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-Induced Cyclooxygenase-2 Expression in Human Lung Epithelial Cells: Involvement of the Phospholipase C-
2, Protein Kinase C-, Tyrosine Kinase, NF-
B-Inducing Kinase, and I-B Kinase 1/2 Pathway1
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induced a dose- and time-dependent increase in
cyclooxygenase-2 (COX-2) expression and PGE2 formation in
human NCI-H292 epithelial cells. Immunofluorescence staining
demonstrated that COX-2 was expressed in cytosol and nuclear envelope.
Tyrosine kinase inhibitors (genistein or herbimycin) or
phosphoinositide-specific phospholipase C inhibitor (U73122) blocked
TNF--induced COX-2 expression. TNF- also stimulated
phosphatidylinositol hydrolysis and protein kinase C (PKC) activity,
and both were abolished by genistein or U73122. The PKC inhibitor,
staurosporine, also inhibited TNF--induced response. The
12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC
activator, also stimulated COX-2 expression, this effect being
inhibited by genistein or herbimycin. NF-
B DNA-protein binding and
COX-2 promoter activity were enhanced by TNF-, and these effects
were inhibited by genistein, U73122, staurosporine, or pyrolidine
dithiocarbamate. TPA stimulated both NF-B DNA-protein binding and
COX-2 promoter activity, these effects being inhibited by genistein,
herbimycin, or pyrolidine dithiocarbamate. The TNF--induced, but not
the TPA-induced, COX-2 promoter activity was inhibited by phospholipase
C-
2 mutants, and the COX-2 promoter activity induced by either agent
was attenuated by dominant-negative mutants of PKC-,
NF-B-inducing kinase, or I-B (inhibitory protein that dissociates
from NF-B) kinase (IKK)1 or 2. IKK activity was stimulated by both
TNF- and TPA, and these effects were inhibited by staurosporine or
herbimycin. These results suggest that, in NCI-H292 epithelial cells,
TNF- might activate phospholipase C-2 via an upstream tyrosine
kinase to induce activation of PKC- and protein tyrosine kinase,
resulting in the activation of NF-B-inducing kinase and IKK1/2, and
NF-B in the COX-2 promoter, then initiation of COX-2 expression and
PGE2 release. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of COX-2 expression requires de novo mRNA and protein
synthesis (7), indicating regulation at the
transcriptional level. Studies on the transcriptional regulation of
COX-2 genes have led to the identification of a number of
transcriptional factors that are mediated through specific
cis-acting elements. Transcriptional activation in response
to extracellular signals involves the regulated assembly of
multiprotein complexes on enhancers and promoters (9). In
the human COX-2 gene, the nucleotide sequence of the 5'-flanking region
contains a canonical TATA box and the consensus sequences of the
NF-B, NF-IL-6 (CCAAT/enhancer-binding protein ß; C/EBPß), and
cAMP response element (CRE) sites in the 275-bp region upstream of the
transcriptional start site (10). Sequences homologous to
the consensus sequences of the NF-B, NF-IL-6, and CRE sites are also
found in the corresponding regions of the mouse (11, 12)
and rat (13) COX-2 gene. The NF-B consensus sites are
found in the mouse COX-2 promoter region and are important in COX-2
mRNA induction by TNF- (12). CRE and C/EBPß act as
positive regulatory elements for COX-2 transcription
(14, 15, 16).
