Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.

Anteby
E. Y.
,
Johnson
R. D.
,
Huang
X.
,
Dryden
D. K.
,
Nelson
D. M.
,
Sadovsky
Y.
(
1998
).
Lipopolysaccharide enhances the transcription of prostaglandin H synthase-2 gene in primary human trophoblasts.
Am. J. Obstet. Gynecol
178
,
469
473
Baeuerle
P. A.
,
Baichwal
V. R.
(
1997
).
NF-B as a frequent target for immunosuppressive and anti-inflammatory molecules.
Advan. Immunol
65
,
111
137
Baeuerle
P. A.
(
1998
).
IB-NF- B structures: at the interface of inflammation control [comment].
Cell
95
,
729
731
Bredt
D. S.
,
Hwang
P. M.
,
Glatt
C. E.
,
Lowenstein
C.
,
Reed
R. R.
,
Snyder
S. H.
(
1991
).
Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.
Nature
351
,
714
718
Callejas
N. A.
,
Castrillo
A.
,
Bosca
L.
,
Martin-Sanz
P.
(
1999
).
Inhibition of prostaglandin synthesis up-regulates cyclooxygenase-2 induced by lipopolysaccharide and peroxisomal proliferators.
J. Pharmacol. Exp. Ther
288
,
1235
1241
Carbo
N.
,
Lopez-Soriano
F. J.
,
Fiers
W.
,
Argiles
J. M.
(
1996
).
Tumour growth results in changes in placental amino acid transport in the rat: a tumour necrosis factor alpha-mediated effect.
Biochem. J
313
,
77
82
Casado
M.
,
Diaz-Guerra
M. J. M.
,
Rodrigo
J.
,
Fernandez
A. P.
,
Bosca
L.
,
Martin-Sanz
P.
(
1997
).
Expression of the calcium-independent cytokine-inducible (iNOS) isoform of nitric oxide synthase in rat placenta.
Biochem. J
324
,
201
207
Chomczynski
P.
,
Sacchi
N.
(
1987
).
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Colville-Nash
P. R.
,
Qureshi
S. S.
,
Willis
D.
,
Willoughby
D. A.
(
1998
).
Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1.
J. Immunol
161
,
978
984
Conrad
K. P.
,
Vill
M.
,
McGuire
P. G.
,
Dail
W. G.
,
Davis
A. K.
(
1993
).
Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi.
FASEB J
7
,
1269
1276
Crofford
L. J.
(
1997
).
COX-1 and COX-2 tissue expression: implications and predictions.
J. Rheumatol
24
,
15
19
DeWitt
D. L.
(
1991
).
Prostaglandin endoperoxide synthase: regulation of enzyme expression.
Biochim. Biophys. Acta
1083
,
121
134
Diaz-Guerra
M. J. M.
,
Velasco
M.
,
Martin-Sanz
P.
,
Bosca
L.
(
1996
).
Evidence for common mechanisms in the transcriptional control of type II nitric oxide synthase in isolated hepatocytes. Requirement of NF-B activation after stimulation with bacterial cell wall products and phorbol esters.
J. Biol. Chem
271
,
30114
30120
Epling
C. L.
,
Stites
D. P.
,
McHugh
T. M.
,
Chong
H. O.
,
Blackwood
L. L.
,
Wara
D. W.
(
1992
).
Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.
Cytometry
13
,
615
620
Feng
L.
,
Sun
W.
,
Xia
Y.
,
Tang
W. W.
,
Chanmugam
P.
,
Soyoola
E.
,
Wilson
C. B.
,
Hwang
D.
(
1993
).
Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.
Arch. Biochem. Biophys
307
,
361
368
Fletcher
B. S.
,
Kujubu
D. A.
,
Perrin
D. M.
,
Herschman
H. R.
(
1992
).
Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase.
J. Biol. Chem
267
,
4338
4344
Galea
E.
,
Reis
D. J.
,
Xu
H.
,
Feinstein
D. L.
(
1995
).
Transientexpression of calcium-independent nitric oxide synthase in blood vessels during brain development.
FASEB J
9
,
1632
1637
Ghosh
S.
,
May
M. J.
,
Kopp
E. B.
(
1998
).
