From the Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two closely related IB
kinases as well as
the upstream kinase, NIK, which integrates interleukin-1
(IL-1
)-
and tumor necrosis factor (TNF)-
-dependent activation of
the transcription factor NF-
B have recently been described. However,
in this emerging pathway the role of previously identified components
of cytokine-induced NF-
B activation, namely
phosphatidylcholine-specific phospholipase C and protein kinase C,
remains unclear. We now show that, in A549 human alveolar epithelial
cells, the activation of a stably transfected
NF-
B-dependent reporter gene by TNF-
and IL-1
is completely blocked by the phosphatidylcholine-specific phospholipase C
inhibitor D609 and the protein kinase C inhibitor RO31-8220. However,
IL-1
-induced I
B
degradation as well as NF-
B nuclear translocation and DNA binding, as determined by Western blot and electro-mobility shift assay, respectively, are not affected by these
inhibitors. A similar effect, although less pronounced, is observed
with the p38 mitogen-activated protein kinase inhibitor SB 203580. On
the basis of these data we propose the existence of a second signaling
pathway induced by IL-1
and TNF-
that is activated in parallel to
the cascade leading to I
B
degradation and is specifically
required for NF-
B-dependent transcriptional competency.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transcription factor nuclear factor-B
(NF-
B)1 plays a key role
in the transcriptional regulation of adhesion molecules, enzymes, and
cytokines involved in chronic inflammatory diseases (reviewed in Ref.
1). In epithelial cells, which play a major role in inflammation,
pro-inflammatory cytokines, such as interleukin (IL)-1
, rapidly
induce NF-
B DNA binding and cause up-regulation of
NF-
B-dependent genes, including cyclooxygenase-2 (2) and inducible nitric-oxide synthase (3). Because the potent
anti-inflammatory effects of glucocorticoids have been linked to a
functional antagonism between the NF-
B subunit p65 and the activated
glucocorticoid receptor (4-6), NF-
B activation pathways have
attracted much attention as potential targets for new anti-inflammatory
strategies.
In resting cells, the inhibitory subunit IB
is bound to the
p50/p65 heterodimer of NF-
B in the cytoplasm. Treatment of cells
with IL-1
or tumor necrosis factor (TNF)-
results in the specific
phosphorylation of two serine residues on I
B
(7) followed by the
ubiquitination (8) and degradation of this subunit (9). This releases
active NF-
B, which then translocates to the nucleus and activates
transcription. Recently, two closely related kinases that directly
phosphorylate I
B
have been described (10-12). In addition, the
upstream kinase, where the IL-1
and TNF-
signaling pathways
converge prior to I
B
phosphorylation, has been identified as a
mitogen-activated protein kinase kinase kinase and named
NF-
B-inducing kinase (13).
At present, it remains unclear where other previously identified
pathways activated by IL-1 and TNF-
feed into this emerging signal transduction cascade. For example, the
phosphatidylcholine-specific phospholipase C (PC-PLC) as part of the
sphingomyelin pathway upstream of the second messenger ceramide has
been implicated in TNF activation of NF-
B (14). However, the signal
transduction pathway leading to nuclear translocation of NF-
B after
TNF stimulation was found to be intact in acidic
sphingomyelinase-deficient mice (15). Protein kinase C (PKC) isoforms
have also been implicated in NF-
B activation. Transfection of a
dominant negative mutant of the atypical isoform PKC-
severely
impaired the activation of a NF-
B-dependent reporter
gene plasmid by sphingomyelin (16), implicating a role of PKC-
downstream of the sphingomyelin pathway. In addition, PKC-
was shown
to phosphorylate I
B
in vitro (17). In contrast, the
expression of highly purified PKC isoenzymes
,
,
,
,
,
and
in vivo failed to induce I
B
phosphorylation (18). However, in vivo studies with constitutively active
isoforms demonstrated novel PKC-
to be a potent inducer of a
NF-
B-dependent reporter gene (19).
