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*
Department of Internal Medicine, Division of Endocrinology, Metabolism and
Pathobiochemistry, University of Tübingen,
Tübingen, Germany.
Institute of Pathology, University of Munich, Munich, Germany.
Medical Center Hospital Bergmannsheil, Bochum, Germany.
Correspondence to Dr. Erwin D. Schleicher, Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, Otfried-Müller-Straße 10, D-72076 Tübingen, Germany. Phone: + +49 7071 29 87599; Fax: + +49 7071 29 5974; E-mail: enschlei{at}med.unituebingen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among many diverse effects, TGF-ß1 promotes the accumulation of extracellular matrix by increasing the synthesis of extracellular matrix components and by reducing matrix degradation. Therefore, chronically elevated expression of TGF-ß1 is suggested to be the major mediator of progressive fibrosis in renal diseases associated with sclerosis and namely in diabetic nephropathy (12,13). The therapeutic effect of inhibiting TGF-ß1 action by injecting neutralizing TGF-ß1 antibodies has been shown in experimental glomerulonephritis and experimental diabetic nephropathy (13,14). These data demonstrate that inhibition of TGF-ß1 activity results in suppression of disease-induced glomerular matrix accumulation.
Little is known about the molecular mechanism of the pathologic dysregulation of TGF-ß1 gene expression in diabetes. Previous studies revealed that the promoter regions of the human TGF-ß1 gene contain consensus sequences for the transcription factor activating protein-1 (AP-1), which mediate the phorbol ester responsiveness of the TGF-ß1 promoter (15,16). Phorbol esters are also known to activate protein kinase C (PKC), and high glucose-induced activation of PKC has been suggested to be involved in the development of diabetic nephropathy (17,18). In particular, hyperglycemiainduced synthesis of extracellular matrix proteins has been shown to be associated with activation of PKC in mesangial cells (19). Furthermore, recent studies have shown that treatment of diabetic rats with an oral PKC-ß inhibitor ameliorates vascular dysfunctions (20). Taken together, these data suggest a possible link between glucose-mediated PKC activation and AP-1—mediated TGF-ß1 gene activation.
The present study was designed for better understanding of the molecular mechanism of the high glucose-induced TGF-ß1 overexpression. We demonstrate that high glucose stimulates TGF-ß1 promoter activity and increases specific binding of AP-1 proteins to the corresponding consensus DNA sequences. Furthermore, we show that PKC- and p38 MAPK-dependent pathways mediate the activation of the TGF-ß1 promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]UTP and [-32P]dATP were from
Hartmann (Braunschweig, Germany); antibodies against AP-1/c-Jun (catalog
number 44X), c-Jun (1694X), JunB (73X), JunD (74X), c-Fos (253X), CREB-1
(186X), ATF-1 (270X), and ATF-2 (187X) were from Santa Cruz Technologies
(Santa Cruz, CA); and antibodies against phospho-c-Jun ser-63 and
phospho-ATF-2 thr-71 were from New England Biolabs (Schwalbach, Germany).
Cell Culture
Mesangial cells isolated from porcine glomeruli were cultured and
characterized as described previously
(21). Human mesangial cells
were cultured as described by the supplier. For experimental purposes, RPMI
1640 containing 6 mM or 30 mM glucose was used and FCS serum was substituted
by 2% Ultroser. For equal osmolarity, NaCl was added.
Preparation of Luciferase Reporter Constructs and Site-Directed
Mutagenesis
The TGF-ß1-luciferase reporter vector pGL3wt was generated by
insertion of nucleotides -453 to +11 of the human TGF-ß1 promoter into
the KpnI and BglII sites of the vector pGL3b, which contains
the firefly (photinus pyralis) luciferase gene. Base substitutions
were made by oligonucleotide-mediated mutagenesis as described previously
(22) with modifications. The
mutagenic primers are shown in Table
1. Clones were tested by restriction enzyme digestion if possible,
and positive clones were sequenced.
