Protective effects of
aspirin against acute myocardial infaction and death in men with unstable angina.
N. Eng. J. Med. 309, 396-403.

The Steering Committee of the Physicians Health Study Research Group (1988).
Preliminary report: findings from the aspirin component of the ongoing physicians
health study. N. Eng. J. Med. 318, 262-264.

Schiff, E., and Mashiach, S. (1992). The Use of Low Dose Aspirin in
Pregnancy. Am. J. Reprod. Immunol. 28, 153-156.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

147

Shen, J., Wanwimolruk, S., Wilson, P.D., Seddon, R.J., and Roberts, MS
(1993). A Clinical Trial of a Slow-Release Formulation of Acetylsalicylic Acid
in Patients at Risk for Preeclampsia. Br. J. Clin. Pharmac. 35, 664-667.

Marnett, LJ. (1992). Aspirin and the potential role of prostaglandins in colon
cancer. Cancer Res. 52, 5575-5589.

Thun, M.J., Namboodiri, M.M., and Heath, CW. (1991). Aspirin use and
reduced risk of fatal colon cancer. N. Eng. J. Med. 325, 1593-1596.

Thun, M.J., Namboodiri, M.M., Calle, E.E., Flanders, W.D., and Heath, CW.
(1993). Aspirin use and risk of fatal cancer. Cancer Res. 53, 1322-1327.

Peleg, 1.1., Maibach, H.T., Brown, S.H., and Wilcox, C.M. (1994). Aspirin and
nonsteroidal anti-inflammatory drug use and the risk of subsequent colorectal
cancer. Arch. Int. Med 154, 394-399.

Rigas, B., Goldman, I.S., and Levine, L. (1993). Altered Eicosanoid levels in
Human Colon Cancer. J. Lab. Clin. Med 122, 518-523.

Smith, B.J., Curtis, J.F., and Eling, TE. (1991). Bioactivation of xenobiotics by
prostaglandin H synthase. Chem. Biol. Interact. 79, 245-264.

Eling, T.E., Thompson, D.C., Foureman, G.L., Curtis, J .F., and Hughes, M.F.
(1990). Prostaglandin H synthase and xenobiotic oxidation. In "Annual Reviews
of Pharmacology and Toxicology", Vol. 30, pp. 1-45. Annual Reviews Inc. , Palo
Alto.

Whitlock, J.P., Jr. (1990). Genetic and molecular aspects of 2,3,7,8-
tetrachlorodibenzo-p-dioxin action. Annu. Rev. Pharmacol. Toxicol. 30, 251-277.

Poland, A., and Knutson, LC. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and
related halogenated aromatic hydrocarbons: examination of the mechanism of
toxicity. Annu. Rev. Phannacol. Toxicol. 22, 517-554.

Landers, J .P., and Bunce, NJ. (1991). The Ah receptor and the mechanism of
dioxin toxicity. Biochem. J. 276, 273-287.

Safe, S.H. (1986). Comparitive toxicology and mechanism of action of
polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol.
Toxicol. 26, 371-399.

Kimbrough, RD, and Jensen, A.A., Eds. (1989). "Halogenated biphenyls,
terphenyls, naphthalenes, dibenzodioxins and related products", 2nd Ed. Elsevier,
New York.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

148

McConnel, E.E., Moore, J.A., Haseman, H.K., and Harris, M.W. (1978). The
comparitive toxicity of chlorinated dibenzo-p—dioxin in mice and guinea pigs.
Toxicol. Appl. Pharmacol. 44, 335-356.

Olson, J.R., Holscher, M.A., and Neal, R.A. (1980). Toxicity of 2,3,7,8-
tetrachlorodibenzo-p—dioxin in the Golden Syrian Hamster. Toxicol. Appl.
Pharmacol. 55, 67-78.

