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*
Medizinische Poliklinik, Division of Nephrology, University of Bonn,
Germany
Division of Nephrology, Medizinische Hochschule Hannover,
Germany.
Correspondence to Dr. Dirk Bokemeyer, Medizinische Poliklinik, University of Bonn, Wilhelmstrasse 35-37, 53111 Bonn, Germany. Phone: +49 228 2872263; Fax: +49 228 2872266; E-mail: bokemeyer{at}uni-bonn.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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Mitogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduce extracellular cues to intracellular responses (2). Because in vitro studies have shown MAP kinases to be an intracellular checkpoint in the control of cellular growth, these might be crucial mediators of cellular proliferation in renal diseases mediated by extracellular stimuli. Extracellular mediators suggested to contribute to the pathogenesis of proliferative glomerulonephritis, e.g., platelet-derived growth factor, are known to stimulate extracellular signal-regulated kinase (ERK), the archetypal MAP kinase, in vitro in a variety of cell lines (1,3, 4, 5, 6, 7). ERK belongs to the group of serine/threonine kinases and regulates the expression of many genes by phosphorylation of several transcription factors (2,8, 9, 10). Binding of extracellular stimuli to G protein-coupled receptors or protein-tyrosine kinase receptors induces the formation of GTP-Ras, which in turn induces the sequential activation of cytoplasmic protein kinases leading to phosphorylation and activation of MEK (MAP kinase/ERK kinase) (9). MEK, the specific activator of ERK, is a dual specificity protein kinase that phosphorylates both threonine and tyrosine regulatory sites in ERK (11). Although an extensive body of data describes the pivotal role of this signaling pathway in the control of cellular proliferation in vitro (2,12, 13, 14, 15), little is known about the role of ERK in physiologic or pathophysiologic conditions or its activation in vivo. Recently, we demonstrated in anti-glomerular basal membrane (GBM) GN rats for the first time an activation of ERK in vivo that accompanied cellular proliferation (16). These data pointed to ERK as a regulator of cellular growth in anti-GBM GN. In contrast to ERK, the more recently described MAP kinases stress-activated protein kinase (SAPK) and p38 MAP kinase are thought to inhibit cell growth and to induce cellular apoptosis (17). However, their role in physiologic or pathophysiologic conditions and their mode of activation in vivo are unknown.
In the present study, we induced a mesangioproliferative GN in rats by injection of anti-Thy1.1 antibody. The early phase of mesangiolysis after induction of the anti-Thy1.1 GN is followed by an overshooting proliferative phase that resembles the human mesangioproliferative GN (18). Recent studies demonstrate that the early (mesangiolysis) (19,20) as well as the late phase (resolution from hypercellularity) (21) is associated with apoptosis. Therefore, this disease model allowed us to examine the role of MAP kinases in the proliferative phase as well as in phases that were associated with apoptosis during the time course of the anti-Thy1.1 GN in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of the Anti-Thy1.1 GN
All animal experiments were approved by the local review boards.
Anti-Thy1.1 mesangial proliferative GN was induced in male Wistar rats
(Charles River, Sulzfeld, Germany) weighing 150 to 160 g by injection of 1
mg/kg monoclonal anti-Thy1.1 antibody (clone OX-7; European Collection of
Animal Cell Cultures, Salisbury, United Kingdom). A total of 20 animals was
sacrificed and nephrectomized 2 h and 6, 10, and 14 d after injection of the
anti-Thy1.1 antibody. A renal cortical section was obtained for light
microscopy. Another cortical section was directly lysed in Triton X-100 buffer
(see below), while the rest of the cortical tissue (about 80% of the whole
cortex) was used to generate a preparation of glomeruli using standard sieving
methods (22) before lysis in
Triton X-100 buffer.