In addition to PC-PLC and PKC, the mitogen-activated protein kinase
(MAPK), p38, has been implicated in NF-B activation, because the
selective inhibitor, SB203580, was able to inhibit the activation of a
NF-
B-dependent reporter gene by TNF-
. However, NF-
B nuclear translocation and DNA binding was unaffected (20). A
similar effect has been observed with the protein-tyrosine kinase (PTK)
inhibitor genistein, which was able to inhibit
lipopolysaccharide-induced activation of NF-
B-dependent
transcription (21).
We have used human type II A549 pneumocyte cells to provide evidence of
a second signaling pathway that is distinct from IB
degradation
and NF-
B nuclear translocation but is required for NF-
B
transcriptional activation by IL-1
and TNF-
.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture-- A549 cells obtained from ECACC (code 86012804) were cultured as previously described (22). Prior to transfection, cells were grown in T-75 culture flasks to 50-60% confluency.
Plasmid Construct--
The NF-B-dependent
reporter, 6NF-
Btkluc, contains three tandem repeats of the sequence
5'-AGC TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GA-3', which harbors two copies of the NF-
B binding site (underlined) upstream of a minimal thymidine kinase promoter (
105 to
+51) driving a luciferase gene as described before (23). Neomycin
resistance was conferred by ligating a HincII
(blunted)/PvuI fragment from pMC1neoPoly(A) (Stratagene,
Cambridge, UK) into the PvuI site of 6NF-
Btkluc
downstream of the luciferase gene. The resulting plasmid was named
6NF-
Btkluc.neo.
Stable Transfection and Luciferase Assay--
Cells were washed
with serum-free medium and incubated with medium containing 8 µg of
plasmid and Tfx50 (Promega, UK) for 2 h. Subsequently, cells were
cultured in fresh medium for 16 h before adding 0.5 mg/ml G-418
(Life Technologies, Inc.). Foci of stable transfected cells developed
after approximately 14 days of culture in the presence of G-418. To
create a heterogeneous population with regard to integration site,
multiple clones were then harvested and used for experiments for
another eight passages while maintained in medium containing 0.5 mg/ml
G-418. Cells were stimulated with IL-1 and TNF-
(R & D Systems,
Oxon, UK) at 1 and 10 ng/ml, respectively. Where used, RO31-8220
(Alexis, Nottingham, UK), SB203580, herbimycin A, and D609 (Calbiochem,
Nottingham, UK) were added 5 min prior to stimulation. Cells were
harvested 6 or 24 h later and assayed for luciferase activity
using a commercially available luciferase reporter gene assay
(Promega).
Semi-quantitative RT-PCR--
RNA isolation, reverse
transcription, PCR primers, conditions, and cycling parameters for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as described
previously (24). Luciferase primers were: 5'-GAG AGC AAC TGC ATA AGG
CTA-3' (forward) and 5'-TAC ATC GAC TGA AAT CCC TGG-3' (reverse)
(accession number M15077). Cycling parameters were: 94 °C, 20 s; 60 °C, 30 s; 72 °C, 30 s. The number of
amplification cycles used was the number necessary to achieve exponential amplification where product formation is proportional to
starting cDNA, and in each case this was determined as described (24). Following amplification, products (10 µl) were run on 2.0%
agarose gels stained with ethidium bromide. After densitometry, data
were expressed as the ratio of luciferase/GAPDH as a percentage of
IL-1 treated as means ± S.E. Because the transfected
luciferase gene has no introns, identical control amplifications were
performed from similar reverse transcriptions in which the reverse
transcriptase had been omitted. In these cases no product was visible,
indicating that any genomic contamination was below detectable levels
(data not shown).
Nuclear Extract Preparation and Assay for DNA Binding of
Transcription Factor--
A549 cells were grown to confluency in
6-well plates and incubated in serum-free medium for 24 h prior to
treatment. Nuclear protein was isolated 1 h after stimulation with
1 ng/ml IL-1 or 10 ng/ml TNF-
as described previously (22). Where
used, inhibitors were added 5 min prior to stimulation. The consensus NF-
B (5'-AGT TGA GGG GAC TTT CCC AGG-3') and Oct-1 (TGT
CGA ATG CAA ATC ACT AGA) probe were obtained from Promega. Specificity was determined by prior addition of 100-fold excess unlabeled consensus
oligonucleotide. Reactions were separated on 7% native acrylamide gels
before vacuum drying and autoradiography.