Henck, J.W., New, M.A., Kociba, R.J., and Rao, KS. (1981). 2,3,7,8-
tetrachloro-p-dioxin: acute oral toxicity in hamsters. Toxicol. Appl. Pharmacol.
59, 405-407.

Roberts, L. (1991). Dioxin risks revisited. Science 251, 624-626.
Roberts, L. (1991). More pieces in the dioxin puzzle. Science 252, 377.
Bailar, LC. (1991). How dangerous is dioxin? N. Eng. J. Med. 324, 260-262.

Israel, D.I., and Whitlock, LP. (1983). Induction of mRNA specific for
cytochrome Pl-450 in wild type and variant mouse hepatoma cells. J. Biol.
Chem. 258, 10390-10394.

Israel, D.I., and Whitlock, J .P. (1984). Regulation of cytochrome P1-450 gene
transcription by 2,3,7 ,8-tetrachlorodibenzo-p-dioxin in wild-type and variant
mouse hepatoma cells. J. Biol. Chem. 259, 5400-5402.

Friling, R.S., Bensimon, A., Tichauer, Y., and Daniel, V. (1990). Xenobiotic-
inducible expression of murine glutathione S-transferase Ya subunit gene is
controlled by an electrophile-responsive element. Proc. Natl. Acad. Sci. USA 87,
6258-6262.

Favreau, L.V., and Pickett, QB. (1991). Transcriptional regulation of the rat
NAD(P)H:quinone reductase gene. Identification of regulatory elements
controlling basal level expression and inducible expression by planar aromatic
compounds and phenolic antioxidants. J. Biol. Chem. 266, 4556-4561.

Jaiswal, A.K. (1991). Human NAD(P)H:Quinone oxidoreductase (NQOl) gene
structure and induction by dioxin. Biochemistry 30, 10647-10653.

Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991). Targets for
dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science
254, 415-418.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

149

Choi, E.J., Toscano, D.G., Ryan, J.A., Riedel, N., and Toscano, W.A., Jr.
( 1991). Dioxin induces transforming growth factor-alpha in human keratinocytes.
J. Biol. Chem. 266, 9591-9597.

Gaido, K.W., Maness, S.C., Leonard, L.S., and Greenlee, W.F. (1992).
2 ,3 ,7, 8-Tetrachlorodibenzo—p-dioxin-dependent regulation of transforming growth
factors-alpha and -beta 2 expression in a human keratinocyte cell line involves
both transcriptional and post-transcriptional control. J. Biol. Chem. 267 , 24591-
24595.

Rifkind, A.B., Gannon, M., and Gross, S.S. (1990). Arachidonic acid
metabolism by dioxin-induced cytochrome P-450: a new hypothesis on the role
of P-450 in dioxin toxicity. Biochem. Biophys. Res. Commun. 172, 1180-1188.

Theobald, H.M., Moore, R.W., Katz, L.B., Pieper, R.O., and Peterson, RE.
(1983). Enhancement of carrageenan and dextran-induced edemas by 2,3,7,8-
tetrachlorodibenzo—p-dioxin and related compounds. J. Pharmacol. Exp. Ther.
225, 576-583.

Knutson, J .C., and Poland, A. (1984). 2,3,7,8-Tetrachlorodibenzo-p-dioxin:
examination of biochemical effects involved in the proliferation and differentiation
of XB cells. J. Cell Physiol. 121, 143-151.

Rifldnd, A.B., and Muschick, H. (1983). Benoxaprofen suppression of
polychlorinated biphenyl toxicity without alteration of mixed function oxidase
function. Nature 303, 524-526.

Quilley, OR, and Rifldnd, AB. (1986). Prostaglandin release by the chick
embryo heart is increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin and by other
cytochrome P-448 inducers. Biochem. Biophys. Res. Commun. 136, 582-589.

Hassid, A. , and Levine, L. (1977). Induction of fatty acid cyclooxygenase
activity in canine kidney cells (MDCK) by benzo(a)pyrene. J. Biol. Chem. 252,
6591-6593.