TNF- is a pleiotropic cytokine, primarily produced by activated
macrophages and some T lymphocytes, that is involved in a wide range of
biological effects, including inflammation, mitogenesis,
differentiation, immune modulation, and antitumor immunity
(17). These activities result from interaction with
specific cell surface receptors expressed in most cell types. Two types
of TNF receptor, TNFR55 and TNFR75, with respective molecular masses of
55 and 75 kDa, have been characterized (18). TNF- is
synthesized as a 26-kDa type II transmembrane proform, which is then
processed by a metalloprotease, resulting in the release of mature
TNF-, consisting of three 17-kDa subunits (19, 20);
this trimeric ligand can bind to TNFR55 or TNFR75, thereby engaging
three receptor chains. Although TNF- has a higher affinity for
TNFR75 than for TNFR55, most of the biological responses of TNF- are
thought to be mediated through TNFR55 (18). Ligand-bound
trimeric TNFR55 recruits TNFR-associated proteins (e.g.,
TNFR-associated death domain protein, Fas-associated death domain
protein, and TNFR-associated factor 2) to its intracytoplasmic domain
to generate intracellular signaling cascades that act via second
messengers, including ceramide, diacylglycerol (DAG), and arachidonic
acid metabolites (21). Although, in the main, not well
understood, certain intracellular signaling pathways by which TNF-
induces COX-2 expression have been proposed; these include the
activation of tyrosine kinase (22) and protein kinase C
(PKC) (23). However, the relationship between these
pathways is unknown. In the present study, we explored the
intracellular signaling pathway involved in TNF--induced COX-2
expression in a human alveolar epithelial cell line, NCI-H292. The
results show that TNF- might activate phospholipase C-2
(PLC-2) via an upstream tyrosine kinase, resulting in activation of
PKC-, protein tyrosine kinase, NF-B-inducing kinase (NIK), I-B
kinase (IKK) 1/2, and NF-B in the COX-2 promoter, which is followed
by COX-2 expression and PGE2 release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Goat polyclonal Abs specific for COX-1 or COX-2 or rabbit
polyclonal Abs specific for the p65, p50, or p52 subunit of NF-B or
for I-B- or IKKß were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Human rTNF- was purchased from R&D Systems
(Minneapolis, MN). RPMI 1640 medium, FCS, penicillin, and streptomycin
were obtained from Life Technologies (Gaithersburg, MD). Staurosporine,
pyrolidine dithiocarbamate (PDTC), and histone III-S were obtained from
Sigma (St. Louis, MO). D609, U73122, U73343, genistein, and herbimycin
were obtained from Calbiochem (San Diego, CA). T4 polynucleotide kinase
was obtained from New England Biolabs (Beverly, MA). Poly(dI-dC), the
PGE2 enzyme immunoassay kit, HRP-labeled donkey
anti-rabbit second Ab, and the enhanced chemiluminescence (ECL)
detecting reagent were obtained from Pharmacia Biotech (Uppsala,
Sweden). Myo-[3H]inositol (23.5 Ci/mmol) and
[-32P]ATP (3000 Ci/mmol) were obtained from
DuPont-New England Nuclear (Boston, MA). FITC-conjugated and
HRP-conjugated rabbit anti-goat IgG Abs were obtained from Cappel
(Aurora, OH). Tfx-50 and the luciferase assay kit were obtained from
Promega (Madison, WI).
Cell culture
NCI-H292 cells, a human alveolar epithelial cell carcinoma, were
obtained from American Type Culture Collection (Manassas, VA) and
cultured in RPMI 1640 supplemented with 10% FCS, 100 U/ml of
penicillin, and 100 µg/ml of streptomycin in six-well plates for
experiments involving COX-2 expression, phosphatidylinositol (PI)
hydrolysis assay, and transfection on 24-mm glass coverslips in 35-mm
dishes for COX-2 immunofluorescence studies, in 6-cm dishes for PKC
activity measurements, or in 10-cm dishes for the NF-B gel-shift
assay.
Plasmids
The COX-2 promoter construct (-459/+9) was a generous gift from
Dr. L. H. Wang (University of Texas, Houston, TX). The PLC-2
mutants were gifts from Dr. T. Kurosaki (Kansai Medical University,
Moriguchi, Japan) (the mutants SH2(N) and SH2(C) in which
Arg564 or Arg672 are
replaced, respectively, by Ala). The dominant-negative mutants were
provided by Dr. A. Altman (La Jolla Institute for Allergy and
Immunology, San Diego, CA) (the PKC- mutant, PKC-/KR), Dr. M.
Karin (University of California, San Diego, CA) (the NIK mutant KKAA),
and Signal Pharmaceutical (San Diego, CA) (the IKK1 mutant KM and the
IKK2 mutant KM). pGEX-I-B1–100(1–100) was a gift from Dr. Nakano
(University of Junteudo, Tokyo, Japan).
Preparation of cell extracts and Western blot analysis of COX-2 and COX-1
Following treatment with TNF- or
12-O-tetradecanoylphorbol 13-acetate (TPA), the cells were
harvested and cell lysates were prepared and subjected to SDS-PAGE
using 7.5% running gels, as described previously (24).
The proteins were transferred to a nitrocellulose membrane, which was
then incubated successively at room temperature with 0.1% milk in TTBS
(50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween-20) for 1
h, with goat Ab specific for COX-2 or COX-1 for 1 h, and with
HRP-labeled anti-goat Ab for 30 min. After each incubation, the
membrane was washed extensively with TTBS. The immunoreactive band was
detected using ECL detection reagent and developed with Hyperfilm-ECL
(Pharmacia Biotech). Quantitative data were obtained using a computing
densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale,
CA). In pretreatment experiments, cells were incubated for 30 min at
37°C with the tyrosine kinase inhibitors, genistein and herbimycin,
the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor,
U73122, or the PKC inhibitor, staurosporine, before addition of
TNF- for 16 h. At the concentrations used, these inhibitors had
no cytotoxic effect on NCI-H292 cells.