Western Blot Analysis-- Confluent A549 cells grown in 6-well plates were stimulated for the indicated times and harvested in 200 µl of lysing buffer (1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mM Tris-base, 75 mM NaCl, 10 mM EDTA, pH 7.4, supplemented with 0.5 mM PMSF, 2 mM sodium orthovanadate, 10 µg/ml leupeptin, 25 µg/ml aprotinin, 1.25 mM NaF, 1 mM sodium pyrophosphate).
Prior to loading onto 10% SDS polyacrylamide gels, samples were denatured by boiling for 5 min. Gels were run at 200 mA for 40 min at 25 °C. Proteins were transferred onto Hybond-ECL nitro-cellulose paper (Amersham, Buckinghamshire, UK) in blotting buffer (20 mM Tris-base, 192 mM glycine, 20% methanol) at 400 mA for 1 h at 25 °C. Membranes were blocked for 1 h with a 5% (w/v) nonfat dry milk solution in TBS/T (10 mM Tris-base, 150 mM NaCl, 0.1% Tween-20) before incubating the filter for 1 h with rabbit polyclonal anti-human I ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Induction of two p50/p65 NF-B DNA binding complexes by IL-1
and TNF-
has previously been shown in these cells (2, 22). To
investigate the effects on
B-dependent transcription, a
NF-
B-dependent reporter, 6NF-
Btkluc.neo, was stably
transfected into A549 cells. As shown in Fig.
1, the PTK inhibitor herbimycin A (25),
the PC-PLC inhibitor D609 (14), the PKC inhibitor RO31-8220 (26), and
the p38 MAPK inhibitor SB203580 (27) inhibited both the IL-1
and
TNF-
stimulation of NF-
B-dependent luciferase
activity at concentrations previously shown to be selectively
effective. The MAPK/extracellular regulated kinase kinase-1/2
inhibitor, PD 098059, at 10 µM, which potently inhibits
downstream activation of the extracellular regulated kinase (ERK)1 and
ERK2 (28), and the PTK inhibitor, genistein (21), at 100 µM had no effect on luciferase activity induced by
IL-1
or TNF-
(data not shown). Table
I depicts the EC50 for the
various inhibitors; results are in the range of previously reported
selectively active concentrations (see references cited above).
Vehicle, 1 µl/ml Me2SO, had no effect on luciferase
activity after IL-1
or TNF-
stimulation. Inhibitors alone had no
effect on luciferase activity (data not shown).
|
|
To examine the effect of these inhibitors on NF-B nuclear
translocation and DNA binding electro mobility shift assays (EMSA) were
performed. IL-1
-induced NF-
B DNA binding was only slightly altered by the nonselective PTK inhibitor herbimycin A, whereas the
potent inhibitors of NF-
B-dependent transcription,
RO31-8220, SB203580, and D609, had no effect (Fig.
2A). Specificity of the complex was shown by competing out the signal with a 100-fold excess of
cold competitor (data not shown). Neither IL-1
nor any of the
inhibitors under investigation had an effect on DNA binding activity of
the noninducible transcription factor Oct-1 (data not shown). Likewise
induction of NF-
B DNA binding by TNF-
was also unaffected by
RO31-8220, D609, and SB203580 (Fig. 2B). Because the changes
in luciferase activity were observed at 24 h and the lack of
change in NF-
B DNA binding was after a 1-h treatment, there remained
the possibility that these drugs exert their effects by changing the
longer term levels of active NF-
B. However, because the reporter
assay produced identical data after a 6-h treatment, this possibility
seemed remote (data not shown). Semi-quantitative RT-PCR was used to
further examine this question. Consistent with the luciferase activity
data, both RO31-8220 and D609 showed total repression of
IL-1
-induced luciferase mRNA following a 1-h incubation (Fig.