NF-B and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu. Rev. Immunol
16
,
225
260
Herschman
H.
,
Gilbert
R.
,
Reddy
S.
,
Xie
W. L.
(
1997
).
Coordinate regulation of the inducible forms of prostaglandin synthase and nitric oxide synthase in fibroblasts and macrophages.
Advan. Exp. Med. Biol
400
,
177
182
Hortelano
S.
,
Genaro
A. M.
,
Bosca
L.
(
1992
).
Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide.
J. Biol. Chem
267
,
24937
24940
Imseis
H. M.
,
Zimmerman
P. D.
,
Samuels
P.
,
Kniss
D. A.
(
1997
).
Tumour necrosis factor-alpha induces cyclo-oxygenase-2 gene expression in first trimester trophoblasts: suppression by glucocorticoids and NSAIDs.
Placenta
18
,
521
526
Inoue
H.
,
Yokoyama
C.
,
Hara
S.
,
Tone
Y.
,
Tanabe
T.
(
1995
).
Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.
J. Biol. Chem
270
,
24965
24971
Janssens
S. P.
,
Simouchi
A.
,
Quertermous
T.
,
Bloch
D. B.
,
Bloch
K. D.
(
1992
).
Cloning and expression of a cDNA encoding human endothelium-derived relating factor/nitric oxide synthase.
J. Biol. Chem
267
,
22694
–.
Kishi
K.
,
Itoh
M.
,
Kanamori
S.
,
Hirashiba
M.
,
Kawai
M.
(
1993
).
Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-II secretion in vitro.
J. Reprod. Fertil
99
,
519
527
Kujubu
D. A.
,
Fletcher
B. S.
,
Varnum
B. C.
,
Lim
R. W.
,
Herschman
H. R.
(
1991
).
TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue.
J. Biol. Chem
266
,
12866
12872
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lamas
S.
,
Marsden
P. A.
,
Li
G. K.
,
Tempst
P.
,
Michel
T.
(
1992
).
Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform.
Proc. Nat. Acad. Sci. USA
89
,
6348
6352
Lim
H.
,
Paria
B. C.
,
Das
S. K.
,
Dinchuk
J. E.
,
Langenbach
R.
,
Trzaskos
J. M.
,
Dey
S. K.
(
1997
).
Multiple female reproductive failures in cyclooxygenase 2-deficient mice.
Cell
91
,
197
208
Lowenstein
C. J.
,
Alley
E. W.
,
Raval
P.
,
Snowman
A. M.
,
Snyder
S. H.
,
Russell
S. W.
,
Murphy
W. J.
(
1993
).
Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide.
Proc. Nat. Acad. Sci. USA
90
,
9730
9734
Lyons
C. R.
,
Orloff
G. J.
,
Cunningham
J. M.
(
1992
).
Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.
J. Biol. Chem
267
,
6370
6374
Majerus
P. W.
(
1998
).
Prostaglandins: critical roles in pregnancy and colon cancer.
Curr. Biol
8
,
87
–.
Martin-Sanz
P.
,
Cascales
M.
,
Bosca
L.
(
1989
).
Glucagon-induced changes in fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase in cultured rat foetal hepatocytes.
Biochem. J
257
,
795
799
Mirochnitchenko
O.
,
Inouye
M.
(
1996
).
Effect of overexpression of human Cu,Zn superoxide dismutase in transgenic mice on macrophage functions.
J. Immunol
156
,
1578
1586
Molnar
M.
,
Suto
T.
,
Toth
T.
,
Hertelendy
F.
(
1994
).
Prolonged blockade of nitric oxide synthesis in gravid rats produces sustained hypertension, proteinuria, thrombocytopenia and intrauterine growth retardation.
Am. J. Obstet. Gynecol
170
,
1458
1466
Moncada
S.
,
Palmer
R. M.
,
Higgs
E. A.
(
1991
).
Nitric oxide: physiology, pathophysiology and pharmacology.
Pharmacol. Rev.
43
,
109
142
Morris
J. K.
,
Richards
J. S.
(
1996
).
An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells.
J. Biol. Chem
271
,
16633
16643
Nguyen
M.