Immunofluorescence staining
NCI-H292 cells grown on coverslips were treated for 16 h
with TNF- or TPA in growth medium, rapidly washed with PBS, then
fixed at room temperature for 10 min with 3.7% paraformaldehyde. After
washing with PBS, the cells were blocked for 15 min with 1% BSA in
TTBS containing 0.1% Triton X-100, then incubated with anti-COX-2
Ab (1:100) for 1 h, washed extensively, and stained for 30 min
with anti-goat IgG-fluorescein (1:200). After further washes, the
coverslips were mounted on glass slides using mounting medium (2%
n-propyl gallate in 60% glycerol, 0.1 M PBS, pH 8).
Optical sections of the immunostained cells were observed and
photographed using a Zeiss Axiovert inverted microscope equipped with a
photoMicroGraph Digitized Integration System (Zeiss, Oberkochen,
Germany).
Measurement of PI hydrolysis
PI hydrolysis was assessed by measuring the accumulation of
[3H]inositol phosphates (IP) in cells labeled
by 24-h incubation in growth medium containing
myo-[3H]inositol (2.5 µCi/ml), as previously
described (25). After incubation, the cells were washed
with physiologic salt solution (PSS: 118 mM NaCl, 4.7 mM KCl, 2.5 mM
CaCl2, 1.2 mM MgCl2, 1.2 mM
KH2PO4, 11 mM glucose, and 20 mM HEPES, pH 7.4) containing 10 mM LiCl
and incubated at 37°C for 20 min. Pretreatment with 10 µM U73122 or
U73343 or 30 µM genistein was performed by adding the reagent to the
PSS 30 min before stimulation with 30 ng/ml of TNF- for 10
min.
PKC activity assay
Cells treated with TNF- for 10 min, 1 h, or 2 h
were harvested, and membrane fractions were prepared and assayed for
PKC activity, as previously described (26); the assay was
performed at 30°C for 5 min in 25 µl of 30 mM Tris-HCl buffer, pH
7.5, containing 6 mM magnesium acetate, 0.12 mM
[-32P]ATP, 0.4 mM
CaCl2, 40 µg/ml of phosphatidylserine, 8
µg/ml of 1,2-dioleoylglycerol, 1 mg/ml of histone III-S, and the
membrane preparation (2.5–5 µg protein). The
Ca2+- and phospholipid-independent activity was
measured under the same conditions in the absence of
Ca2+ and phospholipid and in the presence of 2
mM EGTA.
Preparation of nuclear extracts and the EMSA
Control cells, or cells pretreated with various inhibitors, were
treated with TNF- or TPA for 1 h, then nuclear extracts were
isolated as described previously (24). Briefly, cells were
washed with ice-cold PBS and pelleted. The cell pellet was resuspended
in hypotonic buffer (10 mM HEPES. pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 mM NaF, and 1 mM NaVO4) and incubated
for 15 min on ice before being lysed by the addition of 0.5% Nonidet
P-40, followed by vigorous vortexing for 10 s. The nuclei were
pelleted and resuspended in extraction buffer (20 mM HEPES, pH 7.9, 400
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM NaF,
and 1 mM Na3VO4), then the
tube was vigorously shaken at 4°C for 15 min on a shaking platform.
The nuclear extracts were then centrifuged, and the supernatants were
aliquoted and stored at -80°C.
Oligonucleotides corresponding to the NF-B consensus sequences
in the human COX-2 promoter (underlined:
5'-AGAGTGGGGACTACCCCCTCT-3') were synthesized,
annealed, and end labeled with [-32P]ATP
using T4 polynucleotide kinase. The nuclear extract (6–10 µg) was
incubated at 30°C for 20 min with 1 ng of the
32P-labeled NF-B probe (40,000–60,000 cpm) in
10 µl of binding buffer (1 µg of poly(dI-dC), 15 mM HEPES, pH 7.6,
80 mM NaCl, 1 mM EGTA, 1 mM DTT, and 10% glycerol), as described
previously (24). DNA/nuclear protein complexes were
separated from the DNA probe by electrophoresis on a native 6%
polyacrylamide gel, then the gel was vacuum dried and subjected to
autoradiography using an intensifying screen at -80°C. When
supershift assays were performed, polyclonal Abs specific for p65,
p50, or p52 were added to the nuclear extracts 30 min before
the binding reaction, and the DNA/nuclear protein complexes were
separated on a 4.5% polyacrylamide gel.
