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B
in Active Minimal-Change Nephrotic Syndrome
,
,||
*
Institut National de la Santé et de la
Recherche Médicale Unit 99, Henri Mondor
Hospital, Créteil, France.
Upress B JE 2129, Henri Mondor Hospital,
Créteil, France.
Nephrology Service, Henri Mondor Hospital,
Créteil, France.
Immunology-Biology Service, University of Paris XII,
Créteil, France.
||
Nephrology Service, Armand Trousseau Hospital, Paris, France.
¶
Nephrology Service, Robert Debré Hospital,
Paris, France.
#
Nephrology Service, Necker Hospital for Sick Children, Assistance Publique
des Hôpitaux de Paris, Paris,
France.
Correspondence to Dr. Djillali Sahali, INSERM Unité 99, Hôpital Henri Mondor, AP-HP, 51, avenue du Mal de Lattre-de-Tassigny, 94010, Créteil, France. Phone: 33-1-49-81-35-30; Fax: 33-1-48-98-09-08; E-mail: sahali{at}im3.inserm.fr
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B (NF-B) in the regulation of cytokine expression, its
activity during the relapse and remission phases of steroid-sensitive MCNS was
analyzed. During relapse, nuclear extracts from peripheral blood mononuclear
cells displayed high levels of NF-B DNA-binding activity, consisting
primarily of p50/RelA (p65) complexes. NF-B p65 and IB
proteins were barely detected or not detected in cytosolic fractions during
relapse, in contrast to remission. The lack of expression of IB
protein was associated with downregulation of IB mRNA and
increases in the levels of the mRNA encoding the proteasome 2 subunit
proteolytic pathway. In addition, inhibition of proteasome activity induced
cytosolic accumulation of phosphorylated IB and significant
reductions in the NF-B binding activity in nuclear extracts from
peripheral blood mononuclear cells from patients experiencing relapses. These
results suggest that alterations in the NF-B/IB
regulatory feedback loop may contribute to the immunologic abnormalities that
occur in steroidsensitive MCNS. |
Introduction |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies of T cell compartments in MCNS demonstrated expansion of
CD4+ and CD8+ T cell populations, including those with
the CD45RO memory phenotype
(6,7).
T cell expansion was often associated with increased synthesis of several
cytokines, such as tumor necrosis factor- (TNF-) and
interleukin-13 (IL-13)
(8,9).
Clinical observations and experimental data also suggest that T cells with
Th2-like phenotypes are involved in the pathophysiologic processes of MCNS.
First, the production of IgE is often increased during relapses, independent
of previous atopic manifestations. Second, both cell-mediated immunity and
delayed hypersensitivity, which are characteristic of Th1 cell function, seem
to be defective in relapses
(10). Immune dysfunction seems
not to be restricted to T cells, because levels of cytokines produced
primarily by monocytes, such as IL-1 and IL-8, were increased in relapses. In
contrast, remissions were characterized by downregulation of these cytokines
(11). These findings suggest
that molecular events upstream from cytokine production may be impaired in
MCNS.
Nuclear factor-B (NF-B) plays a key role in the regulation of
cytokine expression, through association with other transcription factors and
protein-protein interactions with coactivator proteins
(12). Because most cytokines
whose levels are increased during relapses and downregulated during remissions
are partly or predominantly regulated by NF-B, we postulated possible
involvement of this regulatory pathway in MCNS.
The human NF-B/Rel family includes five members, i.e.,
NF-B1 (p50), NF-B2 (p52), Re1A (p65), cRel, and RelB, which form
various homo- and heterodimers. Their activity is regulated by IB
proteins (IB, IBß, and Bcl-3)
(12). IB and
IBß specifically interact with RelA (p65), whereas Bcl-3 binds p50
homodimers. In unstimulated immune mononuclear cells, NF-B, which is
primarily composed of p50 and p65, is inactivated in the cytoplasm through
reversible association with IB proteins
(12,13).
After activation, cytosolic IB is phosphorylated by the
IB kinase complex and degraded by the proteasome system
(12). Free NF-B
complexes can subsequently move to the nucleus, where they regulate
NF-B-dependent gene expression. After activation of NF-B,
IB is rapidly resynthesized and sequestrates NF-B in the
cytoplasm, thus switching off the NF-B activity
(12). This negative feedback
occurs despite continuous activation by cytokines or protein kinase activators
and results from transcriptional activation of the IB gene by
NF-B
(14,15,16).