,
Camenisch
T.
,
Snouwaert
J. N.
,
Hicks
E.
,
Coffman
T. M.
,
Anderson
P. A.
,
Malouf
N. N.
,
Koller
B. H.
(
1997
).
The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth.
Nature
390
,
78
81
Nosseri
C.
,
Coppola
S.
,
Ghibelli
L.
(
1994
).
Possible involvement of poly(ADP-ribosyl) polymerase in triggering stress-induced apoptosis.
Exp. Cell. Res
212
,
367
373
Pilbeam
C. C.
,
Kawaguchi
H.
,
Hakeda
Y.
,
Voznesensky
O.
,
Alander
C. B.
,
Raisz
L. G.
(
1993
).
Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells.
J. Biol. Chem
268
,
25643
25649
Royall
J. A.
,
Ischiropoulos
H.
(
1993
).
Evaluation of 2,7 -dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2in cultured endothelial cells.
Arch. Biochem. Biophys
302
,
348
355
Salvemini
D.
,
Settle
S. L.
,
Masferrer
J. L.
,
Seibert
K.
,
Currie
M. G.
,
Needleman
P.
(
1995
).
Regulation of prostaglandin production by nitric oxide; an in vivo analysis.
Br. J. Pharmacol
114
,
1171
1178
Schreiber
E.
,
Matthias
P.
,
Muller
M. M.
,
Schaffner
W.
(
1989
).
Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells.
Nucl. Acids Res
17
,
6419
–.
Spink
J.
,
Cohen
J.
,
Evans
T. J.
(
1995
).
The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. Activation by nuclear factor-B.
J. Biol. Chem
270
,
29541
29547
Sugimoto
Y.
,
Yamasaki
A.
,
Segi
E.
,
Tsuboi
K.
,
Aze
Y.
,
Nishimura
T.
,
Oida
H.
,
Yoshida
N.
,
Tanaka
T.
,
Katsuyama
M.
,
Hasumoto
K.
,
Murata
T.
,
Hirata
M.
,
Ushikubi
F.
,
Negishi
M.
,
Ichikawa
A.
,
Narumiya
S.
(
1997
).
Failure of parturition in mice lacking the prostaglandin F receptor.
Science
277
,
681
683
Swierkosz
T. A.
,
Mitchell
J. A.
,
Warner
T. D.
,
Botting
R. M.
,
Vane
J. R.
(
1995
).
Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids.
Br. J. Pharmacol
114
,
1335
1342
Tawa
N. E. J.
,
Odessey
R.
,
Goldberg
A. L.
(
1997
).
Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.
J. Clin. Invest
100
,
197
203
Terenzi
F.
,
Diaz-Guerra
M. J.
,
Casado
M.
,
Hortelano
S.
,
Leoni
S.
,
Bosca
L.
(
1995
).
Bacterial lipopeptides induce nitric oxide synthase and promote apoptosis through nitric oxide-independent pathways in rat macrophages.
J. Biol. Chem
270
,
6017
6021
Wetzka
B.
,
Nusing
R.
,
Charnock-Jones
D. S.
,
Schafer
W.
,
Zahradnik
H. P.
,
Smith
S. K.
(
1997
).
Cyclooxygenase-1 and-2 in human placenta and placental bed after normal and pre-eclamptic pregnancies.
Human Reprod
12
,
2313
2320
Wunsch
D. M.
,
Anderson
L. D.
,
Pepe
G. J.
,
Albrecht
E. D.
(
1986
).
Regulation of progesterone formation by human placental cells in culture.
Endocrinology
119
,
998
1003
Xie
Q. W.
,
Whisnant
R.
,
Nathan
C.
(
1993
).
Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.
J. Exp. Med
177
,
1779
1784
Xie
Q.
,
Nathan
C.
(
1994
).
The high-output nitric oxide pathway: role and regulation.
J. Leuk. Biol
56
,
576
582
Xie
W. L.
,
Chipman
J. G.
,
Robertson
D. L.
,
Erikson
R. L.
,
Simmons
D. L.
(
1991
).
Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.
Proc. Nat. Acad. Sci. USA
88
,
2692
2696
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