Download Mechanism of NFkB activation by interleukin

2s Biochemical Society Transactions ( 1 996) 24
Mechanism of NFKB activation by interleukin-1 and
tumour necrosis factor in endothelial cells
I +TNF
CI + I L I
112 3 4 516 7 8 9
ANDREW BOWIE, PAUL N. MOYNAGH* and LUKE A.J.
ONEILL
CI
Biochemistry Department, Trinity College Dublin, Ireland and
*Pharmacology Department, University College Dublin, Ireland.
Endothelial cells (ECs) are a key target for the pleiotropic proinflammatory cytokines interleukin-I (ILI) and tumour necrosis
factor (TNF). Activation of ECs by these cytokines leads to altered
gene expression, although the molecular basis of this process is
unclear and remains the subject of much investigation. However, IL1
and TNF potently activate the transcription factor NFKBin many cell
types [I], and this is likely to be a key step in the induction of EC
genes by these cytokines. Evidence from other cell types (T cell lines
in particular) has suggested that the mechanism of activation of
NFKBis common to both cytokines and may involve oxidative stress
(an increase in intracellular reactive oxygen species (ROS) or
hydrogen peroxide) [2, 31. We have tested this oxidative stress
model in ECs using human umbilical vein endothelial cells
(HUVECs) and a recently described immortalised human EC line,
ECV304, which we have previously characterised as a good model
for EC activation by ILI and TNF [4].
NFKB activation was assessed by treating confluent cells with
various stimulants, and isolating nuclear extracts by the method of
Osborn et a1 [5]. Extracts (1 or 2pg protein) were incubated with
[32P] labelled DNA containing the NFKB consensus sequence.
Protein-bound DNA was separated from unbound DNA by native
polyacrylamide gel electrophoresis. The gels were then dried and
autoradiographed. Competition analysis with unlabelled wild-type
and mutant NFKB prode confirmed that protein-DNA complexes
were specific for NFKB.Both ECV304 cells and HUVECs showed a
time-dependent activation of NFKBin response to 10 n d m l ILI or
TNF or 100 ndml phorbol 12-myristate 13-acetate (PMA) that was
detectable from 5-15 min, peaked at 30 min-l h and remained
elevated for up to 48 h after stimulation.
Pyrrolidine dithiocarbamate (PDTC) has been shown to inhibit
NFKBactivation in certain cells, and has been implicated as a general
inhibitor of NFKBthat acts independently of the activating agent and
cell line used [3]. It has been suggested that PDTC inhibition is
mediated through its antioxidant properties, and this has been used as
further evidence for the model of activation by oxidative stress [3].
We have previously shown that PDTC inhibits NFKBactivated by
TNF and PMA, but not ILI, in ECV304 cells [4]. Here, in
HUVECs, NFKB activation by PMA, but not JLI or TNF was
inhibited consistently, at PDTC concentrations of 0.01-10 mM.
These novel findings indicate that PDTC may not in fact inhibit
NFKBindependent of the activating agent.
It is conceivable that the PDTC-sensitivity of TNF and PMA in
ECV304s and PMA in HUVECs is due to an involvement of ROS or
hydrogen peroxide in these pathways, but not those that are
insensitive (ILI in ECV304s and ILI and TNF in HUVECs). To test
this hypothesis, experiments involving hydrogen peroxide were
performed. Since hydrogen peroxide freely diffuses across the cell
membrane, measuring extracellular release is an indication of
ide release bv IL I -.TNF- and
Table 1
PMA- treated ECV304 cells. Hydrogen peroxide release from cells
was measured by the horseradish peroxidase-mediated oxidation of
the fluorescent probe scopoletin to its non-fluorescent oxidised form
[6]. The data is expressed as the mean f S.E.M. for five
experiments, each performed in triplicate. There was no significant
difference between values for stimulated and control cells, either
overall (values below) or in any single experiment. Measured values
varied from 0.9 to 14.9 pmoledmidmg protein.