Six animals were used to examine the effect of treatment with heparin on the activity of renal MAP kinases. Rats were treated with heparin dissolved in phosphate-buffered saline (PBS) (100 U heparin/100 g body wt) or PBS by intraperitoneal micro-osmotic pumps (model Alzet 2001; Charles River) from day 2 to day 5 after injection of the anti-Thy1.1 antibody. The rats were sacrificed and examined on day 6.
Renal Morphology
Tissue for light microscopy was fixed in methyl Carnoy's solution
(23) and embedded in paraffin.
Four-micrometer sections were stained with the periodic acid-Schiff (PAS)
reagent and counterstained with hematoxylin. In PAS-stained sections, the
number of mitoses within 100 glomerular tufts was determined.
A murine IgM monoclonal antibody (clone F7-26) to single-stranded DNA (Alexis Corp. San Diego, CA) was used to detect cells in the early stages of apoptosis (24). In addition, cellular death in renal sections was assayed by the terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling method (25).
Western Blot Analysis
Whole cortical tissue or isolated glomeruli were homogenized in 2 ml of
Triton X-100 lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl chloride, 0.1 mM
sodium orthovanadate) at 4°C. After incubation for 5 min, lysates were
centrifuged at 4°C for 15 min at 10,000 x g. The soluble
lysates were mixed 1:4 with 5x Laemmli buffer and heated for 5 min at
95°C. Soluble lysates (80 µg) were loaded per lane and separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4 and 10%
acrylamide for stacking and resolving gels, respectively. Protein was
transferred to nitrocellulose membranes (pore size, 0.45 µm; Schleicher
& Schuell, Keene, NH) and probed with polyclonal antibodies against the
C-terminal peptide of p42 ERK
(26), p46 SAPK, or p38 MAP
kinase (both from Santa Cruz Biotechnology), polyclonal antibodies against the
phosphorylated regulatory sequences of ERK (Santa Cruz), SAPK, or p38 MAP
kinase (both from New England Biolabs, Beverly, MA), or with a monoclonal
antibody against MEK-1 (Transduction Laboratories, Lexington, KY). The primary
antibodies (diluted 1:1000) were detected using horseradish
peroxidase-conjugated rabbit anti-mouse IgG or horseradish
peroxidase-conjugated protein A and visualized by Amersham ECL system after
extensive washing of the membranes. The intensity of the identified bands was
quantified by densitometry using the BioRad Gel Doc 1000 system with the
software Multi-Analyst (Bio-Rad, Munich, Germany).
Kinase Activity Assays
A total of 400 µg of soluble lysates (as described above) was incubated
for 90 min with 2 µl of polyclonal antibody recognizing p42 ERK
(26), p46 SAPK, or p38 MAP
kinase (both from Santa Cruz). Immuncomplexes were adsorbed to protein
A-Sepharose, washed twice with lysis buffer and twice with kinase buffer (10
mM MgCl2, 20 mM Hepes, pH 7.4, containing 200 µM
Na-orthovanadate), and resuspended in 60 µl of kinase buffer containing 50
µM ATP. The final reaction buffer also contained 2 µg of GST-Elk1 for
ERK- or 2 µg of GST-ATF2 for SAPK and p38 MAP kinase assays. The reaction
was initiated by incubation at 30°C for 45 min. Thereafter, 20 µl of
4x Laemmli buffer was added to terminate the reaction, and samples were
subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
Proteins were then analyzed by Western blot analysis, as described above, with
polyclonal anti-phospho-Elk1 or anti-phospho-ATF2 antiserum (both from New
England Biolabs) recognizing only the phosphorylated forms of Elk1 and ATF2,
respectively.