Levine, L. (1977). Chemical carcinogens stimulate canine kidney (MDCK) cells
to produce prostaglandins. Nature 268, 447-448.

Jones, P.B., Durrin, L.K., Fisher, J.M., and Whitlock, J.F., Jr. (1986). Control
of gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Multiple dioxin-
responsive domains 5’-ward of the cytochrome P1-450 gene. J. Biol. Chem. 261,
6647-6650.

Denison, M.S., Fisher, J .M., and Whitlock, LP. (1988). The DNA recognition
site for the dioxin-Ah receptor complex. J. Biol. Chem. 263, 17221-17224.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

150

Denison, M.S., Fisher, J .M., and Whitlock, LP. (1988). Inducible, receptor-
dependent protein-DNA interactions at a dioxin-responsive transcriptional
enhancer. Proc. Natl. Acad. Sci. USA 85, 2528-2532.

Denison, M.S., Fisher, J .M., and Whitlock, J .P. (1989). Protein-DNA
interaction at recognition sites for the dioxin-Ah receptor complex. J. Biol. Chem.
264, 16478-16482.

Whitlock, J.P., Denison, M.S., Fisher, J.M., and Shen, ES. (1989). Induction
of hepatic cytochrome P450 gene expression by 2,3,7,8-tetrachlorodibenzo-p-
dioxin. Mol. Biol. Med 6, 169-178.

Jones, P., Galeazzi, D., Fisher, J., and Whitlock, LP. (1985). Control of
cytochrome P1-450 gene expression by dioxin. Science 227, 1499-1502.

Miller, A., Israel, D., and Whitlock, J. (1983). Biochemical and genetic analysis
of variant mouse heptoma cells defective in the induction of benzo(a)pyrene-
metabolizing enzyme activity. J. Biol. Chem. 258, 3523-3527.

Rushmore, T.H., King, R.G., Paulson, K.E., and Pickett, CB. (1990).
Regulation of glutathione S-transferase Ya subunit gene expression: identification
of a unique xenobiotic-responsive element controlling inducible expression by
planar aromatic compounds. Proc. Natl. Acad. Sci. U. S. A. 87, 3826-3830.

Favreau, L.V., and Pickett, CB. (1987). Transcriptional regulation of the rat
NAD(P)H:Quinone Reductase. J. Biol. Chem. 268, 19875-19881.

Rushmore, R.H., Morton, M.R., and Pickett, QB. (1991). The antioxidant
responsive element. Activation by oxidative stress and identification of the DNA
consensus sequence required for functional activity. J. Biol. Chem. 266, 11632-
11639.

Rushmore, T.H., and Pickett, QB. (1990). Transcriptional regulation of the rat
glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-
responsive element controlling inducible expression by phenolic antioxidants. J.

Biol. Chem. 265, 14648-14653.

Yao, E., and Denison, M.S. (1992). DNA sequence determinants for binding of
transformed Ah receptor to a dioxin-responsive enhancer. Biochemistry 31, 5060-
5067.

Hoffman, E.C., Reyes, H., Chu, F.F., Sander, F., Conley, L.H., Brooks, B.A.,
and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah
(dioxin) receptor. Science 252, 954-958.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

151

Dolwick, K.M., Schmidt, J.V., Carver, L.A., Swanson, H.I., and Bradfield,
C.A. (1993). Cloning and expression of a human Ah receptor cDNA. Mol.
Pharm. 44, 911-917.

Schmidt, J.V., Carver, L.A., and Bradfield, C.A. (1993). Molecular
characterization of the murine Ahr gene. J. Biol. Chem. 268, 22203-22209.

Dolwick, K.M., Swanson, H.I., and Bradfield, C.A. (1993). In vitro analysis of
Ah receptor domains involved in ligand-activated DNA recognition. Proc. Natl.
Acad. Sci. USA 90, 8566-8570.