Treatment
H 2 0 2 release (pmoles/min/mg protein)
6.7 f 1.6
Control
6.6 i 1.4
10 ng/ml IL1
7.6 h 1.7
10 ng/ml TNF
8.2 f 2.3
100 n g h l PMA
Abbreviations used: EC. endothelial cell: ILl. interleukin-I : NAC,
N-acetyl cysteine; PDTC; p rrolidine dithiocardamate; PMA, phorbol
12-myristate 13-acetate; R&, reactive oxygen species; TNF, tumour
necrosis factor
+IL1
I
+ TNF
10 111 12 13 14115 16 17 18
Fig. 1 Effect of hvdroeen Deroxide on NFKB activ u bv sub-0Dtlmd
doses of ILI and TNF. ECV304s (lanes 1-9) or HUVECs (lanes 1018) were pretreated for 3 h with 0 (lanes I , 2, 6, 10, 1 I and 15),
0.01 (lanes 3. 7. 12 and 16). 0.1 (lanes 4. 8, 13 and 17) or 1 mM
hydrogen peroxide (lanes 51'9, 14'and 18) before stimulation with
medium (C, control) or I ng/ml JLI or TNF for 1 h. Nuclear extracts
were assessed for the presence of NFKB. Retarded protein-DNA
complexes are shown. Representativeof four experiments.
intracellular levels. Table 1 shows that there was no significant
increase in hvdroeen Deroxide release from ECV304 cells uDon
exposure to I i l , T-NF or PMA. This suggests that the sensitiviti of
the TNF and PMA pathways to PDTC in ECV304s is not due to an
involvement of hydrogen peroxide in these pathways. Treatment of
ECV304 cells or HUVECs with 0.01-10 mM hydrogen peroxide
alone failed to activate NFKB.In addition, Figure 1 shows that not
only was hydrogen peroxide unable to potentiate ILI- or TNFactivated NFKB in either ECV304 cells or HUVECs, but in fact
higher doses proved inhibitory . Hydrogen peroxide has been shown
to activate NFKBin some cell types [2] but not others [7,8]. Bradley
er a1 have also shown it to be ineffective in ECs [9]. The results here
suggest that hydrogen peroxide is unlikely to be important in either
the PDTC-sensitive or PDTC-insensitive activation of NFKBin ECs.
The effect of another antioxidant, N-acetyl cysteine (NAC), on
NFKBactivation was also determined in order to ascertain whether a
similar pattern of inhibition to PDTC would be observed. NAC is a
glutathione precursor and ROS scavenger that is unrelated in
structure to PDTC. It has been shown to inhibit NFKBin certain cell
types [2, 101. Here, NAC (1-40 mM) had no effect on ILI-, TNF- or
PMA-activated NFKB in either ECV304s or HUVECs, although
inhibition of TNF-activated NFKB in Jurkat T cells was seen, as
reported by Schreck er a1 [4]. Since NAC had no inhibitory effect on
any of the NFKBactivating agents in the ECs it is unlikely that ROS
are involved in NFKB activation in ECs. Additionally, since NAC did
not give a similar pattern of inhibition to PDTC, it is likely that the
differential inhibition by PDTC may be independent of its antioxidant
properties. The precise target for PDTC remains to be determined.
In summary, these results demonstrate that PDTC does not inhibit
NFKB independently of the activating agent in endothelial cells.
Hydrogen peroxide or ROS are unlikely to be involved in either the
PDTC-sensitive or the PDTC-insensitive pathways to NFKB
activation in endothelial cells, and hence the oxidative stress model
for NFKB activation may not apply to ILI and TNF in endothelial
cells. Futhermore, the basis of the differential inhibition by PDTC in
endothelial cells may be independent of its antioxidant properties
since another antioxidant, NAC, had no inhibitory effect on IL1-,
TNF- or PMA-activated NFKB.
Work was supported by FORBAIRT and The Irish Heart Foundation.
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