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Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.
Correspondence to Dr. Gunter Wolf, University of Hamburg, University Hospital Eppendorf, Department of Medicine, Division of Nephrology and Osteology, Pavilion 61, Martinistrasse 52, D-20246 Hamburg, Germany. Phone: +49 40 42803 5011; Fax: +49 40 42803 5186; E-mail: WOLF{at}UKE.unihamburg.de
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Recent studies have shown that oxygen radicals may stimulate mitogen-activated protein (MAP) kinases in various nonrenal cell lines (reviewed in reference (5). For example, in rat vascular smooth muscle cells (VSMC), p44/42 MAP kinase is activated by superoxide anions (6). Furthermore, in rat neuronal cells, p44/42 MAP kinase is stimulated by reactive oxygen species and nitric oxide (7).
The present study was undertaken to test whether MAP kinase activation provides a signal transduction link between AngII-induced intracellular generation of reactive oxygen products and the subsequent induction of p27Kip1 and cellular hypertrophy. Our data provide evidence that AngII-induced O2. formation activates p44/42 MAP kinase, as well as p38 MAP kinase, in proximal tubular cells. However, only activation of p44/42 MAP kinase is responsible for enhanced p27Kip1 protein expression and cellular hypertrophy.
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Measurement of O2. Generation in Intact
Cells
Measurement of O2. generation in intact cells was
performed as described previously, using the lucigenin method
(4). Cells were stimulated with
10-7 M AngII (Sigma, Deisenhofen, Germany) for 4 h with or without
preincubation for 1 h with 10-5 M of the specific MAP kinase kinase
1 (MEK1) inhibitor PD98059 [2-(2'-amino-3'-
methoxyphenyl)oxanaphthalen-4-one]
(10) or the p38-specific
inhibitor SB203580
[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole]
(both from Calbiochem, Bad Soden, Germany). Cells were slightly trypsinated
and collected by centrifugation. The pellet was then washed in a modified
Krebs buffer containing 130 mM NaCl, 5 mM Kcl, 1 mM MgCl2, 1.5 mM
CaCl2, 1 mM K2HPO4, and 20 mM Hepes, pH 7.4,
and centrifuged. After washing, cells were resuspended in Krebs buffer
containing 10 mM glucose and 1 mg/ml bovine serum albumin. Measurement of
O2. generation was performed in a luminometer (LB 9501;
Berthold, Wildbad, Germany) and started by addition of 100 µl of lucigenin
(final concentration 500 µM; Sigma). For measurement of
O2. generation, 5 x 106
LLC-PK1 and 107 MCT cells were used. Photon emission was
counted every 15 s for up to 15 min. A buffer blank (reaction buffer with
lucigenin, but without cells) was subtracted from each reading. To calculate
the amount of O2. synthesized by intact cells, a
standard curve was generated as described previously
(4), and total photon emissions
at 10 min were converted into nM O2..
Western Blot Analysis
Cells were grown in 50-ml flasks in DMEM with 10% FCS. At approximately 70
to 80% confluence, the medium was changed to serum-free DMEM for 24 h and
cells were subsequently stimulated with a single dose of 10-7 M
AngII for 2 min up to 24 h. Some cells were also treated with 10-5
to 2 x 10-4 M H2O2. To test the effect
of AngII-mediated O2. on the expression of MAP kinases
and p27Kip1, 5 x 10-6 M of the antioxidant
N-acetylcysteine (NAC) or 10-5 M of the flavoprotein
inhibitor diphenylene iodinium (DIP) was included in some experiments
(4). In addition, cells were
coincubated with 10-5 M of PD98059 or SB203580. At the end of the
stimulation period, cells were washed with phosphate-buffered saline (PBS) and
lysed with 100 µl of lysis buffer (2% sodium dodecyl sulfate [SDS], 60 mM
Tris-HCl, pH 6.8), and the lysates were cleared by centrifugation. Fifty
micrograms of protein (measured by a modification of the Lowry method) were
denatured by addition of 5% glycerol, 5% mercaptoethanol, and 0.03% bromphenol
blue, and then boiling for 10 min. After centrifugation, the supernatants were
loaded onto a 12% SDS-polyacrylamide gel. A low molecular weight marker
(RainbowmarkerTM; Amersham-Buchler, Braunschweig, Germany) served as
molecular weight standard. After electrophoresis, proteins were electroblotted
semidry for 1 h at 0.8 mA/cm2 to a polyvinylidene difluoride
membrane (Hybond P, Amersham). Selected membranes were stained with Ponceau S
to ensure equal loading and transfer of proteins. The blots were blocked in 5%
nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature.