In NF-B (p65) translocation studies, both cytosolic and nuclear
extracts were used, while only cytosolic extracts were used in
I-B- degradation studies. The extracts were subjected to SDS-PAGE
using a 10% running gel, and immunoblot analysis was performed as
described above.
Determination of PGE2 concentrations
Following treatment of cells with TNF- for 4, 8, 16, or
24 h, PGE2 levels in the culture medium were
measured using an enzyme immunoassay kit from Amersham Pharmacia
Biotech (Piscataway, NJ).
Transient transfection and luciferase assay
NCI-H292 cells, grown in six-well plates, were transfected with
the human COX-2 firefly luciferase (LUC) plasmid, pGS459 (-459/+9),
using Tfx-50 (Promega), according to the manufacturer’s
recommendations. Briefly, reporter DNA (0.4 µg) and ß-galactosidase
DNA (0.1 µg) were mixed with 2.25 µl of Tfx-50 in 1 ml of
serum-free RPMI 1640. The plasmid pRK containing the ß-galactosidase
gene driven by the constitutively active SV40 promoter was used to
normalize the transfection efficiency. After 10–15-min incubation at
room temperature, the mixture was applied to the cells. One hour later,
1 ml of RPMI 1640 containing 20% FCS was added; from this point, the
cells were grown in medium containing 10% FCS. The following day,
cells were exposed to 30 ng/ml of TNF- or 1 µM TPA for 6 h,
then cell extracts were prepared and luciferase (Promega), and
ß-galactosidase activity were measured. The luciferase activity of
each well was normalized to the ß-galactosidase activity. In
dominant-negative mutant experiments, cells were cotransfected with
reporter and ß-galactosidase and either the PLC-2 mutant or
dominant-negative PKC-, NIK, IKK1, or IKK2 mutant or the empty
vector.
In vitro IKK activity assay
Following 10-min treatment with TNF- or TPA or 30-min
pretreatment with staurosporine or herbimycin before addition of
TNF- or TPA, cells were rapidly washed with PBS, then lysed with
ice-cold lysis buffer, as described above, and the IKK proteins
immunoprecipitated. Fifty micrograms of total cell extract were
incubated for 1 h at 4°C with 0.5 µg of anti-IKKß
Ab, and the Ab-bound protein was collected using protein A-Sepharose
CL-4B beads (Sigma). The beads were then washed three times with lysis
buffer without Triton X-100 and incubated for 30 min at 30°C in 20
µl of kinase reaction mixture containing 20 mM HEPES, pH 7.4, 5 mM
MgCl2, 5 mM MnCl2, 0.1 mM
Na3VO4, 1 mM DTT, 1 µg of bacterially expressed
GST-I-B1–100(1–100), and 10 µM
[-32P]ATP. The reaction was stopped by the
addition of Laemmli buffer and subjected to 10% SDS-PAGE,
phosphorylated-GST-I-B1–100(1–100) being visualized by
autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced COX-2 expression in NCI-H292 cells
When the cells were exposed to 10 ng/ml of IL-1ß, TNF-, or
IFN-, 1 µg/ml of LPS, or 1 µM TPA, only TNF- and TPA
stimulated COX-2 expression (data not shown). TNF- induced COX-2
expression in a concentration- and time-dependent manner. For an
exposure period of 16 h, maximum COX-2 expression was obtained
using 30 ng/ml of TNF- (Fig. 1
),
while, when cells were treated with 30 ng/ml of TNF- for various
times, COX-2 expression was significant at 4 h and maximal at
16 h, remaining at this level for up to at least 24 h. COX-1
expression was not affected by these treatments (Fig. 1). Fig. 2 shows the concentration- and
time-dependent production of PGE2 in response to
TNF-. The basal release of PGE2 was 61.6
pg/µg of total protein. After 16-h treatment with 1, 3, 10, or 30
ng/ml of TNF-, this rose to 140, 194, 274, or 323 pg/µg protein,
respectively (Fig. 2A), while, after treatment with 30 ng/ml
of TNF- for 4, 8, 12, or 24 h, PGE2
release was 81, 102, 279, or 475 pg/µg protein, respectively (Fig. 2B). NS-398 (COX-2 inhibitor) (10 µM) (27)
attenuated the PGE2 release induced by 16-h
treatment with 30 ng/ml of TNF- (323 pg/µg protein) to 102 pg/µg
protein. Induction of COX-2 by TNF- was further demonstrated by
immunofluorescence staining. As shown in Fig. 3, no COX-2 expression was seen in the
basal state (Fig. 3B), but was apparent in the cytosol and
nuclear envelope following TNF- treatment (Fig. 3D). In
the following COX-2 expression experiments, the cells were treated with
30 ng/ml of TNF- for 16 h; under these conditions, both
transcriptional (actinomycin D) and translational (cycloheximide)
inhibitors blocked the TNF--induced COX-2 expression (data not
shown).