Statistical Analyses
Values are expressed as means ± SEM. Statistical analyses were
performed using the t test. P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated the in vivo activity of MAP kinases both in the renal cortex and in isolated glomeruli at 2 h and on days 6, 10, and 14 after induction of anti-Thy1.1 GN. An anti-ERK Western blot analysis that separates unphosphorylated from phosphorylated protein forms (bands with delayed mobility indicate phosphorylated ERK), as well as anti-phospho-ERK Western blot analysis were used (Figures 2A and 3A, top and middle panels). The anti-phospho-ERK Western blot analysis identified only ERK forms that were phosphorylated within its regulatory sequence. In addition, an immune complex ERK activity assay was performed (Figures 2A and 3A, bottom panels, and Figure 2B). In crude cortical lysates, we detected a slight increase in the activation of ERK at 2 h with a maximal activation at days 6 and 10. At later time points, a decrease in ERK activity was detectable (Figure 2). More significantly, in isolated glomeruli an even more pronounced increase in ERK activity was detected with a maximal activation at day 6, indicating that the glomeruli were the major source of the increased cortical ERK activity in anti-Thy1.1 GN (Figure 3).
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In additional experiments, the activity of SAPK was examined by Western blot analysis and immune complex assays. As shown in Figure 4, a moderate increase in SAPK activity was detectable in whole cortical lysates as well as in isolated glomeruli with a time course similar to the described ERK activation. However, in isolated glomeruli (Figure 4B) the magnitude of SAPK activation, especially with regard to the results of the SAPK assay (Figure 4B, bottom panel), was less prominent than the activation of ERK. The more obvious phosphorylation of glomerular SAPK (Figure 4B, top panel) might be due to a phosphorylation of only one of both regulatory residues that is not sufficient for SAPK activation. Such a mechanism has been described for the ERK cascade in the case of only moderate ERK activating signaling (27). Equal loading of cortical and glomerular proteins was confirmed by reprobing immunoblots with a polyclonal antiserum detecting total SAPK (Figure 4A, bottom panel, and Figure 4B, middle panel).
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In contrast to ERK and SAPK, the p38 MAP kinase followed a different pattern of activation throughout the course of the present study. As shown in Figure 5, no increase in p38 MAP kinase activity was notable at any time point examined in cortical or glomerular tissue compared with control animals. Rather, a significant decrease in p38 MAP kinase activity was detectable as early as 2 h after induction of anti-Thy 1.1 GN. On day 6, the p38 MAP kinase activity was almost completely suppressed in whole cortical lysates (Figure 5A) as well as in isolated glomeruli (Figure 5B, top panel). However, at later time points p38 MAP kinase activity recovered to control levels. Equal loading of cortical and glomerular proteins was confirmed by reprobing immunoblots with an polyclonal anti-p38 MAP kinase antiserum (Figure 5, A and B, bottom panels).
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Expression of MAP Kinase Kinases in Anti-Thy1.1 GN
Previous in vitro
(28,29)
and in vivo (16)
studies suggested a protein upregulation of the ERK-activating kinases MEK1
and MEK2 to be important in the long-term regulation of ERK. To examine
whether this mechanism is also involved in the observed ERK activation, we
assayed the MEK protein expression by Western blot analysis in the anti-Thy1.1
GN. As shown in Figure 6, an
increase in MEK1 and more dramatically MEK2 protein levels was observed in
animals with anti-Thy1.1 GN in whole cortical samples and even more pronounced
in isolated glomeruli. Thus, in a manner similar to ERK activation, MEK
protein levels were found elevated principally in glomeruli. Furthermore, the
time course of MEK1 and MEK2 expression was similar to that of ERK
activation.