We report for the first time that, during MCNS relapses, strong persistent
stimulation of NF-B activity was observed in PBMC, whereas the
expression of IB was hardly or not detected. Downregulation of
IB protein was associated with low levels of IB
mRNA and increases in the levels of proteasome 2 subunit (2P)
mRNA. The inhibition of proteasome activity in PBMC during relapse
concomitantly induced significant reduction of NF-B binding activity
and accumulation of phosphorylated IB. In contrast, remission
was associated with downregulation of NF-B and stabilization of
IB protein. These new data suggest that subtle alterations of
the NF-B pathway might contribute to immune abnormalities underlying
the pathophysiologic processes of MCNS.
|
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All patients (children and adults) exhibited proteinuria of > 3 g/24 h and severe hypoproteinemia. Serum albumin levels were not available for all patients at the time of blood sampling.
Relapse was defined by a sudden onset of the nephrotic syndrome (proteinuria with at least 3+ protein levels, as assessed by urine dipsticks, for 3 d consecutively) in a patient previously free of proteinuria, regardless of therapy. In all cases, the diagnosis of nephrotic relapse was established at the time of blood sampling. Remission was defined by the disappearance of the nephrotic syndrome, with proteinuria of <0.5 g/24 h for 13 patients and 0.6 g/24 h for one.
The clinical and laboratory characteristics of the patients with MCNS are summarized in Table 1. Informed consent was obtained from the parents and whenever possible from the pediatric patients, as well as from adult patients and normal volunteers.
|
Purification of PBMC and T Cell Subsets
PBMC were purified through a Ficoll/Hypaque density gradient (Eurobio,
France). A CD4+ T cell-enriched population was collected by
immunomagnetic negative selection, using a cocktail of haptenconjugated CD8-,
CD11b-, CD16-, CD19-, CD36-, and CD56-specific antibodies and magnetic
cell-sorting microbeads coupled to an anti-hapten monoclonal antibody
(Miltenyi Biotech, Auburn, CA). The purity of the preparation was 90 to 95%,
as assessed by flow cytometric analysis using FITC-conjugated CD2-, CD4-,
CD19-, and CD8-specific antibodies.
Electromobility Shift Assays
Cytosolic and nuclear fractions were prepared essentially as described
previously (18). Protein
concentrations were assayed using the Bio-Rad dye reagent (Bio-Rad, Richmond,
CA), following the instructions provided by the manufacturer.
The double-stranded oligonucleotide probes (100 ng), with the consensus
(sc-2505) and mutant (sc-2511) NF-B sequences (Santa Cruz
Biotechnology, Santa Cruz, CA), were labeled with [
-32P]ATP
(3000 Ci/mmol) and purified on Chromaspin 30 columns (Clontech, Palo Alto,
CA). Binding assays were performed for 30 min at 4°C with gentle shaking,
using 10 to 20 µg of nuclear extracts, 4 µg of poly(dI-dC) in 20 µl
of binding buffer [10 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithiothreitol,
0.2 mM ethylenediaminetetraacetate, 12% glycerol, 1 mM levels of protease
inhibitors], 0.1% Nonidet P-40, and 50,000 cpm of NFB probe. The
samples were loaded onto a native 5% acrylamide gel in 0.5x TBE buffer
(90 mM Tris-HCl, pH 8.3, 90 mM borate, 4 mM ethylenediaminetetraacetate).
Migration of the samples was performed at 4°C for 2 h. The subunit
composition of DNA-protein complexes containing NF-B was determined by
preincubation of nuclear extracts with 2 µg of polyclonal antibodies raised
against p50 (sc-7178X), p65 (RelA) (sc-7151X), cRel (sc-278X), RelB (sc-226x),
or p52 (sc-298; Santa Cruz Biotechnology), for 1 h before addition of the
probe. Gels were dried and analyzed, after overnight exposure, using a
PhosphorImager (Storm 840; Molecular Dynamics, Sunnyvale, CA). Band shifts
were quantified using ImageQuant analysis software (version 1.11). Similar
electromobility shift assays were performed using the interferon-
(IFN-)-activated consensus site (GAS) and the IFN--stimulated
response element (ISRE) consensus binding sites (Santa Cruz
Biotechnology).
Western Blotting
Polyclonal antibodies raised against human NF-B p65 (sc-109), Sp1
(sc-420), and IB (sc-209 or sc-371) were obtained from Santa
Cruz Biotechnology. Polyclonal anti-actin antibody (A 2066) was purchased from
Sigma Chemical Co. (St. Louis, MO). Cytoplasmic extracts (50 µg of protein)
were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes by electroblotting. Immunoblotting
and detection (ECL system; Amersham, Buckinghamshire, UK) were performed
according to the instructions provided by the manufacturer.