Watson, A.J., and Hankinson, O. (1992). Dioxin- and Ah receptor-dependent
protein binding to xenobiotic responsive elements and G-rich DNA studied by in
vivo footprinting. J. Biol. Ozem. 267, 6874-6878.

Morgan, J .E., and Whitlock, J .P. (1992). Transcription-dependent and
transcription-independent nucleosomal disruption induced by dioxin. Proc. Natl.
Acad. Sci. USA 89, 11622-11626.

Elferink, C.J., and Whitlock, J.P. (1990). 2,3,7,8-tetrachlorodibenzo-p-dioxin-
inducible, Ah receptor-mediated bending of enhancer DNA. J. Biol. Chem. 265 ,
5718-5721.

Durrin, L.K., and Whitlock, J.P. (1989). 2,3,7,8-tetrachlorodibenzo-p—dioxin-
inducible aryl hydrocarbon receptor-mediated change in CYP1A1 chromatin
structure occurs independently of transcription. Mol. Cell. Biol. 9, 5733-5737.

Smith, W.L., DeWitt, D.L., Shimokawa, T., Kraemer, S.A., and Meade, EA.
(1990). Molecular basis for the inhibition of prostanoid biosynthesis by
nonsteroidal anti-inflammatory agents. Stroke 21, IV24-IV28.

Sambrook, J ., Fritsch, ER, and Maniatis, T. (1989). "Molecular Cloning: A
Laboratory Manual”, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor.

Feinburg, A.P., and Vogelstein, B. (1984). A technique for radioloabeling DNA
restriction endonuclease fragments to high specific activity. Anal. Biochem. 137,
266-267.

Kraemer, S.A., Meade, S.A., and DeWitt, D.L. (1992). Prostaglandin
endoperoxide synthase gene structure: identification of the transcriptional start site
and 5’-flanking regulator sequences. Arch. Biochem. Biophys. 293, 391-400.

Sanger, P., Nicklen, S., and Coulson, A.R. (1977). DNA sequences with chain
terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

152

Devereux, J ., Haeberli, P., and Smithies, O. (1984). A comprehensive set of
sequence analysis programs for the VAX. NAR 12, 387-395.

Fletcher, B.S., Kujubu, D.A., Perrin, D.M., and Herschman, HR. (1992).
Structure of the mitogen-inducible T1810 gene and demonstration that the TIS10-
encoded protein is a functional prostaglandin G/H synthase. J. Biol. Chem. 267 ,
4338-4344.

Bradford, M.M. (1976). A Rapid and Sensitive Method for the Quantitation of
Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.
Anal. Biochem. 72, 248-254.

Breathnach, R., and Chambon, P. (1981). Organization and expression of
eukaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349-383.

Bucher, P., and Tifonov, EN. (1986). Compliation and analysis of eukaryotic
Pol 11 promoter seqeunces. NAR 14, 10009-10026.

McKnight, S., and Tijan, R. (1986). Transcriptional selectivity of viral genes in
mammalian cells. Cell 46, 795-805.

Jones, N.C., Rigby, P.W.J., and Ziff, EB. (1988). Trans-acting protein factors
and the regulation of eukaryotic transcription from studies on DNA tumor
viruses. Genes Dev. 2, 267-281.

Li, R., Knight, J.D., Jackson, S.P., Tijan, R., and Botchan, MR. (1991). Direct
interaction between Spl and the BPV enhancer E2 protein mediates synergistic
activation of transcription. Cell 65, 493-505.

Kadonaga, J.T., Jones, K.A., and Tijan, R. (1986). Promoter-specific activation
of RNA polymerase II transcription by Spl. NBS 11, 20-23.

Pugh, BR, and Tijan, R. (1990). Mechanism of trnascriptional activation by
Spl: Evidence for coactivators. Cell 61, 1187-1197.