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Transfection Methods
Mesangial cells were transfected with Superfect according to the
instructions of the supplier. One d before transfection, 2.0 x
105 cells/well were seeded in six-well plates with 2 ml of growth
medium. For one well, 2 µg total of DNA and 100 µl of RPMI 1640 without
supplements were premixed, 10 µl of Superfect was added, and the samples
were mixed for 10 s. After 10 min, the samples were diluted with 600 µl of
growth medium and the total volume was transferred to the cells. After 3 h,
the different culture media with or without inhibitors were added and cells
were harvested after 24 h. For transfection experiments with PMA, we used the
Ca3(PO4)2-DNA-coprecipitation-method
(23) because Superfect
interfered with PMA. For one well, 4 µg total of DNA, 36 µl of
H2O, 160 µl of Hepes-buffered saline 5/4 (50 mM Hepes-KOH pH
7.05, 172.5 mM NaCl, 6.25 mM KCl, 875 µM Na2HPO4, 7
mM Glucose), and 10.4 µl of 2.5 M CaCl2 were mixed for 20 s,
incubated for 20 min, and added to the medium. After 4 h, the medium was
replaced by 1 ml of 10% glycerol in growth medium and cells were incubated for
3 min. Then the glycerol medium was removed and the culture medium was added.
After 20 h, the cells were stimulated with 0.5 µM PMA and harvested after 9
h. For normalization of transfection efficiencies, we cotransfected 0.25 µg
of pSV-ß-galactosidase control vector. To study the effect of PMA, we
used 0.5 µg pRL-TK vector, which contains the seapansy (renilla
reniformis) luciferase gene under control of the herpes simplex virus
thymidine kinase promoter, because the pSV- ß-galactosidase vector
responds to PMA.
Reporter Gene Assays
Transfected cells were washed once with phosphate-buffered saline,
incubated with 150 µl of lysis buffer from the ß-galactosidase assay
for 30 min, and harvested. After 2 min of centrifugation, the supernatants
were stored at -80°C or immediately used for the measurements.
ß-Galactosidase and firefly luciferase activities were determined
according to the instructions of the manufacturers. Co-transfected firefly and
seapansy luciferase activities were assayed with the dual-luciferase reporter
assay system. Chemiluminescence was determined with a Magic Lite Analyzer from
Ciba Corning (Fernwald, Germany). All transfection experiments were repeated
at least three times.
Electrophoretic Mobility Shift Assay
Cells (6.0 x 106) were seeded onto 15-cm culture dishes
with 15 ml of culture medium and incubated with 6 mM or 30 mM glucose for 24 h
or 0.5 µM PMA for 6 h before harvesting. Nuclear proteins were prepared as
described recently (22).
Appropriate synthetic oligonucleotides (see
Table 1) were end-labeled with
[-32P]dATP (3000 Ci/mM) and Klenow enzyme and were incubated
with up to 13 µg of nuclear protein in 20 µl of 7 mM Hepes-KOH (pH 7.9),
100 mM KCl, 3.6 mM MgCl2, and 10% glycerol on ice for 20 min.
Poly[d(I-C)] (0.05 mg/ml) was added as unspecific competitor. The samples were
run on a 5% nondenaturing polyacrylamide gel in a buffer containing 25 mM
Tris-HCl (pH 8.0), 190 mM glycine, and 1 mM ethylenediaminetetraacetate. Gels
were dried and analyzed by autoradiography. In supershift experiments, 4 µg
of specific antibody were incubated with the binding reaction mixture for 1 h
on ice before adding the radiolabeled DNA fragment.
Western Blots
Nuclear extracts of mesangial cells were separated by sodium dodecyl
sulfate polyacrylamide (7.5%) gel electrophoresis. Proteins were transferred
to nitrocellulose by semidry electroblotting (transfer buffer, 48 mM Tris, 39
mM glycine, 0.0375% sodium dodecyl sulfate, 20% (vol/vol) methanol). Then
nitrocellulose membranes were blocked with
NaCl/ethylenediaminetetraacetate/Triton (NET) buffer (150 mM NaCl, 50 mM
Tris/HCl [pH 7.4], 5 mM EDTA, 0.05% Triton X-100, 0.25% gelatin) and incubated
with the first antibody (diluted 1:1000 in NET) overnight at 4°C. After
the membranes were washed with NET, they were incubated with horseradish
peroxidase-conjugated anti-rabbit or anti-goat IgG for 1 h at room
temperature. Visualization of immunocomplexes was performed by enhanced
chemiluminescence as described
(24).
Statistical Analysis
Results presented are derived from at least three independent experiments.
