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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 5, 2001 as doi:10.1096/fj.00-0518fje. |
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* Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany;
Abteilung für Innere Medizin, Universität Münster, Münster, Germany.
3Correspondence: Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität Theodor-Stern-Kai 7, D 60590 Frankfurt am Main, Germany. E-mail: Pfeilschifter{at}em.uni-frankfurt.de
SPECIFIC AIM
Evidence is accumulating that NO affects important signaling pathways and, on a more long-term scale, modulates gene transcription in mesangial cells (MC), thus perpetuating proinflammatory responses during glomerulonephritis. We sought to identify target genes and to decipher possible signaling mechanisms that lead to up- or down-regulation of genes by NO.
PRINCIPAL FINDINGS
1. Identification of MIP-2 as an NO-regulated gene
To examine the effects of NO on the transcription
pattern, rat MC were treated with IL-1ß (2 nM) and IL-1ß plus
diethylenetriamine nitric oxide (DETA-NO, 0.5 mM) for 24 h.
Comparison of the intensities of the resulting cDNA patterns showed a
prominent band that was obviously induced by IL-1ß and further
enhanced when DETA-NO was present during the incubation period.
Reamplification, purification and direct sequencing identified the
fragment as a homologue of the GRO chemokine family (CINC-2ß and
MIP-2).
IL-1ß strongly induced MIP-2 mRNA expression, an effect that was markedly enhanced by DETA-NO. In a further approach, cytokine-induced endogenous NO production by MC was blocked by using the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). It is remarkable that L-NMMA drastically attenuated IL-1ß-induced MIP-2 expression, which suggests a role for endogenously produced NO in mediating cytokine-induced MIP-2 expression in MC. Moreover, administration of an NO donor markedly up-regulated MIP-2 mRNA levels. Also cytokine-induced MIP-2 protein levels were potentiated by exogenously given NO. Blockage of endogenous NO production by L-NMMA markedly reduced MIP-2 expression. The NO donor alone also increased MIP-2 expression by MC significantly but to a much lesser extent than IL-1ß (0.08±0.01 ng/ml vs. 0.79±0.02 ng/ml at 24 h).
2. Cloning of the MIP-2 promoter and effects of IL-1ß
and NO on promoter activity
We cloned two genomic DNA fragments of 770 and 2000 bp
length that were adjacent directly to the 5'-region of the published
rat MIP-2 cDNA. The transcriptional start site of the rat MIP-2 gene
was examined by primer extension. We could localize the site of
initiation of mRNA synthesis at a distance of 28 bp from the putative
TATA-box. A fragment of the MIP-2 5'-flanking region that represents
766 bp of the MIP-2 promoter and contains putative binding sites for
the transcription factors AP-1 (-329–322), NF-IL-6 (-218–210), and
NF
B (-75–65) was fused to the luciferase gene and is further
referred to as pGL3-MIP2. The cloned genomic rat DNA in pGL3-MIP2
represents 766 bp upstream of the transcriptional start site and 64 bp
of the first exon. In MC transiently transfected with pGL3-MIP2
stimulation with IL-1ß (0.5 nM), DETA-NO (0.5 mM), or SNAP (0.5 mM)
yielded a comparable induction of luciferase activity (Fig. 1A
). A co-incubation with IL-1ß plus DETA-NO further
enhanced, in an additive manner, MIP-2 promoter activity. Co-incubation
of cells with IL-1ß and L-NMMA markedly reduced MIP-2
promoter activity, which clearly shows that endogenously produced NO
transcriptionally up-regulates MIP-2 gene expression also (Fig. 1A
).
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Two deletions of the MIP-2 promoter in pGL3-MIP2 were constructed to
obtain plasmids lacking sequences 5' of the putative AP-1 site and the
AP-1 site itself (pGL3-MIP2
-370 and pGL3-MIP2-253, respectively)
as shown in Fig. 1B
. With pGL3-MIP2-253 as a template,
site-directed mutagenesis was performed to disrupt the putative binding
sites for NF-IL6 and NFB, which resulted in plasmids
pGL3-MIP2-253a and pGL3-MIP2-253b, respectively. Whereas deletion
of the sequences 5' of the putative AP-1 and the AP-1 site itself
exerted no significant changes in inducibility by DETA-NO, disruption
of either the NF-IL6 or the NF-B site completely abolished
inducibility by DETA-NO (Fig. 1B
). These data indicate that
both NF-B and NF-IL6 are essential for the NO-mediated MIP-2
transcription.