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Effect of an Antiproliferative Treatment with Heparin on MAP Kinase
Activities in Anti-Thy1.1 GN
To examine the functional relevance of the observed changes in MAP kinase
activities throughout the time course of the present study, we treated the
animals with heparin (250 U/100 g body wt) from day 2 to day 5 after injection
of the anti-Thy1.1 antibody. On day 6, the rats were sacrificed and renal MAP
kinase activities were examined as described above. As expected, heparin
reduced the glomerular proliferation at day 6 as detected by a reduction of
glomerular mitotic figures (Figure
7). As shown in Figure
8A and summarized in Figure
8B, heparin treatment induced a decrease in ERK activity on day 6
compared to animals treated with PBS. In addition, p38 MAP kinase activation,
which was markedly reduced in animals receiving PBS, returned to control
levels in animals treated with heparin as detected by anti-phospho-p38 MAP
kinase Western blot analysis (Figure
9, top panel). Equal loading of glomerular lysates was confirmed
by reprobing immunoblots with an antiserum detecting total p38 MAP kinase
(Figure 9, middle panel). These
results were confirmed by p38 MAP kinase immunocomplex assay using ATF2 as the
substrate (Figure 9, bottom
panel)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extracellular stimuli such as cytokines bind to their receptors at the cell membrane, inducing the interaction of multiple intracellular signaling molecules and sequential induction of cytoplasmic protein kinases leading to the activation of MAP kinases. ERK is activated by the upstream kinase MEK. In vitro studies suggest that the protein levels of MEK are tightly regulated and are involved in the long-term regulation of ERK (28,29). The present study demonstrates an upregulation of MEK protein levels in rats with anti-Thy1.1 GN, thereby increasing the amount of MEK protein available for ERK activation. The elevation of MEK protein levels did correlate with the observed increases in ERK activity. It is therefore likely that the elevated MEK protein levels are contributing to ERK activation in vivo. A similar link between MEK protein levels and ERK activity was demonstrated in anti-GBM GN (16).
Other MAP kinases such as SAPK and p38 MAP kinase are suggested to inhibit cellular proliferation and to induce apoptosis (17,33). Although only moderate changes in SAPK activity were detectable throughout the course of the present study, our data demonstrate a dramatic decrease in p38 MAP kinase activity at day 6 that returned to basal levels by day 14. The observed decreases in p38 MAP kinase activity were accompanied by increased glomerular proliferation. Furthermore, heparin treatment prevented the decrease in p38 MAP kinase activity in anti-Thy GN. To our knowledge, these data demonstrate for the first time such a deactivation of p38 MAP kinase in proliferative diseases in vivo. It is therefore conceivable that the changes in p38 MAP kinase activity might contribute to the increased glomerular cell proliferation in anti-Thy1.1 GN.
Two hours after injection of the anti-Thy1.1 antibody, a significant increase in glomerular apoptosis was demonstrated. However, no decrease in the activity of ERK, which is suggested to prevent apoptosis (17), was detectable. As mentioned above, increases in SAPK or p38 MAP kinase activity are suggested to induce apoptosis (17). Although we detected a mild increase in SAPK activity at 2 h, a similar degree of SAPK activity was observed at later time points (6 d), where the frequency of apoptotic figures had decreased markedly. Furthermore, no increase in p38 MAP kinase activity was detected at 2 h. We therefore conclude that the intracellular regulation of apoptosis in early anti-Thy1.1 GN involves signaling pathways other than MAP kinase cascades.
The initial increase in glomerular monocytes and macrophages in the anti-Thy1.1 GN is known to be followed by a decrease in the number of these infiltrating cells at later time points (18). Because of the kinetics of the observed changes in MAP kinase activities, it seems likely that the resident glomerular cells rather than the infiltrating cells are the site with altered intracellular MAP kinase signaling. Additional studies will be needed to define to what extent glomerular mesangial cells, endothelial cells, or podocytes contribute to the changes in glomerular ERK and p38 MAP kinase activities.
In conclusion, our data suggest that the ERK cascade as well as the p38 MAP kinase are involved in the intracellular regulation of glomerular proliferation in anti-Thy1.1 GN. In addition, the upregulation of MEK1 and MEK2 protein levels seems to play a role in the control of ERK activity in vivo. In contrast to the close correlation of the activity of MAP kinases to changes of glomerular cell proliferation, the intracellular signal transduction by MAP kinase cascades does not seem to be responsible for glomerular apoptosis in early anti-Thy1.1 GN. The presented data provide new insights into the pathogenesis of glomerular proliferation in response to immune injury and might therefore point to new therapeutic strategies in proliferative glomerulonephritis.
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Acknowledgments |
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References |
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