Smale, S.T., Schmidt, M.C., Berk, A.J., and Baltimore, D. (1990).
Transcriptional activation by Spl as directed through TATA or initiator: Specific
requirement for mammalian transcription factor IID. Proc. Natl. Acad. Sci. USA
87, 4509-4515.

Angel, P., Imagawa, M., Shiu, R., Stein, B., Imbra, R., Rahmsdorf, H., Jonat,
C., Herrlich, P., and Karin, M. (1987). Phorbol ester-inducible genes contain a
common cis element recognized by a TPA-modulated trans-acting factor. Cell 49,
729-739.

157.

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

153

Sakai, D., Helms, S., Carlstedt-Duke, J., Gustafsson, Jan-A., Rottman, P.M.,
and Yamamoto, KR. (1988). Hormone-mediated repression: a negative
glucocorticoid response element from the bovine prolactin gene. Genes Dev. 2,
1144-1154.

Akerblom, I.E., Slater, E.P., Beato, M., Baxter, J.D., and Mellon, P.L. (1988).
Negative regulation by glucocorticoids through interference with a CAMP
responsive enhancer. Science 241, 350-353.

Imagawa, M., Chiu, R., and Karin, M. (1987). Transcription factor AP-2
mediates induction by two different signal-transduction pathways: Protein Kinase
C and CAMP. Cell 51, 251-260.

Shimokawa, T., and Smith, W.L. (1992). Prostaglandin Endoperoxide synthase:
The aspirin acetylation region. J. Biol. Chem. 267, 12387-12392.

Herschman, H.R., Fletcher, B.S., and Kujubu, DA. (1993). TIS 10, a mitogen-
inducible glucocorticoid-inhibited gene that encodes a 2nd prostaglandin
synthase/cyclooxygenase enzyme. J. Lipid Mediat. 6, 89-99.

Sirois, J., Simmons, D.L., and Richards, J. (1992). Hormonal regulation of
mesenger ribonucleic acid encoding a novel isoform of prostaglandin
endoperoxide H synthase in rat preovulatory follicles. J. Biol. Chem. 267, 11586-
11592.

Brannon, T.S., North, A.J., Wells, L.B., and Shaul, P.W. (1994). Prostacyclin
Synthesis in Ovine Pulmonary Artery is Developmenatally Regulated by Changes
in Cyclooxygenase-1 Gene Expression. J. Clin. Invest. 93, 2230-2235.

Roesler, W.J., Vandenbark, G.R., and Hanson, R.W. (1988). Cyclic AMP and
the induction of eukaryotic gene transcription. J. Biol. Chem. 263, 9063-9066.

Shaw, G., and Kamen, R. (1986). A conserved AU sequence from the 3’
untranslated region of GM-CSF mRNA mediates selective degradation. Cell 46,
659-667.

Brewer, G. (1991). An A+U-Rich Element RNA-Binding Factor Regulates c-myc
mRNA stability In Vitro. Mol. Cell. Biol. 11, 2460-2466.

Boulder, B., Thompson, P., Keyes, J ., Hagerty, K., and Crawford, D. (1988).
Assay of a ribonuclease that preferentially hydrolyses mRN As containing cytokin-
derived UA-rich instability sequences. Biochem. Biophys. Res. Commun. 152,
973-980.

168.

169.

170.

171.

172.

173.

174.

175.

176.

177.

178.

154

Ristimaeki, A., Garfinkel, S., Wessendorf, J., Maciag, T., and Hla, T. (1994).
Induction of Cyclooxygenase-2 by Interleuldn-l alpha. J. Biol. Chem. 269,
11769-11775. .

Diaz, A., Reginato, A.M., and Jimenez, S.A. (1992). Alternative splicing of
human prostaglandin G/H synthase mRNA and evidence of differential regulation
of the resulting transcripts by transforming growth factor 81, Interleukin 16, and
Tumor Necrosis Factor 0:. J. Biol. Chem. 267, 10816-10822.