The following antibodies were used in a 1:1000 dilution: a mouse monoclonal
anti-p27Kip1 antibody (Transduction Laboratories, Lexington, KY), a
rabbit anti-p38 MAP kinase antiserum, a rabbit polyclonal anti-p44/42 MAP
kinases antibody, a monoclonal mouse antibody generated against
phospho-specific p38 MAP kinase (Thr180/Tyr182), and a monoclonal mouse
phospho-specific p44/42 MAP kinase (Thr202/Tyr204) antibody (all from New
England Biolabs, Beverly, MA). Incubations were carried out for 1 h at room
temperature. The blots were then washed once for 15 min and twice for 5 min in
PBS containing 0.1% Tween 20. After incubation with an appropriate secondary
antibody coupled to horseradish peroxidase, blots were washed again for 25 min
and the detection was carried out using the ECL system (Amersham). Selected
blots were washed and reprobed with an antibody against ß-actin (Sigma)
to control for small variations in protein loading and transfer. Exposed films
were scanned with Fluor-STM Multi-Imager (Bio-Rad Laboratories, Hercules,
CA), and data were analyzed with the computer program Multi-AnalystTM
from Bio-Rad. Signal intensities in control lanes were arbitrarily assigned a
value of 1.00. Western blots were repeated 3 to 5 times with qualitatively
similar results.
Immunoprecipitation
Quiescent cells at approximately 80% confluence were stimulated with
10-7 M AngII for up to 24 h. Cells were rinsed with PBS and then
lysed in 100 µl of immunoprecipitation buffer (1% Triton X-100, 150 mM
NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM
ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetra-acetic
acid, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl
fluoride, and 0.5% Nonidet P-40) by leaving the dishes on ice for 30 min. The
cells were scraped from the dish and the lysates were cleared by
centrifugation. For immunoprecipitations, lysates were incubated with 900
µl of the immunoprecipitation buffer and 5 µl of either p44/42 MAP
kinase or p38 MAP kinase rabbit polyclonal non-phospho-specific antibodies for
1 h on ice with slight agitation
(11). Fifty microliters of
Staphylococcus aureus Cowan strain (PansorbinTM; Calbiochem) was
added, and tubes were shaken for another 30 min on ice. The immunoprecipitates
were washed six times with immunoprecipitation buffer and subjected to Western
blot analysis, using the mouse monoclonal anti-phosphotyrosine antibody PY20
(Transduction Laboratories) as first antibody
(11). Incubation with the
secondary antibody and detection were carried out as described above.
Immunoprecipitation experiments were independently performed three times with
qualitatively similar results.
p44/42 MAP Kinase Assay
p44/42 MAP kinase activity was nonradioactively measured, applying a kit
from New England Biolabs that detects the specific Ser383 phosphorylation of
the p44/42 MAP kinase substrate Elk-1. In brief, cells were stimulated for 2
min up to 24 h with 10-7 M AngII or 5 x 10-5 M
H2O2. As a specificity control, some cells were also
coincubated with 10-5 M PD98059, 5 x 10-6 M NAC,
or 10-5 M DIP. After rinsing in PBS, cells were lysed in lysis
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
ethyleneglycol-bis (ß-aminoethyl ether)-N,N'-tetra-acetic
acid, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1% Triton
X-100, and 1 µg/ml leupeptin). After centrifugation, cell lysates were
incubated with an immobilized phospho-p44/42 MAP kinase monoclonal antibody.
For selected experiments, an antibody against total MAP kinase was also used.
After washing, pellets were incubated in kinase buffer (25 mM Tris-HCl, pH
7.5, 5 mM ß-glycerophosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, 10 mM MgCl2, and 200 µM ATP) with
2 µg of Elk-1 protein for 30 min at 30°C. The reaction was terminated
by addition of electrophoresis sample buffer and boiling. After
SDS-polyacrylamide gel electrophoresis and Western blotting, phosphorylated
Elk-1 protein was detected with a specific anti-phosphorylated Elk-1
monoclonal antibody (New England Biolabs). p44/42 MAP kinase assays were
independently performed four times with qualitatively similar results.