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-induced COX-2 expression and activation of PKC by TNF-
To study the intracellular signaling pathway involved in
TNF--induced COX-2 expression, the tyrosine kinase inhibitors,
genistein and herbimycin (28, 29), were used. When cells
were pretreated for 30 min with 10–100 µM genistein or 1 µM
herbimycin, TNF--induced COX-2 expression was inhibited. The extent
of inhibition seen using 10, 50, or 100 µM genistein was 49%, 93%,
or 97%, respectively, while that using 1 µM herbimycin was 87%
(Fig. 4A). When cells were
pretreated with 10 µM U73122 (a PI-PLC inhibitor) (30),
TNF--induced COX-2 expression was inhibited by 86%, while 10 µM
U73343 (an inactive analogue of U73122), 100 µM D609 (a PC-PLC
inhibitor) (31), or 100 µM propranolol (a phosphatidate
phosphohydrolase inhibitor) (32) had no effect (Fig. 4B).
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-induced COX-2 expression was inhibited by U73122,
indicating involvement of the PI-PLC pathway, TNF--induced PI
hydrolysis was measured. Following 10-min treatment of cells with 30
ng/ml of TNF-, a 5.5-fold increase in IP formation was seen; this
effect was less marked after 1 h, although the values were still
higher than basal (Fig. 5A).
The TNF--induced IP formation seen after 10-min treatment was
inhibited by 96% or 94% by 10 µM U73122 or 30 µM genistein,
respectively, while 10 µM U73343 had no effect (Fig. 5B),
indicating that, in NCI-H292 cells, TNF- stimulates the PI-PLC
pathway by activating tyrosine kinase.
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for various times. As shown in
Fig. 6A, membrane PKC activity
increased 3-fold after treatment with TNF- for 10 min; after 2
h, the effect was slightly less, although still higher than basal (Fig. 6A). U73122 (10 µM) or genistein (30 µM) inhibited
the TNF--induced increase in PKC activity by 90% and 83%,
respectively, while 10 µM U73343 had no effect (Fig. 6B).
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was involved in the
regulation of TNF--induced COX-2 expression, staurosporine, a PKC
inhibitor (33), was used. Following pretreatment of cells
with 30 or 100 nM staurosporine, TNF--induced COX-2 expression was
inhibited by 64% or 82%, respectively (Fig. 7). Other PKC inhibitors, such as Ro
31-8220 or calphostin C, were not tested in this study because of
cytotoxicity seen after 16 h of incubation. Because PKC had been
shown to be involved, the effect of direct TPA-mediated PKC activation
on COX-2 expression was examined. TPA (1 µM) also induced a
time-dependent increase in COX-2 expression, which was significant at
4 h, maximal at 16 h, then declined after 24 h (Fig. 8A). COX-1 expression was not
affected by TPA treatment. Induction of COX-2 expression by TPA was
also demonstrated by immunofluorescence staining (Fig. 3
F).
When cells were pretreated with 10–100 µM genistein or 1
µM herbimycin, TPA-induced COX-2 expression was inhibited, the
extent of inhibition being 49%, 79%, or 90% using 10, 30, or 100
µM genistein, respectively, and 81% using 1 µM herbimycin (Fig. 8B). TPA-induced COX-2 expression was inhibited by
staurosporine (data not shown).
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B induction in the nuclei of TNF--stimulated NCI-H292
cells, and inhibition by PDTC, genistein, U73122, or staurosporine
PDTC, an antioxidant, caused dose-dependent inhibition of TNF--
or TPA-induced COX-2 expression and induction of NF-B
DNA-protein-binding activity (Fig. 9; see
Fig. 11, A and E), 100 and 200 µM PDTC,
respectively, inhibiting the TNF--induced COX-2 expression by 26%
or 55% and the TPA response by 69% and 77% (Fig. 9).
