(Received for publication, November 20, 1995; and in revised form, February 22, 1996)
From the
Glomerular mesangial cells produce reactive oxygen intermediates
when stimulated by interleukin-1 (IL-1) or tumor necrosis factor.
Recent observations suggest that reactive oxygen intermediates may play
a role in IL-1 and tumor necrosis factor signaling and may up-regulate
gene expression. We therefore evaluated the effects of antioxidants on
IL-1-induced cyclooxygenase-2 (Cox-2) and inducible nitric-oxide
synthase (iNOS) expression in rat mesangial cells. The oxidant
scavenger, pyrrolidine dithiocarbamate (PDTC), inhibited iNOS
expression at the transcriptional level, since PDTC abolished iNOS mRNA
accumulation. In contrast, PDTC inhibited Cox-2 expression at the
post-transcriptional level, since PDTC did not affect IL-1
-induced
Cox-2 mRNA levels but inhibited Cox-2 protein expression and
prostaglandin E
production. Another antioxidant, rotenone,
which inhibits reactive oxygen intermediate production by inhibiting
the mitochondrial electron transport system, did not inhibit
IL-1
-induced iNOS and Cox-2 mRNA expression but inhibited iNOS and
Cox-2 protein expression, suggesting a post-transcriptional target for
the inhibition of iNOS and Cox-2 expression induced by IL-1
. These
results suggest that not only transcriptional regulation but also
post-transcriptional mechanisms are involved in redox-sensitive
inhibition of cytokine induced Cox-2 and iNOS expression. These results
suggest a novel approach for intervention in cytokine-mediated
inflammatory processes.
Interleukin-1 (IL-1) ()is a cytokine which mediates a
variety of processes in host defense, such as inflammation and the
cellular response to injury(1) . During glomerular
inflammation, cytokines from infiltrating macrophages and activated
mesangial cells may act to sustain and promote glomerular damage. We
have previously demonstrated that IL-1
induces cyclooxygenase-2
(Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases
in proinflammatory mediators, PGE
(2) and
NO(3) , in rat mesangial cells. The molecular signaling
mechanisms by which IL-1
induces Cox-2 and iNOS includes
transcriptional activation of these genes to produce increased levels
of mRNA species which are ``unstable.'' This mRNA is then
translated into protein and degraded. These intracellular events are
therefore potentially subject to regulation at the transcriptional or
post-transcriptional level. Furthermore, the factors which control
message stability and translational efficiency are not well understood.
Mesangial cells produce reactive oxygen intermediates (ROI) with
stimulation by endotoxin and cytokines, including IL-1 and tumor
necrosis factor(4) . ROI are produced during various
electron-transfer reactions. When generated in excess, ROI can damage
cells by peroxidizing lipids and disrupting proteins and nucleic acids.
However, ROI may exert signaling functions and regulate gene expression
at moderate
concentrations(5, 6, 7, 8, 9, 10) .
During glomerular inflammation, ROI from activated mesangial cells may
act as signaling molecules. We have therefore evaluated the mechanisms
by which some antioxidants can influence the expression of the
proinflammatory genes, Cox-2 and iNOS when they are up-regulated by the
cytokine IL-1.
Figure 1:
Effect of PDTC on
nitrite (A) and PGE production (B). Cells
were pretreated with PDTC for 1 h and then stimulated with IL-1
(50 units/ml) for 24 h. The stable metabolite of NO, nitrite, in the
medium was measured by the Griess reaction. PGE
in the
medium was determined by gas chromatography-mass spectrometry.
,
basal;
, IL-1.
Figure 2:
Effect of PDTC on iNOS protein (A) and iNOS mRNA expression (B). Cells were
pretreated with PDTC for 1 h and then stimulated with IL-1 (50
units/ml). Cells were harvested at 24 h for Western blot analysis (A) and at 12 h for Northern blot analysis (B),
respectively. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Figure 3:
Effect of PDTC on NF-B activation.
Cells were pretreated with PDTC for 1 h and then stimulated with
IL-1
(50 units/ml) for 30 min. Nuclear protein was extracted as
described under ``Experimental Procedures'' and used for
electrophoretic mobility shift assay. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Figure 4:
Effect of PDTC on Cox-2 mRNA (A)
and Cox-2 protein (B) expression. Cells were pretreated with
PDTC for 1 h and then stimulated with IL-1 (50 units/ml). Cells
were harvested at 3 h for Northern blot analysis (A) and at 24
h for Western blot analysis (B),
respectively.
Figure 5:
Effect of rotenone on nitrite (A)
and PGE production (B). Cells were pretreated with
rotenone for 1 h and then stimulated with IL-1
(50 units/ml) for
24 h. The stable metabolite of NO, nitrite, in the medium was measured
by the Griess reaction. PGE
in the medium was determined by
gas chromatography-mass spectroscopy.
, basal;
,
IL-1.
Figure 6:
Effect
of rotenone on Cox-2 mRNA (A) and Cox-2 protein (B)
expression. Cells were pretreated with rotenone for 1 h and then
stimulated with IL-1 (50 units/ml). Cells were harvested at 3 h
for Northern blot analysis (A) and at 24 h for Western blot
analysis (B), respectively. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Figure 7:
Effect of rotenone on iNOS mRNA (A) and iNOS protein (B) expression. Cells were
pretreated with rotenone for 1 h and then stimulated with IL-1 (50
units/ml). Cells were harvested at 12 h for Northern blot analysis (A) and at 24 h for Western blot analysis (B),
respectively.