Semiquantitative Reverse Transcription-PCR
Total RNA was isolated from PBMC using an RNeasy kit (Qiagen, Chatsworth,
CA). The primer sequences and main PCR characteristics are indicated in
Table 2. The forward primer of
each pair was labeled with the 6-Fam dye, except for the TNF- and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Reverse
transcription-PCR (RT-PCR) was performed in a 9600 Perkin Elmer apparatus,
using a RT-PCR access kit (Promega, Madison, WI). The amplified products were
detected in an Applied Biosystems 373A automated DNA sequencer (Applied
Biosystems, Foster City, CA) and quantified using the GeneScan program. For
five patients who were studied in both relapse and remission, the expression
of IB and 2P mRNA was analyzed by semiquantitative
RT-PCR, using 2 µg of total RNA
(19). After Southern blotting,
PCR products were detected with specific internal oligonucleotides. The
expression of GAPDH mRNA was analyzed in parallel, as a control.
|
Quantification of TNF- mRNA by Quantitative RT-PCR
The level of TNF- mRNA expression was analyzed by RT as described
above. Quantitative PCR was performed using a LightCycler (Roche Molecular
Biochemicals, Welwyn Garden City, UK). The samples (2 µl of the RT reaction
mixture, corresponding to 20 ng of total RNA) were amplified in a 20-µl of
the RT reaction mixture, corresponding to 20 ng of total RNA) were amplified
in a 20-µl reaction mixture containing 0.5 mM levels of each primer and
1x LightCycler DNA master SYBR green buffer (Roche Molecular
Biochemicals). Carryover was prevented by using dUTP instead of dTTP, with
heat-labile uracil DNA glycosylase. A standard curve was prepared by using
dilutions of RNA prepared from the pUc9-TNF- plasmid. The TNF-
primers amplified a 391-bp sequence (Table
2). The PCR was initiated by denaturation at 95°C for 2 min,
followed by 40 three-step cycles (95°C for 1 s, 60°C for 10 s, and
72°C for 24 s). The relative value for each sample was calculated using
LightCycler analysis software. All PCR findings were normalized to GAPDH
expression, to control for variations in the RT reactions.
Incubation of PBMC with the Proteasome Inhibitor MG132
PBMC were suspended in complete RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum, 50 µg/ml penicillin, and 100 µg/ml
streptomycin, in a humidified incubator containing 5% CO2, at a
concentration of 2 x 106 cells/ml. Cells were divided into
two equal fractions and incubated overnight at 37°C in the absence
(control) or the presence of 10 µM levels of the proteasome inhibitor MG132
(carbobenzoxyl-leucinyl-leucinyl-leucinal-H; Calbiochem, San Diego, CA). After
incubation, cells were pelleted by centrifugation at 1200 x g
for 10 min and washed three times with ice-cold phosphate-buffered saline, and
protein extracts were prepared.
Incubation with Serum
To assess the effects of serum on NF-B activation, PBMC were
incubated with autologous serum from patients experiencing MCNS relapses.
Normal autologous human serum was used as a control. Nuclear and cytoplasmic
extracts were prepared as described above.
Incubation with IFN-
Normal PBMC were cocultured with IFN- (Schering Plough, Kenilworth,
NJ) at a concentration of 1000 IU/ml and were then processed as described
above.
Statistical Analyses
Results were analyzed using ANOVA. Statistical significance was determined
using the nonparametric Mann-Whitney U test.
|
Results |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B DNA-Binding Activity in Nuclear Extracts and Loss of
Cytoplasmic IB Protein in PBMC from Patients Experiencing MCNS
RelapsesB DNA-binding activity in
nuclear extracts and the expression of IB in cytoplasmic
preparations derived from the same PBMC samples (11 relapses and eight
remissions) and four normal control samples. All PBMC nuclear extracts from
patients with nephrotic relapses who were not receiving steroids exhibited
high levels of NF-B DNA-binding activity
(Figure 1A), whereas the
expression of IB protein was barely detectable in the
corresponding cytosolic extracts (Figure
1B). In contrast, the NF-B DNA-binding activity was barely
or not detectable in nuclear extracts from patients experiencing remission,
and IB protein was easily identified. The specificity of the
NF-B binding was demonstrated by loss of the band shifts in the
presence of an excess of unlabeled or mutant NF-B probe. Moreover, no
displacement of NF-B complexes was obtained in the presence of an
unlabeled activator protein-1-specific oligonucleotide
(Figure 1C). Immunoblotting of
nuclear extracts demonstrated no reactivity with anti-actin antibodies,
excluding the possibility of cross-contamination by cytoplasmic fractions
(data not shown).