2C). These data indicate that the changes in luciferase
expression were the result of immediate changes in
B-dependent transcription and not due to effects on p50
or p65 expression or luciferase translation.
|
Because NF-B DNA binding was unaffected, these data suggest that
phosphorylation and subsequent degradation of I
B
would also be
unaffected by these compounds. The time course of IL-1
-induced I
B
degradation and resynthesis is shown in Fig.
3. The I
B
signal is lost by 15 min
post-stimulation except for a retarded band indicating phosphorylated
but as yet undegraded I
B
. RO31-8220 and SB203580 appeared to have
little effect on loss of I
B
, whereas D609 seemed to result in a
marginally reduced loss of I
B
. These data are consistent with the
EMSA data indicating no substantial effect of these compounds on
NF-
B activation. By contrast both D609 and RO31-8220 delayed, by 30 and 60 min, respectively, the reappearance of IL-1
-induced I
B
,
whereas SB203580 had no obvious effect.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A significant step toward understanding the mechanism of NF-B
activation was the recent identification of two related I
B
kinases (IKK
and IKK
) and the upstream kinase, NF-
B-inducing kinase, integrating IL-1
- and TNF-
-induced NF-
B activation (reviewed in Refs. 11, 29, and 30). However, because the pathways
leading to I
B
phosphorylation and subsequent degradation are
characterized in some detail now, the role of other previously identified components of cytokine-mediated NF-
B activation is becoming less clear. By employing a NF-
B-dependent
luciferase reporter gene stably transfected into the human alveolar
epithelial cell line A549, the compounds D609, RO31-8220, and SB203580
selective for the sphingomyelin, PKC, and p38 MAPK pathways,
respectively, were shown to be potent inhibitors of
NF-
B-dependent transcription after IL-1
or TNF-
stimulation. However, neither of these inhibitors had an effect on
I
B
degradation or NF-
B nuclear translocation and DNA binding.
We therefore propose the existence of a second pathway triggered by
TNF-
and IL-1
in parallel to I
B
degradation, which confers
NF-
B transcriptional competency.
In the same assay system the PTK inhibitor herbimycin A was able to
partially inhibit both the luciferase activity and DNA binding as
determined by EMSA. The PTK inhibitor genistein, previously reported to
inhibit NF-B transactivation induced by lipopolysaccharide in a
pro-monocytic cell line (21), had no effect on
NF-
B-dependent transcription in our model. The PTK
p59fyn is a possible target for these inhibitors, because it
was shown to be involved in NF-
B-mediated activation of the HIV long
terminal repeat promoter (31). Discrepant findings with different PTK inhibitors as shown here for genistein and herbimycin A were recently described for the regulation of inducible nitric-oxide synthase mRNA in primary rat hepatocytes involving the PTK
pp60c-src (25), suggesting different target
proteins for each inhibitor.
In contrast to the partial effect exercised by herbimycin A and
SB203580, the PC-PLC inhibitor D609 completely abolished
NF-B-dependent reporter gene activation by IL-1
and
TNF
at doses of 50 µg/ml. The sphingomyelin pathway with its
second mediator ceramide has previously been implicated in
TNF-
-induced NF-
B activation (14). At doses of 100 µg/ml, D609
completely inhibited PC-PLC; however, doses of 250 µg/ml only
partially affected NF-
B DNA binding (14). In contrast, the
EC50 for the D609 effect on NF-
B-dependent
transcription in this study is similar to the one calculated from the
dose response curve for TNF-
-activated PC-PLC (14). Taking the data
concerning the time course of I
B
degradation presented here into
account, we conclude that the inhibitory effect of D609 on NF-
B
activation is only marginally due to the inhibition of I
B
degradation and nuclear translocation. More importantly, D609, at doses
that abolish PC-PLC activity completely, inhibits
NF-
B-dependent transcription induced by IL-1
and
TNF-
. The delayed resynthesis of I
B
observed here supports
this view, because the I
B
promoter contains multiple NF-
B
sites responsible for conferring TNF-
inducibility (32, 33).