Transient Transfection with Phosphothioate Oligonucleotides
Transient transfection was carried out using phosphothioate-modified sense
and antisense p44/42 MAP kinase oligonucleotides (antisense: 5'-GCC GCC
GCC GCC GCC AT-3'; sense: 5'-ATG GCG GCG GCG GCG GC-3')
(12). Cells (approximately 50
to 60% confluence) were transfected with 1 µg of oligonucleotides per
milliliter in DMEM without FCS using LipofectinTM (Life Technologies-BRL,
Gaithersburg, MD), as recommended by the manufacturer. Cells were incubated
for 16 h. After 16 h, the transfection medium was removed and cells were
incubated in serum-free DMEM with or without 10-7 M AngII for
another 18 h. Western blots for phosphorylated MAP kinase, p27Kip1,
and ß-actin were performed by probing consecutively the same membrane as
described above.
[3H]Leucine Incorporation
Incorporation of [3H]leucine into de novo synthesized
proteins was used as a reliable parameter of cell hypertrophy. We have
previously found in extensive studies a close correlation of
[3H]leucine incorporation with other parameters of cell hypertrophy
(1,2,3).
Cells were seeded in 24-well plates (105 cells/well) and rested in
serum-free DMEM for 24 h. Quiescent cells were stimulated with a single dose
of 10-7 M AngII or control medium in the presence or absence of
10-5 M of PD98059 or SB203580. Some cells were also incubated with
5 x 10-5 H2O2 with and without PD98059
or SB203580. Additional cells were transiently transfected with p44/42 MAP
kinase sense or antisense phosphothioate-modified oligonucleotides (0.5 µg
oligonucleotides/well) as described above before stimulation with AngII or
H2O2. Total stimulation time with mediators was 18 h.
After 6 h of incubation, 5 mCi [3H]leucine (142 Ci/mmol; Amersham)
was added per well. At the end of the incubation period, cells were washed
with PBS and proteins were precipitated with 10% ice-cold TCA. Proteins were
redissolved in 0.5 M NaOH containing 0.1% Triton X-100, and radioactivity was
measured by liquid scintillation spectroscopy after adding 5 ml of
scintillation cocktail (1).
Statistical Analyses
All values are presented as means ± SEM. Statistical significance
among multiple groups was tested with nonparametric Kruskal—Wallis test.
Individual groups were then tested using the Wilcoxon—Mann—Whitney
test. A P value of <0.05 was considered significant.
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Applying phospho-specific anti-p38 MAP kinase antibodies revealed that AngII also induced phosphorylation of this kinase in MCT cells (Figure 3), as well as in LLC-PK1 cells (data not shown). Although this AngII-mediated phosphorylation of p38 MAP kinase exhibited a similar time pattern as the activation of p44/42 MAP kinase, it was weaker than phosphorylation of the p44/42 MAP kinase (quantification of densitometric values are given in Table 1).
Because we have previously observed that AngII stimulates the intracellular generation of O2[UNK] through the activation of membrane-bound NADP(H) oxidase (4), we investigated whether reactive oxygen products are involved in the AngII-induced phosphorylation of p44/42 MAP kinases. As demonstrated in Figure 4, preincubation of the cells for 18 h with the flavoprotein inhibitor DIP completely inhibited AngII-stimulated phosphorylation of p44/42 MAP kinases at 10 min in MCT cells. In addition, preincubation with the antioxidant NAC partly abolished this phosphorylation. Both agents also inhibited the later (after 12 h) AngII-induced phosphorylation (data not shown). However, neither DIP nor NAC influenced the total expression of 42 MAP kinase, indicating that the inhibitors were not toxic for the cells (Figure 4). Similar effects were also observed in LLC-PK1 cells (data not shown) (for quantitative values, see Table 1).
To further investigate the role of reactive oxygen species, MCT cells were treated with 5 x 10-5 M exogenous H2O2. Figure 5 and Table 1 reveal that this concentration of H2O2 also induces a biphasic phosphorylation pattern of p44/42 MAP kinases with an early peak at 2 to 10 min and a later phosphorylation at 4 h. However, in contrast to AngII-induced phosphorylation, the second phosphorylation peak induced by H2O2 occurred earlier.
MAP Kinase Activity
Although it is generally agreed that phosphorylation of MAP kinases leads
to enzymatic activity, we actually measured MAP kinase activity in our system
using the detection of phosphorylated Elk-1, a specific substrate for p44/42
MAP kinase. A single dose of 10-7 M AngII also induces a biphasic
phosphorylation of Elk-1 in MCT (Figure
6A) and LLC-PK1 cells (data not shown). Coincubation
with PD98059 inhibited the AngII-induced early as well as late phosphorylation
of Elk-1 in MCT cells (Figure
6B). This phosphorylation also was attenuated in the presence of
DIP as well as NAC (data not shown). A quantification of data from
Figure 6, A and B, is presented
in Table 1. The first MAP
kinase activity peak using Elk-1 as substrate could also be detected if total
p44/42 MAP kinases instead of phosphorylated MAP kinases were
immunoprecipitated (Figure 6C).