Figure 8: Effects of PDTC and rotenone as general inhibitors of translation. In vitro translation was carried out with rabbit reticulocyte lysate. Lane 1 shows no RNA added; lane 2 standard RNA from kit; lanes 3-5, PDTC at concentrations indicated under ``Experimental Procedures''; and lanes 6 and 7, rotenone.
Figure 9:
Effect of PDTC and rotenone on the
temporal expression of Cox-2 and iNOS mesangial cells were incubated
with IL-1 with and without PDTC and rotenone for the times
indicated. Immunoblots were performed on lysates and probed for Cox-2 (A) and iNOS (B). This figure shows the results of
quantitative densitometry performed on the
blots.
In this study, we have demonstrated that two mechanistically
distinct antioxidants, PDTC and rotenone, inhibit Cox-2 protein
expression and PGE production. Inhibition of Cox-2 is
likely to be unrelated to alterations in its gene transcription,
because neither compounds significantly reduced the level of the Cox-2
gene transcripts. Neither of these compounds directly inhibited the
catalytic activity of Cox enzyme in crude microsomal preparations in vitro. These data suggest that these two antioxidants
inhibit the Cox-2 expression at a post-transcriptional level.
Recent
observations have demonstrated that proinflammatory cytokines, IL-1 and
tumor necrosis factor, increase the production of ROI in mesangial
cells(4) . The mitochondrial electron transport system is one
of the major sources for cellular ROI generation. Schultz-Oschoff et al.(10) showed that tumor necrosis factor
-induced cytotoxicity and NF-
B activation were abolished by
the mitochondrial electron transport system inhibitor, rotenone.
Changes in redox status have been reported to modulate the activation
of transcription factors, such as NF-
B (7, 8, 9, 10) and
AP-1(5, 6) . Thus it is proposed that these cytokines
may mediate their effects in part via ROI. However, several lines of
evidence suggest that the redox status of the cell can affect
post-transcriptional events in the
cell(13, 14, 15) .
Redox status has been
shown to regulate mRNA stability and modulate translation in a cell
free system(13, 14, 15) . Our data suggests
that similar post-transcriptional events might be involved in the
regulation of Cox-2 expression in vivo. Cox-2 and iNOS mRNA
have ``AUUUA'' motifs in their 3`-untranslated
regions(16, 17) . This AU-rich element has been
considered to be a mRNA instability determinant. We have shown that
IL-1 stabilizes the Cox-2 message by phosphorylation of cytosolic
factors which bind to the AUUUA-rich 3`-untranslated region in rat
mesangial cells(18) . Furthermore, some of these AU-rich motif
binding factors are known to be redox-sensitive (15) . However,
changes in stability of Cox-2 mRNA may not account for the effect of
the antioxidants PDTC and rotenone, since the steady state levels of
Cox-2 transcripts were not different in the control cells (IL-1
treated) and the antioxidant-treated (IL-1
plus antioxidant)
cells. Inhibition of translation is more consistent with our
observations, since the antioxidants did not inhibit Cox-2 mRNA
expression but inhibited Cox-2 protein expression. Eukaryotic
translation is regulated by many eukaryotic initiation factors and RNA
binding proteins. One of the eukaryotic initiation factors, eukaryotic
initiation factor-2, changes its function with redox status as well as
phosphorylation(14) . Redox status is also known to regulate
the RNA-protein interaction of the iron-responsive element binding
protein, which binds to the 5`-untranslated region of ferritin mRNA and
3`-untranslated region of the transferrin receptor mRNA. These cellular
events control the translation of ferritin mRNA and stability of the
transferrin receptor mRNA(13) . Thus the change in redox status
might regulate directly or indirectly some eukaryotic initiation
factors and/or RNA binding proteins which regulate the translation
and/or stability of Cox-2 mRNA.
In the case of iNOS, PDTC and
rotenone appeared to inhibit IL-1 induced iNOS expression by the
different mechanisms. PDTC inhibited iNOS expression at the
transcriptional level, since PDTC inhibited iNOS mRNA accumulation.
However, we cannot exclude the possibility that PDTC also affects
translational efficiency of the iNOS gene since the drug produced a
reduction in iNOS mRNA. It has been demonstrated that PDTC inhibits
NF-B activation in many cell types and that the promoter for the
iNOS gene has a
B binding consensus. However, consistent with the
recent observation by Rovin(19) , our data demonstrates that
PDTC does not inhibit IL-1
-induced NF-
B activation in
mesangial cells at concentrations which inhibit PGE
and NO
formation. Thus PDTC appeared to inhibit the iNOS mRNA expression by
some other unknown mechanisms. In contrast to PDTC, rotenone inhibited
iNOS expression at a post-transcriptional level, since it did not
inhibit iNOS mRNA expression but suppressed iNOS protein expression.
In summary, we have shown that two mechanistically different
antioxidants, PDTC and rotenone, inhibit IL-1-induced Cox-2 and
iNOS expression in rat mesangial cells. Our data suggests that a change
in cellular redox status may influence gene expression at multiple
levels which include transcription and post-transcriptional events.
Neither PDTC nor rotenone appeared to be general inhibitors of
translation (Fig. 8) but their effects appeared to be restricted
to a subset of messages which include Cox-2 and iNOS. Furthermore, the
effects of these agents was simply to reduce the magnitude of the
increase of protein expression with an effect on the time to peak
expression. These results may suggest a potentially novel mechanistic
approach to therapeutically intervene and down-regulate the biologic
effects of the proinflammatory genes, iNOS and Cox-2, in glomerular
inflammation. The observation that the 3`-untranslated region of many
``unstable'' messages carry motifs that may regulate
translational efficiency by reversible binding of cytosolic or nuclear
factors raise the possibility that some antioxidants may influence,
either directly or indirectly, these RNA-protein interactions.