|
Lack of DNA-Binding Activities Induced by IFN- or - in
Nuclear Extracts
Viral infections elicit high levels of the endogenous cytokines IFN-
and IFN-. Both IFN forms induce signal transduction cascades and
transactivation of target genes bearing specific response elements, such as
GAS and ISRE. To determine whether the NF-B activation in MCNS might
result from viral infection, nuclear extracts were analyzed for GAS-and
ISRE-binding activity. No DNA-protein complexes were detected by mobility gel
shift assays in PBMC nuclear extracts from MCNS relapses, whereas control PBMC
cocultured with IFN- displayed clear binding activity with the GAS/ISRE
consensus oligonucleotides (Figure
2). These results suggest that NF-B activation did not
result from viral infection.
|
Evidence that NF-B Complexes Activated in MCNS Consist Mainly
of p50 and p65 Subunits
The subunit composition of the NF-B-DNA complexes was analyzed by
preincubation of nuclear extracts with antibodies raised against p50, p52, p65
(RelA), cRel, RelB, or Bcl-3, before addition of the NF-B probe. The
complexes were supershifted with both p50- and p65-specific antibodies but not
with those for cRel, p52, RelB, or Bcl-3, suggesting that, in active MCNS, the
NF-B complexes were predominantly composed of p50-p65 (RelA)
heterodimers or p502 or p652 homodimers
(Figure 3A). Supershifting was
incomplete, as reported by others
(20), possibly because of
steric hindrance. In a control experiment, we analyzed the NF-B
activity for five adult patients with nephrotic syndrome related to MN.
NF-B DNA-binding activity was barely detected in nuclear PBMC from
these patients (Figure 3B). For
patient 5, the very low level of NF-B binding likely corresponded to
basal activity (13). However,
no supershift could be identified. Quantification of NF-B band shifts
demonstrated that the activation level for patients with MCNS was consistently
several orders of magnitude higher than that observed for patients with MN
(Figure 3C).
|
Virtual Absence of IB and NF-B p65 Proteins from
the Cytoplasm of PBMC during Relapse
We further compared the cytoplasmic expression of IB and
NF-B p65 proteins in PBMC from normal and patient samples.
IB was expressed at similar levels by two healthy control
subjects and two patients experiencing remission, whereas it was not detected
in PBMC from five patients with MCNS relapses
(Figure 4). All samples
displayed similar patterns for cytoplasmic NF-B p65 and
IB proteins. These results suggest that the NF-B binding
activity in cells from patients with nephrotic relapses (Figures
1A and
3B) results from the
disappearance of IB and the translocation of p65 in the nucleus.
Conversely, the cytoplasmic levels of NF-B p65 and IB
were normalized in remission.
|
Contributions of Both CD4+ and Non-CD4+ T Cells
to NF-B Activity during Relapse
To address whether NF-B activity was selectively increased in the
CD4+ T cell subset, we performed immunomagnetic negative selection
of CD4+ T cells from PBMC from four patients with MCNS relapses who
were not receiving steroids. Nuclear extracts were prepared and assayed for
NF-B activity. The highest level of NF-B binding was observed in
non-CD4+ T cell populations
(Figure 5).
|
Similar Levels of NF-B Activation in Relapses with and without
Steroid Treatment
To assess whether patients experiencing MCNS relapses while receiving
steroids and those experiencing relapses while not receiving steroids exhibit
differences in NF-B activation, we further studied three patients who
experienced relapses while receiving steroids and five patients who
experienced relapses while no longer receiving this treatment. The patients in
the two groups exhibited similar high levels of NF-B activity, which
had been reduced to basal levels during remission
(Figure 6). These results
suggest that MCNS relapses were associated with increased NF-B
activity, regardless of steroid treatment at the time of relapse.
|
Effects of MCNS Relapse Serum on NF-B Activity
To determine whether the serum of patients with MCNS relapses was involved
in the upregulation of NF-B activity, PBMC were incubated overnight
with control or MCNS sera. Normal control PBMC incubated with MCNS sera did
not display significant NF-B DNA-binding activity
(Figure 7). Surprisingly, when
PBMC from patients with active MCNS were incubated with sera from patients
with nephrotic relapses or normal sera, we observed that the NF-B
activity was downregulated in the presence of sera from patients with MCNS
relapses but not normal sera. These results suggest that NF-B
activation precedes mononuclear cell activation and cytokine production.