Means ± SEM were calculated and groups of data were compared using
t test. Statistical significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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High Glucose Increased the Activity of the Human TGF-ß1
Promoter
Transfection experiments revealed a high glucose-induced stimulation of the
human TGF-ß1 promoter fragment -453/+11 as assayed by luciferase
activity. The promoter activity was significantly increased 1.8- to 1.9-fold
after 24 h of incubation with high glucose compared with cells grown in 6 mM
glucose (Table 2). This high
glucose-induced activation was similar in porcine mesangial cells and in human
mesangial cells. For further experiments, porcine mesangial cells were
used.
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Effect of the High Glucose Concentrations and PMA on TGF-ß1
Promoter Activity and Localization of the Responsible cis-Regulatory
Elements
To study whether the two AP-1 binding sites -418/-412 and -371/-364 mediate
the high glucose effect, we mutated both promoter regions by site-directed
mutagenesis (Figure 2B). As
shown in Figure 3A, mutation in
AP-1 box A or box B or in both boxes completely prevented the high
glucoseinduced increase of the TGF-ß1 promoter activity and reduced the
promoter activity in both glucose conditions below control levels. The lowest
remaining promoter activity was found after mutation of both AP-1 sites
(approximately 20% in normal glucose and in high glucose condition), whereas
mutation of AP-1 box B had the lowest effect on promoter activity
(approximately 50%). Furthermore, we examined the regulatory function of one
GC-box, which represents a high-affinity stimulating protein 1 (Sp 1) binding
site. Mutation in this GC-box (Figure
2B) reduced the promoter activity in normal and high glucose
conditions; however, the high glucose-induced stimulation was essentially
unaffected (Figure 3A).
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Because addition of PMA increased TGF-ß1 mRNA levels in mesangial cells, we studied whether PMA induced an increase in TGF-ß1 promoter activity and whether AP-1 binding sites are involved in this activation. Stimulation with PMA led to a 2.3-fold increase of the wild-type TGF-ß1 promoter activity in the mesangial cells (Figure 3B). Mutation in the AP-1 box A and box B or in both boxes prevented the activation by the phorbol ester completely. These results clearly indicate an important role of both AP-1 boxes in hyperglycemia- and PMA-induced activation of the TGF-ß1 promoter and further emphasize that these cis-regulatory elements are responsible for an AP-1—mediated activation.
Effect of Inhibitors of AP-1 and Sp1 on the Glucose-Induced
Stimulation of the TGF-ß1 Promoter
To elucidate further the role of the transcription factors AP-1 and Sp1 in
high glucose-induced TGF-ß1 promoter activation, transfection experiments
with the known inhibitors of AP-1 and Sp1, curcumin
(25) and mithramycin
(26), were performed
(Figure 4). In the presence of
curcumin, the high glucose effect was prevented. Addition of mithramycin did
not abolish the high glucose effect. These data further support the functional
participation of AP-1 in the regulation of TGF-ß1 promoter activity by
high glucose.
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Effect of Elevated Glucose Concentrations on the Amount of AP-1
Proteins
To study the regulation of the activity of AP-1 proteins in mesangial cells
by high glucose, we determined in Western blot analysis whether high glucose
alters nuclear levels of AP-1 proteins. As shown in
Figure 5, A through D, no
significant differences of c-Jun, JunB, JunD, and ATF-2 could be detected
after incubation of mesangial cells with high glucose for 40 h. In Western
blots with nuclear extracts from cells cultured with high glucose for 24 h,
the same results were obtained (data not shown). In contrast, the amount of
c-Fos was significantly increased in nuclear extracts after high glucose
incubation for 24 h (Figure 5E)
and returned to basal levels after 40 h of high glucose incubation (data not
shown). Stimulation of mesangial cells with PMA also led to an increased level
of c-Fos (Figure 5E).