3. Expression of glomerular iNOS and MIP-2 mRNAs in
vivo
As an in vivo model system, we used Thy1.1
glomerulonephritis, a rat model of mesangioproliferative
glomerulonephritis. Northern blot analysis demonstrated a marked
expression of iNOS in isolated glomeruli 1 h after the anti-Thy1.1
antibody injection and maximal expression occurred at 2–4 h.
Glomerular MIP-2 mRNA expression could be demonstrated in glomeruli from anti-Thy 1 nephritic rats 1 h after the disease had been induced with peak levels at 2 h.
Simultaneously, MIP-2 positive cells were detected by
immunohistochemistry in glomeruli from rats with anti-Thy 1
glomerulonephritis between 1 and 24 h after induction of disease.
MIP-2 positive cells were localized mainly in glomerular capillary
lumina (Fig. 2
).
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4. Effect of iNOS-specific inhibitors on glomerular MIP-2
expression and granulocyte infiltration
The i.v. administration of L-NIL or 1400W, two
specific iNOS inhibitors resulted in a marked reduction in glomerular
MIP-2 mRNA expression after both 2 and 4 h of nephritis; likewise,
although less pronounced glomerular MIP-2 expression was reduced at
2 h when another specific inhibitor of iNOS, 1400W, was used.
In anti-Thy 1 nephritic rats receiving L-NIL or 1400W,
there was a reduction in MIP-2– positive cells within glomeruli at 2
and 4 h of the disease. In parallel, the number of infiltrating
granulocytes declined markedly (up to 94%) in anti-Thy1 nephritic rats
after treatment with L-NIL or 1400W at 2 and 4 h (Fig. 2)
.
CONCLUSIONS AND SIGNIFICANCE
An intriguing new aspect of nitric oxide functions is its potential to modulate gene expression, which thus establishes delayed but long-lasting changes in the cellular phenotype.
Here we report the NO-dependent up-regulation of MIP-2 in vitro in cultured MC and in vivo in a model of mesangioproliferative glomerulonephritis, which is characterized by high-output NO formation. MIP-2 is a C-X-C chemokine and triggers the attraction of polymorphonuclear leukocytes.
We observed that NO potently triggers MIP-2 mRNA and protein expression
in MC. By cloning the 5'-flanking region of the rat MIP-2 gene and
reporter gene assays, we suggest that elevation of MIP-2 mRNA levels by
NO is caused by transcriptional activation of the MIP-2 gene. However,
whereas NO and IL-1ß induce MIP-2 promoter activity and mRNA
steady-state levels to a comparable extent, IL-1ß is far more
effective in inducing MIP-2 protein expression. This difference points
to additional actions of IL-1ß that are not shared by NO. Serial and
site-directed deletion mutants of MIP-2 luciferase reporter genes
transfected into MC demonstrate that the binding sites for both NF-IL6
and NFB are essential for the activity of the rat MIP-2 promoter in
response to NO. By contrast, deletion of the AP-1 site has no
significant influence on NO-induced promoter activity.
To study the relevance of NO-dependent MIP-2 expression in vivo, we used the rat model of anti-Thy 1 glomerulonephritis. This model is characterized by a first phase of rapid mesangiolysis and a second phase of compensatory proliferation of MC. We observed an early induction of iNOS mRNA expression in glomeruli of nephritic rats. A role for NO in mediating mesangiolysis via apoptotic and necrotic mechanisms has been postulated from in vitro and in vivo studies. Chemokine expression during anti-Thy 1 nephritis has so far been detected only for MCP-1 and RANTES, both CC chemokines that attract monocytes/macrophages. In both cases blocking of chemokine action by antibodies or inhibitors lowered influx of monocytes/macrophages, indicating a functional relevance of MCP-1 and RANTES during anti-Thy 1 glomerulonephritis.
We observed a reduction of MIP-2 expression and subsequent infiltration of neutrophils in anti-Thy1 nephritic rats when NO synthesis was blocked, which indicates that MIP-2 is an important target for glomerular NO, required for neutrophil recruitment in mesangioproliferative glomerulonephritis. Expression of MIP-2 and iNOS are initial steps in anti-Thy1 nephritis and NO produced by neutrophils and MC may serve to rapidly amplify leukocyte recruitment and to initiate mesangiolysis. Down-regulation of MIP-2 by L-NIL may at least in part explain the beneficial effect of NOS inhibitors observed in the early phases of glomerulonephritis.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0518fje ; to cite this
article, use FASEB J. (January 6, 2001)
10.1096/fj.00-0518fje 
2 Both authors contributed equally to this work.
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P < 0.05 vs. IL-1ß.
B) Each 0.4 µg of plasmids pGL3MIP2, pGL3,-MIP2
, tumor necrosis factor