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B activation after treatment with TNF- was
examined. Nuclear extracts prepared from NCI-H292 cells were assayed by
EMSA for activated NF-B. In nonstimulated NCI-H292 cells, one faint
NF-B-specific DNA-protein complex was identified. TNF- rapidly
(10 min) activated NF-B, similar activation being seen after 1 and
24 h (Fig. 10A). For
subsequent EMSA experiments, cells were treated with TNF- for 1
h. To identify the specific subunits involved in the formation of the
banding pattern of the NF-B dimer after TNF- stimulation,
supershift assays were performed in the presence of Abs specific for
the p65, p50, or p52 subunit. As shown in Fig. 10B,
incubation with anti-p65 or anti-p50 Abs induced a supershift
(arrows a and b, respectively), but no shift occurred in the presence
of anti-p52 Ab. Thus, our data demonstrate the presence of the
p65/p50 NF-B heterodimer in NCI-H292 cells. To characterize the
proteins involved in NF-B activation, the amount of p65 in cytosolic
and nuclear extracts was assayed by Western blotting. As shown in Fig. 10C, p65 was rapidly (10 min) translocated from the cytosol
to the nuclear compartment in stimulated cells. Because the amount of
NF-B protein released to migrate to the nucleus is thought to be
proportional to the degradation of I-B, the I-B- protein level
in the cytosol was also measured. As shown in Fig. 10C,
TNF- rapidly induced complete degradation of I-B-, but the
level was restored after 1 h of TNF- treatment.
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-elicited activation of
NF-B-specific DNA-protein complex formation was inhibited (Fig. 11, A and B).
Following exposure of cells to 1 µM TPA, rapid activation (10 min) of
NF-B was seen; this was sustained for 24 h (Fig. 11C) and inhibited by 30 µM genistein (Fig. 11D).
Induction of COX-2 promoter activity by TNF- and the inhibitory
effect of PDTC, genistein, herbimycin, U73122, staurosporine, PLC-2
mutants, or dominant-negative mutants of PKC-, NIK, or IKK1/2
To further study the involvement of the PI-PLC-dependent PKC
pathway in TNF--induced COX-2 expression, transient transfections
were performed using the human COX-2 promoter-luciferase construct,
pGS459 (-459/+9) (34). This construct contains both
upstream (-447/-438) and downstream (-223/-214) NF-B sites in
the COX-2 promoter. Treatment with 30 ng/ml TNF- or 1 µM TPA led
to a 3.2- or 5.2-fold increase, respectively, in COX-2 promoter
activity, these effects being inhibited by 64% or 54%, respectively,
by 100 µM PDTC (Fig. 12A).
These data further indicate that NF-B is responsible for mediating
TNF-- or TPA-induced COX-2 expression in NCI-H292 cells. The
TNF--induced COX-2 promoter activity was blocked by genistein,
herbimycin, U73122, or staurosporine, while the TPA-induced activity
was inhibited by genistein or herbimycin (Fig. 12A). In
cotransfection experiments, the induction of COX-2 promoter activity by
TNF- was inhibited by the mutant PLC-2 SH2(N) or PLC-2 SH2(C)
(Fig. 12B), or by the dominant-negative mutants PLC-/KR,
NIK (KKAA), or IKK1/2 (KM) (Fig. 12C), while that induced by
TPA was inhibited by the dominant-negative mutants PKC-/KR, NIK
(KKAA), or IKK1 or 2 (KM), but not by the mutant PLC-2 SH2(N) or
PLC-2 SH2(C) (Fig. 12, B and C).
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or TPA, and inhibitory
effect of herbimycin
The endogenous IKK complex was isolated by immunoprecipitation
with anti-IKKß Ab and tested for in vitro kinase activity. As
shown in Fig. 13, both TNF- and TPA
induced IKK activation, and these effects were inhibited by
staurosporine and herbimycin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced COX-2
expression in NCI-H292 cells and that this occurred in the cytosol and
nuclear envelope, as demonstrated by immunofluorescence staining. The
immunofluorescence results are consistent with previous findings that
COX is detected in the nuclear envelope and endoplasmic reticulum
(35), and with the observation of cytosolic
PLA2 (phosholipase A2 from honey bee
venom) translocation to the nuclear envelope and endoplasmic
reticulum (36, 37). Two NF-B consensus sites are found
in the promoter region of the human COX-2 gene (34), these
being the NF-B-5' site (-447/-438) and the NF-B-3' site
(-223/-214). The NF-B-5' site has been shown to be involved in
TNF--mediated COX-2 induction in a mouse osteoblast cell line
(12). The NF-B-3' site, in concert with the NF-IL-6 and
CRE sites, may play a role in facilitating the induction of COX-2 by
LPS and phorbol ester in bovine aortic endothelial cells
(38). In NCI-H292 cells, TNF- increased the levels of
the NF-B-specific DNA-protein complex in nuclear extracts (Fig. 10A). The NF-B blocker, PDTC, partially inhibited
TNF--induced COX-2 expression and NF-B-specific DNA-protein
binding (Figs. 9 and 11A). Induction of COX-2 promoter
activity by TNF- or TPA was also partially blocked by PDTC (Fig. 12). These results indicate that NF-B is indeed responsible for the
TNF--mediated induction of COX-2 expression in NCI-H292 cells.