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High Glucose-Induced Activation of the p38 Pathway Is Inhibited by SB
203580 and Bisindolylmaleimide I
In addition to enhanced gene expression, the transcriptional activity of
AP-1 proteins may be regulated by site-specific phosphorylation by the
stress-activated kinases JNK and p38 MAPK. Therefore, immunoblotting was
performed with nuclear extracts from mesangial cells cultured with normal and
high glucose for 24 h, and phosphorylated AP-1 proteins were detected using a
phospho-specific c-Jun antibody and a phospho-specific ATF-2 antibody
(Figure 6A). Stimulation of
mesangial cells with 10 µg/ml anisomycin, which is known to activate both
JNK and p38 MAPK (27),
resulted in an additional band of the estimated molecular mass of 46 kD
corresponding to the expected size of the phosphorylated c-Jun
(Figure 6A). No bands with
similar size were detected in nuclear extracts from normal and high glucose
conditioned cells with the phospho-Jun ser-63 antibody, indicating the absence
of this phosphorylated form of c-Jun. The lower band detected with the
phospho-specific c-Jun antibody could be explained by less phosphorylated
forms of c-Jun, but the amount of these proteins was similar in normal and
high glucose conditioned cells, whereas treatment with 10 µg/ml anisomycin
increased the intensity of this band. In contrast, the amount of a
phosphorylated form of ATF-2 was markedly increased by high glucose,
indicating an activation of the p38 MAPK pathway by high glucose. Stimulation
of the cells with high glucose for 40 h yielded the same results (data not
shown).
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To elucidate the signal transduction pathways activated by high glucose potentially leading to enhanced gene activation of TGF-ß1, we studied the effect of the PKC inhibitor bisindolylmaleimide and p38 MAPK inhibitor SB 203580 on the activity of the p38 MAPK pathway by quantification of the phosphorylated form of the endogenous substrate ATF-2. In nuclear extracts of mesangial cells, phosphorylated ATF-2 and ATF-2 protein were detected by immunoblotting and quantified (Figure 6B). High glucose increased the level of the phosphorylated forms twofold, whereas 1 µM SB 203580 and 50 and 500 nM bisindolylmaleimide prevented this increase. These data show that addition of either one of the substances is sufficient to inhibit the activation of p38 MAPK by high glucose in mesangial cells.
The High Glucose-Induced Activation of the TGF-ß1 Promoter Is
Prevented by Inhibitors of PKC and p38 MAPK
The results obtained with the phospho-specific ATF-2 antibody suggest a
participation of activated p38 MAPK in the high glucose-stimulated TGF-ß1
gene expression. To study whether these signaling pathways are functionally
involved in TGF-ß1 promoter activation, we transfected and cultured
mesangial cells in the presence of the inhibitors of the p38 MAPK or the PKC
pathway. Addition of 1 and 10 µM SB 203580 as well as 50 and 500 nM
bisindolylmaleimide I prevented the activation of the TGF-ß1 promoter,
whereas the promoter activity in normoglycemic conditions was unaffected
(Figure 7). The results clearly
indicate a participation of both the p38 MAPK and the PKC pathway in the
activation of the TGF-ß1 promoter by ambient high glucose
concentrations.
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Enhanced Binding of Nuclear Proteins from Cells Cultured in High
Glucose to AP-1 Boxes A and B of the TGF-ß1 Promoter
Our data show that both AP-1 boxes are functionally involved in mediating
the high glucose effect. To evaluate whether this effect is mediated by
increased binding of AP-1 to the corresponding DNA consensus sequences in the
TGF-ß1 promoter region, we performed electrophoretic mobility shift
assays with wild-type or mutated oligonucleotides
(Table 1). In nuclear extracts
from cells cultured in high glucose concentrations for 24 h, an enhanced
binding to the AP-1 B and to a much lesser extent to the AP-1 A fragments was
observed (Figure 8A, lanes 2
and 5). Similar results were obtained with nuclear extracts from cells
cultured for 40 h with high glucose (data not shown). The specificity of the
binding was assessed by using the mutated oligonucleotides. As shown in
Figure 8A (lanes 3 and 6),
upper bands (arrows) disappeared almost completely whereas the bands marked
with a bracket remained unchanged, indicating unspecific binding of nuclear
proteins. Furthermore, after addition of an excess of cold wild-type
nucleotides, these bands were still apparent (AP-1 B:
Figure 9B, lanes 6 and 7; AP-1
A: data not shown). Binding of nuclear proteins to the oligonucleotide
containing a high affinity Sp1 binding site was not influenced by high glucose
(Figure 8B, lane 2). With the
mutated Sp1 oligonucleotide, only a very weak binding was detectable
(Figure 8B, lane 4); the
shifted band disappeared completely after addition of an excess of cold
wild-type oligonucleotide. These results indicate that the response to high
glucose is regulated by enhanced binding of proteins.