|
Lack of Correlation between NF-B Activation and TNF-
mRNA Levels
To investigate whether the NF-B activation in MCNS relapses was
induced by cytokines such as TNF- (a potent inducer of NF-B
activity), we analyzed TNF- mRNA expression levels, under nonstimulated
conditions, by quantitative RT-PCR. Paired data were obtained for five
patients who exhibited high levels of NF-B activity. The TNF-
mRNA levels were higher in relapse than in remission for two patients, whereas
no significant difference was detected between the relapse and remission
phases for three patients (Figure
8). These results suggest that the production of TNF- does
not fully account for the NF-B activation detected in these
patients.
|
Low Levels of IB mRNA in PBMC from Patients with Active
MCNS
Because IB protein was barely detected or not detected for
patients with MCNS relapses, IB mRNA expression levels were
analyzed by RT-PCR. The IB PCR primers amplified a PCR product
of 569 bp, corresponding to mature mRNA, in all samples
(19). IB mRNA
levels were reduced for most patients (>50%) experiencing relapses,
compared with normal control subjects (P < 0.01) and patients
experiencing remissions (P < 0.001)
(Figure 9). In contrast,
IB mRNA levels were increased severalfold for patients
experiencing remission, compared with normal control subjects (P <
0.005). For all samples, IB primers did not amplify a 921-bp
product, corresponding to an unspliced form of IB mRNA,
suggesting normal maturation of this transcript during relapse
(19).
|
Upregulation of 2P mRNA during Active MCNS
Although IB mRNA levels were reduced during relapse, these
results might not entirely account for the virtual lack of IB
protein expression. Previous studies demonstrated that stimulation of the
proteasome proteolytic pathway was associated with an increase in the
mRNA-encoding components of this system, such as 2P
(21). Therefore, we determined
2P mRNA levels during the relapse and remission phases of MCNS. As
demonstrated in Figure 10A,
2P mRNA levels were significantly increased during relapse, compared
with those measured during remission and those for normal groups (P
< 0.01). These results demonstrated that the downregulation of
IB was associated with the upregulation of 2P mRNA during
relapse.
|
In addition, the expression of IB and 2P was analyzed
for five patients in both relapse and remission, by using RT-PCR followed by
Southern blotting. The IB mRNA levels were lower, whereas the
2P mRNA levels was higher, in relapse
(Figure 10B). Together, the
increase in 2P mRNA levels and the decrease in IB mRNA
levels led to sharp reduction of the IB/2P ratio in
relapse, compared with remission (Figure
10C). This ratio was significantly increased in remission,
compared with the control group.
Effects of Proteasome Inhibition on NF-B Activity and on
IB Protein Expression in Active MCNS
To further investigate whether the inhibition of proteasome activity could
affect NF-B activity, PBMC from four patients who experienced MCNS
relapses while not receiving steroids were incubated with the proteasome
inhibitor MG132. MG132 clearly inhibited NF-B binding activity, and no
supershift could be detected (Figure
11A). IB was concomitantly stabilized, as suggested
by the accumulation of its phosphorylated form. In contrast, no NF-B
activity was detected in supershift experiments with nuclear extracts from
cells from patients experiencing remissions while receiving steroids.
Furthermore, preincubation with MG132 did not induce accumulation of
phosphorylated IB (Figure
11B), further indicating that IB is stabilized
during remission. Together, these results strongly suggest that both
transcriptional and post-transcriptional mechanisms are involved in the
downregulation of IB in MCNS.
|
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding
activity in nuclear extracts. In contrast, the binding activity returned to
basal levels among patients experiencing remission. We also demonstrated that
no DNA-binding activity for the IFN-- or --responsive elements
could be detected in the same nuclear extracts, suggesting that viral
infection was not responsible for the upregulation of NF-B in MCNS.
Moreover, the typical absence of cellular infiltrates and Ig deposits in the
kidney argues against an inflammatory process, in contrast to conditions
prevailing in other glomerular diseases
(22).
We observed that NF-B activation was not induced by sera from
patients with active disease, which unexpectedly seemed to downregulate this
activity. Moreover, the upregulation of NF-B activity was not closely
related to increased production of TNF-, a known inducer of NF-B
activity. Therefore, it seems that the increase in NF-B activity
results primarily from molecular events that occur at early stages in the
pathophysiologic processes of MCNS. Nevertheless, we cannot exclude, at least
for some patients, the possibility that increased release of TNF-
and/or other cytokines contributes to NF-B activation.