Importantly, this hypothesis would also explain recent findings in
sphingomyelinase-deficient mouse embryonic fibroblasts, where
TNF-
-induced I
B
degradation and NF-
B nuclear translocation was unaffected (15). Based on these findings, Zumbansen and Stoffel
(15) questioned any role for acidic sphingomyelinase, which is
downstream of PC-PLC (14). However, NF-
B transactivation competency
was not investigated and may be the crucial step mediated by PC-PLC and
sphingomyelinase in response to TNF-
. The specificity of D609 for
PC-PLC has recently been challenged by data showing the inhibition of
platelet-derived growth factor-activated phospholipase D as well as
PC-PLC (34). However, TNF-
-induced phopholipase D activity was not
affected by D609 (14), raising the possibility of a platelet-derived
growth factor-specific effect.
Data concerning the role of PKC isoforms in TNF-- or IL-1
-induced
NF-
B activation have been conflicting. Here we find that the
PKC-inhibitor RO31-8220 was able to completely block
NF-
B-dependent transcription yet failed to block NF-
B
nuclear translocation and DNA binding or I
B
degradation. The
marked delay in IL-1
-dependent I
B
resynthesis
further supports the hypothesis of a selective effect on NF-
B
transcriptional competency. However, evidence for the involvement of
PKC isoforms is only indirect, because RO31-8220 is not selective for
PKC isoenzymes (36, 37). Both MAPK-activated protein kinase 1
and
p70 S6 kinase, which are activated in response to growth factors and
phorbol esters, are also inhibited (36). Whether other kinases
activated by cytokines are inhibited remains unclear. The
cross-reactivity toward MAPK-activated protein kinase 1
was examined
by testing the MAPK/extracellular regulated kinase kinase-1/2 specific
inhibitor PD 098059, which blocks the upstream activation of ERK1 and
ERK2. PD 098059 had no effect on NF-
B-dependent
transcription.
In summary, PC-PLC and PKC isoforms appear to be involved in a TNF--
and IL-1
-induced pathway of NF-
B transcriptional activation that
is distinct from the signaling pathway leading to I
B
degradation, NF-
B nuclear translocation, and DNA binding. Because
cytokine-mediated phosphorylation of NF-
B subunits is shown to occur
for p65 after TNF-
stimulation of HeLa cells (38) and a serine
kinase, which specifically phosphorylates NF-
B subunits and not
I
B
, has been described (39), we speculate that both D609 and
RO31-8220 may prevent p65 phosphorylation and lead to reduced
transcriptional activity. Furthermore, Mercurio et al. (12)
identified a RelA kinase activity that was associated with the IKK
signalsome and supports the hypothesis that
cytokine-dependent phosphorylation of p65 (RelA) may be
required for transcriptional activity.
However, regulated phosphorylation of NF-B subunits has been tested
for the p38 inhibitor SB203580 (20), and no change in NF-
B subunit
phosphorylation was detected. Because the inhibition of
NF-
B-dependent transcription by SB 203580 was the least
pronounced effect observed in this study, future work needs to
specifically address the effect of D609 and RO31-8220 on NF-
B
subunit phosphorylation.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from Glaxo Wellcome and the European Commission (Biomed II).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Holder of a Deutsche Forschungsgemeinschaft scholarship.
¶ Funded by the Medical Research Council, UK.
To whom correspondence should be addressed. Tel.:
44-171-352-8121, Ext. 3027; Fax: 44-171-351-8126; E-mail:
robert.newton{at}ic.ac.uk.
1
The abbreviations used are: NF-B, nuclear
factor-
B; EMSA, electromobility shift assay; ERK, extracellular
regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IKK,
I
B kinase; IL, interleukin; MAPK, mitogen-activated protein kinase;
PC-PLC, phosphatidylcholine-specific phospholipase C; PKC, protein
kinase C; PTK, protein-tyrosine kinase; RT, reverse transcriptase; PCR, polymerase chain reaction; TNF, tumor necrosis factor; PMSF,
phenylmethylsulfonyl fluoride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|