However, another transcriptional factor, NF-IL-6, might also be
involved, as reported in MC3T3-E1 cells (12). TNF- did
increase C/EBP-specific DNA-protein binding in NCI-H292 cells (data not
shown). EMSA studies showed rapid activation (10 min) of NF-B in
response to TNF- stimulation (Fig. 10A) and the parallel
translocation of p65 into the nucleus. Complete degradation of
I-B- was also seen (Fig. 10C), as reported in RAW
264.7 cells exposed to LPS (39), and seen in A549 cells
exposed to TNF- or IL-1ß (40). The supershift assay
demonstrated the presence of the p65/p50 NF-B heterodimer in
NCI-H292 cells (Fig. 10B), in contrast to the p50/p50
homodimer and p65/p50 heterodimer found in human hepatic stellate cells
(41).
We demonstrated that the PKC inhibitor, staurosporine, inhibited the
TNF--mediated induction of COX-2 expression in a dose-dependent
manner, indicating that PKC activation is an obligatory event in
TNF--mediated COX-2 expression in these cells. This was further
confirmed by the result that the dominant-negative PKC- mutant,
PKC-/KR, inhibited the TNF--induced COX-2 promoter activity (Fig. 12B). TNF- caused PKC activation, this phenomenon
occurring after 10-min treatment and lasting for 1 h (Fig. 4A). PKC is activated by the physiological activator, DAG,
which can be generated either directly, by the action of PLC, or
indirectly, by a pathway involving the production of phosphatidic acid
by phospholipase D, followed by a dephosphorylation reaction catalyzed
by phosphatidate phosphohydrolase. Normally, the PLC involved in the
production of DAG is PI-PLC, but PC-PLC can also be involved (42, 43). The PI-PLC inhibitor, U73122, inhibited TNF--induced
COX-2 expression, whereas the PC-PLC inhibitor, D609, or the
phosphatidate phosphohydrolase inhibitor, propranolol, did not. The
inactive analogue, U73343, had no effect. TNF- also stimulated PI
hydrolysis, this effect being blocked by U73122, but not by U73343.
Genistein also blocked TNF--induced PI hydrolysis, indicating that
the PI-PLC involved might be PLC-, because PLC- is a SH2
domain-containing protein that utilizes this module to link
phosphotyrosine-containing sequences in a receptor protein or
cytoplasmic protein tyrosine kinase to PI hydrolysis (44).
Structurally, PLC- has a putative pleckstrin homology domain at its
amino terminus, which is followed by the two conserved parts of the
catalytic domain, separated by two tandem SH2 domains and an SH3 domain
(45). Both the N-terminal SH2 (SH2(N)) and the C-terminal
SH2 (SH2(C)) domain mutants of PLC-2 were not functional in surface
IgM-induced apoptosis in B cells lacking wild-type PLC-2
(46). In the present study, despite the existence of
endogenous PLC-2, either mutant inhibited TNF--, but not
TPA-induced COX-2 promoter activity (Fig. 12B), indicating
the possible involvement of PLC-2 in TNF--induced COX-2
expression in NCI-H292 cells. Thus, TNF- might act through the
PI-PLC-2 pathway, but not through the PC-PLC or PC-phospholipase D
pathway, to induce PKC activation in NCI-H292 cells. Genistein blocked
TNF--induced PKC activation (Fig. 6B), again indicating
the requirement for an initial protein tyrosine phosphorylation event
in this PKC activation process.
Because PKC had been shown to be involved in the TNF- effect, direct
activation of PKC by TPA was tested and found to induce COX-2
expression, as shown both by Western blotting (Fig. 8A) and
immunofluorescence staining (Fig. 3F). This TPA-induced
COX-2 expression was inhibited in a dose-dependent manner by genistein
or herbimycin (Fig. 8B), as was the TNF--induced COX-2
expression (Fig. 4A). TPA also stimulated NF-B
DNA-protein binding and COX-2 promoter activity, and these effects were
also inhibited by genistein or herbimycin (Figs. 11D and
12A), as was the TNF--induced activation of NF-B and
COX-2 promoter activity (Figs. 11B and 12A).
These results suggest that protein tyrosine phosphorylation might also
occur downstream of PKC in the induction of NF-B activation. Further
demonstration was discussed below.