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Identification of Nuclear Proteins Binding to AP-1 Box B of the
TGF-ß1 Promoter by Specific Anti—AP-1 Antibodies
To identify the proteins involved in high glucose-induced AP-1 activation
mobility, we performed shift experiments with antibodies specific for several
AP-1 proteins. In experiments with AP-1 box A only, the anti—AP-1
antibody attenuated the binding of nuclear proteins, characterizing the AP-1
box A as a weak AP-1 binding site (data not shown). In experiments with AP-1
box B, the presence of anti—AP-1, anti-JunD, or anti—c-Fos
antibodies caused an almost complete disappearance of the specific shifted
band in normal and in high glucose-treated cells
(Figure 9A, arrow), whereas
addition of anti-JunB antibody (Figure
9A, lanes 3 and 8) had little effect. Noteworthy is that the high
glucose-induced increase in binding of AP-1 was prevented by anti—AP-1
antibody. To detect supershifted bands, we extended the run time of
electrophoresis and increased the exposure time. Under these conditions, the
binding of JunD and c-Fos to the AP-1 box B is obvious
(Figure 9C). Because the data
described above indicate an involvement of the p38 MAPK in the high
glucose-induced activation of the TGF-ß1 promoter and the
AP-1—related proteins CREB-1, ATF-1, and ATF-2 are activated by this
pathway, we studied the binding of these transcription factors to the AP-1
boxes. Specific antibodies, however, could not detect any of these proteins in
the DNA binding complex derived from high glucose-stimulated mesangial cells
(Figure 9B, data for AP-1 box A
are not shown). In conclusion, the supershift analyses identified AP-1 box A
as a weak and box B as a stronger AP-1 binding site and JunD and c-Fos as
component of the complex bound by AP-1 box B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several laboratories showed an increased TGF-ß1 mRNA level in cultured mesangial cells when stimulated with glucose (7,8). Our present observation of a 1.9-fold increase of the TGF-ß1 promoter activity after 24 h of exposure to high glucose is in accordance with the elevation of the TGF-ß1 mRNA and protein levels after 48 h of glucose stimulation (21). Moreover, the data suggest that high glucose stimulates TGF-ß1 gene expression rather than influences TGF-ß1 mRNA stability. Furthermore, by mutation of the AP-1 binding sites, we could demonstrate the important role of both AP-1 binding sites in mediating the high glucose effect. The finding that mutations of one AP-1 site reduced TGF-ß1 promoter activity under control levels suggests a participation of the AP-1 binding sites in the regulation of basal promoter activity. Our results that mutation in both AP-1 binding sites diminished the promoter activity more than mutation in one AP-1 box indicate a cooperative effect of the two binding sites in the regulation of the promoter activity in basal and hyperglycemic conditions (28). The AP-1 boxes are located in the promoter region -453 to -323, which also is responsible for phorbol ester responsiveness and autoinduction via AP-1 proteins (16). Because AP-1 proteins are activated by a variety of stimuli, the role of these two AP-1 sites is not restricted to high glucose. Recently, Hoffman et al. (29) reported a high glucose-induced de novo synthesis of TGF-ß1 mRNA in murine mesangial cells and an induction of the murine TGF-ß1 promoter activity by high glucose after 24 h. However, the regulation of the glucose responsiveness of the murine TGF-ß1 promoter cannot be explained by AP-1 sites because the glucose-responsive region of the mouse promoter was localized between -835 and -406 upstream of the first transcriptional start site, where no AP-1 binding sites reside (30).
The finding that high glucose stimulates the human TGF-ß1 promoter activity via AP-1 binding sites is strongly supported by the glucose-induced enhanced binding of nuclear proteins to both AP-1 sites. The AP-1 proteins JunD and c-Fos were identified in the protein complex binding to AP-1 box B, and the enhanced expression of c-Fos by high glucose suggests the involvement of this protein in the glucose-dependent activation of the TGF-ß1 promoter. However, both AP-1 proteins may also participate in basal promoter regulation. The observed weak DNA binding activity of AP-1 box A alone, which can be explained by the nontypical T A switch at position 7 of the AP-1 consensus sequence (Figure 2A), may indicate that further promoter sequences, e.g., AP-1 box B, are necessary for the function of AP-1 box A. Recently, increased binding of AP-1 to a synthetic oligonucleotide containing two PMA responsive elements in mesangial cells cultured in high glucose was detected after 3 d of high glucose conditioning (31). However, we used AP-1 binding consensus sequences of the TGF-ß1 promoter, which may exert a higher affinity for the high glucose-induced protein complexes.