Xie, W., Merrill, J.R., Bradshaw, W.S., and Simmons, D.L. (1993). Structural
determination and promoter analysis of the chicken mitogen-inducible
prostaglandin G/H synthase gene and genetic mapping of the murine homolog.
Arch. Biochem. Biophys. 300, 247-252.

Sirois, J., Levy, L.O., Simmons, D.L., and Richards, LS. (1993).
Characterization and hormonal regulation of the promoter of the rat prostaglandin

endoperoxide synthase 2 gene in granulosa cells. Identification of functional and
protein-binding regions. J. Biol. Chem. 268, 12199-12206.

Sirois, J ., and Richards, LS. (1993). Transcriptional regulation of the rat

prostaglandin endoperoxide synthase 2 gene in granulosa cells. J. Biol. Chem.
268, 21931-21938.

Akira, S., and Kishimoto, T. (1993). IL-6 and NF-IL6 in acute-phase response
and viral infection. Immunol. Rev 127, 25-50.

Grove, M., and Plumb, M. (1993). C/EBP, NF-KB, and c-Ets family members
and transcriptional regulation of the cell-specific and inducible macrophage
inflammatory protein 1 alpha immediate early gene. Mol. Cell. Biol. 13, 5276-
5289.

Macleod, K., Leprince, D., and Stehelin, D. (1992). The Ets gene family. TIBS
17, 251-256.

Meade, E.A., Smith, W.L., and DeWitt, D.L. (1993). Expression of the murine
prostaglandin (PGH) synthase-1 and PGH synthase-2 isozymes in cos-1 cells. J.
Lipid Mediat. 6, 119-129.

Bedard, Pierre-A., Alcorta, D., Simmons, D.L., Luk, Ka-C., and Erikson, R.L.
( 1987). Constitutive expression of a gene encoding a polypeptide homologous to

biologically active human platelet protein in Rous sarcoma virus-transformed
fibroblasts. Proc. Natl. Acad. Sci. USA 84, 6715-6719.

Fisher, J., Wu, L., Denison, M., and Whitlock, J. (1990). Organization and
function of a dioxin-responsive enhancer. J. Biol. Chem. 265, 9676-9681.

179.

180.

181.

182.

183.

184.

185.

186.

187.

188.

189.

155

Shen, B.S., and Whitlock, J.P. (1992). Protein-DNA interactions at a dioxin-
responsive enhancer. J. Biol. Chem. 267, 6815-6819.

Pendurthi, U.R., Okino, S.T., and Tukey, RH. (1993). Accumulation of the
Nuclear Dioxin (Ah) Receptor and Transcripitonal Activation of the Mouse
Cypla-l and Cypla-2 Genes. Arch. Biochem. Biophys. 306, 65-69.

Handler, J.A., Danilowicz, R.M., and Eling, T. (1990). Mitogenic signaling by
epidermal growth factor (EGF), but not platelet-derived growth factor, requires
arachidonic acid metabolism in BALB/c 3T3 cells. J. Biol. Chem. 265, 3669-
3673.

Ye, R.D., Wun, T.C., and Sadler, J .E. (1987). cDNA cloning and expression in
Esherichia coli of a plasminogen activator inhibitor from human placenta. J. Biol.
Chem. 262, 3718-3725.

Schleuning, W.D., Medcalf, R.L., Hession, C., Rothenbuhler, R., Shaw, A.,
and Kruithof, E.K. (1987). Plasminogen activator inhibitor 2: regulation of gene
transcription during phorbol ester-mediated differentiation of U937 human
histiocytic lymphoma cells. Mol. Cell. Biol. 7, 4564-4567.

Nishida, T., Nishino, N., Takano, M., Kawai, K., Bando, K., Masui, Y., Nakai,
S., and Hirai, Y. (1987). cDNA cloning of IL-la and IL-lB from mRNA of
U937 cells. Biochem. Biophys. Res. Commun. 143, 345-352.