In nonstimulated cells, NF-B dimers are present as cytoplasmic
latent complexes due to the binding of specific inhibitors, the
I-Bs, which mask their nuclear localization signal. Upon stimulation
by proinflammatory cytokines, the I-Bs are rapidly
phosphorylated at two conserved NH2-terminal serines, this
posttranslational modification being rapidly followed by their
polyubiquitination and proteasomal degradation (47, 48, 49).
This results in the unmasking of the nuclear localization signal in
NF-B dimers, which is followed by their translocation to the
nucleus, binding to specific DNA sites (B sites), and targeting gene
activation. The protein kinase that phosphorylates I-Bs in response
to proinflammatory stimuli has been identified biochemically and
molecularly (50, 51, 52, 53, 54). Named IKK, it exists as a complex,
termed the IKK signalsome, which is composed of at least three
subunits, IKK1 (IKK), IKK2 (IKKß), and IKK (55).
IKK1 and IKK2 are very similar protein kinases that act as the
catalytic subunits of the complex (50, 51, 52, 53, 54). In mammalian
cells, IKK1 and IKK2 form a stable heterodimer that is tightly
associated with IKK, a regulatory subunit (56). The
IKKs bind NIK (52, 54), a member of the mitogen-activated
protein kinase kinase kinase family that interacts with TNFR-associated
factor 2, thus linking I-B degradation and NF-B activation to the
TNFR complex (57). NIK activates and phosphorylates IKK
in cotransfection experiments, but is unable to phosphorylate IKKß
(58). However, both IKK and IKKß activity are
reported to be regulated by NIK (59). The physiological
function of IKK1 and IKK2 is still unclear. Initially, overexpression
of catalytically inactive forms of IKK1 and IKK2 that blocked IKK and
NF-B activation suggested that both subunits play similar, and
possibly redundant, roles in I-B phosphorylation and NF-B
activation (50). Recent studies have shown that IKK2, and
not IKK1, is the target for proinflammatory stimuli and plays the major
role in IKK activation and induction of NF-B activity (60, 61). However, our results show that TNF--induced COX-2
promoter activity in NCI-H292 epithelial cells was inhibited by the
dominant-negative mutants NIK (KKAA), IKK1 (KM), or IKK2 (KM). This is
consistent with the findings that IKK1 (KM, AA, or KA) and the IKK2
(KM, AA, or KA) mutant inhibit TNF--induced B-dependent
transcription in HeLa and 293 cells (53, 54). TPA-induced
COX-2 promoter activity was also inhibited by the dominant-negative
mutant for NIK, IKK1, or IKK2, indicating that NIK and IKK1/2 are
involved in the downstream of PKC activation in COX-2 expression
induction. IKK activity was stimulated by both TNF- and TPA and
inhibited by staurosporine and herbimycin (Fig. 13), indicating that
tyrosine kinase activation occurs downstream of PKC in IKK activation.
This tyrosine kinase has been demonstrated to be Src family member,
c-Src, or Lyn. Both TNF- and TPA induced c-Src or Lyn activation,
these effects being inhibited by staurosporine and herbimycin
(unpublished data). Wild-type NIK induced COX-2 promoter activity in
NCI-H292 cells, and this effect was not affected by either
staurosporine or herbimycin (data not shown), confirming that NIK was
involved in the downstream of TNF--induced PKC and tyrosine kinase
activation. The present finding of the involvement of IKK1/2 in
PKC-induced COX-2 expression contrasts with the results that PKC
activates IKKß, but not IKK, in 293 cells, and only IKKß is the
target of PKC in T lymphocytes (62, 63).
In summary, the signaling pathway involved in TNF--induced COX-2
expression in human NCI-H292 epithelial cells has been explored.
TNF- might activate PLC-2 via an upstream protein tyrosine kinase
to induce activation of PKC-, protein tyrosine kinase, NIK, IKK1/2,
and NF-B in the COX-2 promoter, followed by initiation of COX-2
expression and PGE2 release.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ching-Chow Chen, Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan.
3 Abbreviations used in this paper: COX, cyclooxygenase; C/EBPß, CCAAT/enhancer-binding protein ß; CRE, cAMP response element; DAG, diacylglycerol; I-B, inhibitory protein that dissociates from NF-B; IKK, I-B kinase; IP, inositol phosphate; NIK, NF-B-inducing kinase; PC-PLC, phosphatidylcholine-specific phospholipase; PDTC, pyrolidine dithiocarbamate; PI, phosphatidylinositol; PI-PLC, phosphoinositide-specific phospholipase C; PKC, protein kinase C; PLC, phospholipase C; TPA, 12-O-tetradecanoylphorbol 13-acetate.
Received for publication December 1, 1999. Accepted for publication June 15, 2000.
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