The present study supports the suggested involvement of PKC activation in
the development of diabetic nephropathy
(17,18).
However, the detailed mechanism of the link between hyperglycemia-induced PKC
activation and TGF-ß1 gene activation has not been reported. Three lines
of evidence support a PKC involvement in TGF-ß1 promoter regulation by
high glucose. First, activation of the TGF-ß1 promoter by the PKC
activator PMA and glucose was mediated via the same AP-1 binding sites.
Second, application of the PKC inhibitor bisindolylmaleimide I prevented the
high glucose-induced increase of promoter activity. Third, high glucose and
PMA led to a transient increase of c-Fos, which is in line with the reports of
Kreisberg et al.
(32). Particularly, the PKC
ß isoform seems to be involved in mediating high glucose effects on
TGF-ß1 gene expression and deposition of matrix components
(18,33).
The concentration of the PKC inhibitor bisindolylmaleimide I used in the
present study shows high selectivity for the PKC , ß1, and ß2
isoforms (34). Because the PKC
ß2 isoform could not be detected in our mesangial cells by Western
blotting (S. Facchin and E. Schleicher, unpublished results), participation of
the PKC and ß1 isoforms is very likely in mediating the high
glucose-induced TGF-ß1 gene expression.
The involvement of p38 MAPK in the upregulation of TGF-ß1 promoter activity by high glucose was shown by three independent results. p38 MAPK is activated by high glucose as demonstrated by enhanced phosphorylation of its endogenous substrate ATF-2 (35); this phosphorylation is prevented by the p38 MAPK inhibitor SB 203580 and the PKC inhibitor bisindolylmaleimide I, and the identical concentrations of these inhibitors suppress the upregulation of TGF-ß1 promoter activity by high glucose. The activation of p38 MAPK by high glucose was also demonstrated in mesangial-like smooth muscle cells (36). Furthermore, our data suggest that PKC is involved in the activation of p38 MAPK. The activated p38 MAPK pathway may mediate the enhanced binding of AP-1 to the high glucose-responsive AP-1 sites of the TGF-ß1 promoter by increasing their DNA binding activity or inducing protein-protein interactions, thereby leading to enhanced transcriptional activity (35). However, the AP-1—related transcription factors and substrates of p38 MAPK ATF-1, ATF-2, and CREB-1 could not be detected in the protein complex binding to the AP-1 sites of the TGF-ß1 promoter. Therefore, an as yet unidentified protein may be the p38 MAPK substrate, which is involved in the glucose response of the TGF-ß1 promoter, whereas the recently reported increased stabilization of mRNA by p38 MAPK (37) also provides a possible link to the enhanced binding of transcription factors to the glucose-responsive sites of the TGF-ß1 promoter.
In conclusion, our results indicate that hyperglycemia stimulates human TGF-ß1 gene expression via two adjacent AP-1 binding sites in cultured mesangial cells. The increased TGF-ß1 promoter activity is mediated by enhanced binding activity of AP-1 and is regulated by PKC- and p38 MAPK-dependent pathways. Taken together, our data link hyperglycemia and the enhanced expression of the prosclerotic cytokine TGF-ß1 on a molecular basis, providing an improved insight into the pathogenetic mechanism of the development of diabetic nephropathy.
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Acknowledgments |
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 M. A. Friese, J. Wischhusen, W. Wick, M. Weiler, G. Eisele, A. Steinle, and M. Weller RNA Interference Targeting Transforming Growth Factor-{beta} Enhances NKG2D-Mediated Antiglioma Immune Response, Inhibits Glioma Cell Migration and Invasiveness, and Abrogates Tumorigenicity In vivo Cancer Res., October 15, 2004; 64(20): 7596 - 7603. [Abstract] [Full Text] [PDF]
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 C. Weigert, K. Brodbeck, H. Staiger, C. Kausch, F. Machicao, H. U. Haring, and E. D. Schleicher Palmitate, but Not Unsaturated Fatty Acids, Induces the Expression of Interleukin-6 in Human Myotubes through Proteasome-dependent Activation of Nuclear Factor-{kappa}B J. Biol. Chem., June 4, 2004; 279(23): 23942 - 23952. [Abstract] [Full Text] [PDF]
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