We demonstrated concomitant downregulation of IB protein for
these patients, which seemed to be responsible for the sustained NF-B
activation. The downregulation of IB occurred at the mRNA and
protein levels. The low IB mRNA levels during relapse were not
expected, because the activation of NF-B is known to stimulate
transcription of the IB gene under physiologic conditions
(15,16).
Two mechanisms may account for this observation, i.e., lack of
induction of IB gene transcription or increased IB
mRNA decay. The first mechanism has been described in the experimental model
of monocyte adherence, where, in contrast to several cytokine genes, the basal
rates of IB gene transcription are not changed after NF-B
activation (19). Therefore,
the nuclear processing of IB mRNA, independent of the
transcription rate, was suggested to be a determinant in the regulation of
IB protein turnover
(19). The low IB
mRNA levels during relapse may also be attributable to increased
destabilization, the second mechanism. Like that of many short-lived mRNA,
including those encoding cytokines and proto-oncogenes, the
3'-untranslated region of IB mRNA contains multiple
AU-rich destabilizing sequence elements (ARE), which are involved in rapid
mRNA decay
(19,23).
This rapid decay of ARE-containing mRNA is mediated by a 20S protein complex
(24), which was recently
partly identified (25). It
includes the ARE-binding protein AUF1, heat shock protein 70 (hsp70), the
translation initiation factor elf4G, and poly(A)-binding protein.
Ubiquitin-proteasome controls the activity of this protein complex and thus
IB mRNA decay.
Lower IB mRNA levels alone do not fully account for the lack
of detection of IB protein for most patients with MCNS who are
experiencing nephrotic relapses. In fact, it has been demonstrated that the
degradation of IB protein is dependent on the proteasome complex
(12). Our data suggest that
the increased proteasome activity in these patients might also be involved in
IB protein downregulation during relapses. The latter hypothesis
is supported by the fact that the degradation of IB was blocked
in the presence of the proteasome inhibitor MG132, as suggested by
accumulation of the IB phosphorylated form.
The mechanisms by which glucocorticoids (the first-line therapy for MCNS)
induce remission are not understood. Most cytokine genes that are upregulated
in MCNS do not carry a glucocorticoid-responsive element, but their promoter
exhibits binding sites for NF-B
(26). Therefore,
glucocorticoid-induced inhibition of NF-B may explain the
downregulation of many cytokines during remission. This effect relies on a
combination of mechanisms. Similar to NF-B, the glucocorticoid receptor
is maintained in an inactive state in the cytoplasm, complexed to hsp90, which
acts as a "protein chaperon" and prevents the targeting of the
glucocorticoid receptor to the nucleus
(27). After binding to
glucocorticoids, the glucocorticoid receptor dissociates from hsp90 and moves
into the nucleus, where it downregulates NF-B activity essentially by
preventing NF-B from binding to DNA
(28,29)
and/or by increasing transcription of the IB gene
(30). The high levels of
IB mRNA in remission support the latter hypothesis.
There was a striking correlation between the activation of NF-B and
the course of the disease. This was not the case for MN, which is
characterized by a nephrotic syndrome similar to MCNS but for which no
NF-B activation was demonstrated in our cases.
Dysregulation of NF-B activity was previously reported for some
immune-mediated diseases, such as systemic sclerosis
(31). In contrast to MCNS,
patients with active systemic lupus erythematosus exhibit decreased
NF-B activity associated with a lack of p65 subunit protein expression
in nuclear and whole-lymphocyte extracts
(32). This activity remains
weak with time and is independent of the disease index activity and steroid
therapy. The regulation of IB has not yet been reported.
The restoration of IB protein and the associated inhibition
of NF-B in remission argue against a constitutive defect of
IB in MCNS, as reported for Hodgkin's disease
(20,33).
In Hodgkin cells, the IB protein is either truncated or
functionally defective or displays a very short half-life because of excessive
phosphorylation, whereas the IB mRNA levels remain normal. In
addition, constitutive activation of NF-B in some Hodgkin cell lines
results from the release of a soluble factor
(33).
In conclusion, this study supports an important role for the NF-B
pathway in the pathogenesis of MCNS. The downregulation of IB
mRNA and protein levels during relapse involves both transcriptional and
post-transcriptional mechanisms and suggests alteration of the
NF-B/IB autoregulatory feedback loop.
|
Acknowledgments |
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|
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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