Ushikubi, F., Aiba, Y., Nakamura, K., Namba, T., Hirata, M., Mazda, 0.,
Katsura, Y., and Narumiya, S. (1993). Thromboxane A2 receptor is highly
expressed in mouse immature thymocytes and mediates DNA fragmentation and
apoptosis. J. Exp. Med. 178, 1825-1830.

Lundberg, K., Gronvik, K.O., Goldschmidt, T.J., Klareskog, L., and Dencker,
L. (1990). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters intrathymic T-cell
development in mice. Chem. Biol. Interact. 74, 179-193.

Lundberg , K. (1991). Dexamethasone and 2 , 3 ,7, 8-tetrachlorodibenzo-p-dioxin can
induce thymic atrophy by different mechanisms in mice. Biochem. Biophys. Res.
Commun. 178, 16-23.

Kerkvliet, N.I., and Brauner, LA. (1990). Flow cytometric analysis of
lymphocyte subpopulations in the spleen and thymus of mice exposed to an acute
immunosuppressive dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
Environ. Res. 52, 146-154.

Nagarkatti, P.S., Sweeney, G.D., Gauldie, J., and Clark, DA. (1984).
Sensitivity to suppression of cytotoxic T cell generation by 2,3,7,8-

190.

191.

192.

193.

194.

195.

196.

197.

156

tetrachlorodibenzo-p-dioxin (TCDD) is dependent on the Ah genotype of the
murine host. Toxicol. Appl. Pharmacol. 72, 169-176.

Cook, J.C., Dold, K.M., and Greenlee, W.F. (1987). An in vitro model for
studying the toxicity of 2,3,7 ,8-tetrachlorodibenzo-p-dioxin to human thymus.
Toxicol. Appl. Pharmacol. 89, 256-268.

Habenicht, A.J.R., Dresel, H.A., Goerig, M., Weber, Joerg-A., Stoehr, M.,
Glomset, J.A., Ross, R., and Schettler, G. (1986). Low density lipoprotein
receptor-dependent prostaglandin synthesis in swiss 3T3 cells stimulated by
platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 83, 1344-1348.

Raz, A., Wyche, A., Fagan, D., and Needleman, P. (1989). The cell biology of
Fibroblast Cyclooxygenase. In "Advancces in Experimental Medicine and
Biology”: "Renal Eicosanoids" (M.J. Dunn, C. Patrono, and G.A. Cinotti, Eds.),
Vol. 259, pp. 1-21. Plenum Press, New York.

Oshima, T. , Yoshimoto, T., Yamamoto, S., Kumegawa, M. , Yokoyama, C. , and
Tanabe, T. (1991). CAMP-dependent induction of fatty acid cyclooxygenase
mRNA in mouse osteoblastic cells (MC3T3-E1). J. Biol. Chem. 266, 13621-
13626.

Hla, T., and Maciag, T. (1991). Cyclooxygenase Gene Expression is down-
regulated by Heparin-binding (Acidic Fibroblast) Growth Factor-1 in human
endothelial cells. J. Biol. Chem. 266, 24059-24063.

Simonson, M.S., Wolfe, J.A., Konieczkowski, M., Sedor, J .r, and Dunn, M.J.
(1991). Regulation of prostaglandin endoperoxide synthase gene expression in
cultured rat mesangial cells: induction by serum via a protein kinase-C-dependent
mechanism. Mol. Endocrinol. 5, 441-451.

Wu, K.K., Sanduja, R., Tsai, A., Ferhanoglu, B., and Loose-Mitchell, BS.
(1991). Asprin inhibits interleukin l-induced prostaglandin H synthase expression

_ in cultured endothelial cells. Proc. Natl. Acad. Sci. USA 88, 2384-2387.

Xu, X., Ohashi, K., Sanduja, S.K., Ruan, K., Wang, L., and Wu, K.K. (1993).
Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated
transfer of prostglandin H synthase cDNA. J. Clin. Invest. 91, 1843-1849.