However, we have not used this antibody for detection of the second peak,
because we only wanted to test that the source of the antibody for MAP kinase
immunoprecipitation is not critical for our substrate assay. Furthermore, in
data not shown, we found a biphasic Elk-1 phosphorylation after challenge of
MCT cells with 5 x 10-5 M H2O2.
Effect of MAP Kinases on p27Kip 1 Expression
We have shown previously that AngII-induced cell cycle arrest and
concomitant hypertrophy of proximal tubular cells depend on p27Kip
1, which is induced by oxygen anions
(3,4).
To further determine whether phosphorylated MAP kinases play a role as
potential signaling intermediates between AngII-induced oxygen radicals and
p27Kip 1 expression, we inhibited upstream kinases with the specific MEK1
inhibitor PD98059 (10). As
shown in Figure 7A, PD98059
completely inhibits AngII-induced p27Kip 1 expression in MCT cells,
whereas the inhibitor had no significant effect on p27Kip 1
baseline expression in the absence of AngII. Identical effects were detected
in LLC-PK1 cells (for quantitative values, see
Table 1). In contrast,
SB203580, a direct inhibitor of p38 MAP kinase, failed to abolish
AngII-induced p27Kip 1 expression in both cell lines
(Figure 7B and
Table 1).
Because PD98059 could theoretically prevent AngII-mediated induction of p27Kip 1 by interfering with the upstream induction of O2[UNK] generation, we investigated potential effects of this compound on AngII-induced oxygen radical formation. As shown in Figure 8 and summarized in Table 2, PD98059 as well as SB203580 did not inhibit AngII-induced synthesis of O2[UNK]. In contrast, the kinase inhibitors rather stimulated production of reactive oxygen species (Table 2).
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To inhibit p44/42 MAP kinase by a method other than applying PD98059, cells were transiently transfected with specific antisense oligonucleotides derived from sequences previously shown to inhibit p44/42 MAP kinase expression (12). As shown in Figure 9, transient transfection of LLC-PK1 cells with p44/42 MAP kinase antisense, but not sense, phosphothioate-modified oligonucleotides inhibited AngII-induced p27Kip 1 expression (densitometric values are summarized in Table 1). Transient transfection with antisense oligonucleotides almost completely blocked expression of p44/42 MAP kinase (Figure 9). Reincubation of the membrane with an antibody against ß-actin revealed that these effects were not due to unequal loading or transfer of proteins (Figure 9). Similar results were also obtained with MCT cells (blots are not shown; for values, see Table 1).
Exogenous H2O2 (5 x 10-5 M to 10-4 M) also stimulates expression of p27Kip 1 in LLC-PK1 (Figure 10A) and MCT cells (data not shown). This induction was abolished in the presence of PD98059 (Figure 10B).
De Novo Protein Synthesis
We have previously shown that incorporation of [3H]leucine into
proteins is an exact parameter of AngII-induced cell hypertrophy in proximal
tubular cells
(1,2,3,4).
Consequently, this parameter was used to investigate the effects of MAP kinase
inhibition on AngII-induced hypertrophy. As demonstrated in
Table 3, AngII-stimulated
protein synthesis in the two tubular cell lines was blocked in the presence of
PD98059. This inhibitor had no significant effect on baseline
[3H]leucine incorporation. Furthermore, transfection of MCT as well
as LLC-PK1 cells with MAP kinase antisense, but not sense,
oligonucleotides attenuated AngII-stimulated hypertrophy
(Table 3). Interestingly,
transfection with antisense oligonucleotides stimulated leucine incorporation
in MCT, but not in LLC-PK1 cells compared with controls. Although
the mechanisms of this remain unclear, it is likely caused by unspecific
processes induced by transfection.
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We and others have demonstrated previously that AngII stimulates hypertrophy of cultured proximal tubular cells from various species (1,2,16,17). Other studies by our group have demonstrated that AngII-mediated hypertrophy of proximal tubular cells occurs in the G1 phase of the cell cycle and that this G1 arrest depends on AngII-stimulated induction of p27Kip 1 (3). AngII-stimulated activation of membrane NAD(P)H oxidase, resulting in O2. generation, plays a pivotal role in p27Kip 1 and induction of cellular hypertrophy in proximal tubular cells (4). The observation in other cell systems that reactive oxygen species may activate MAP kinases (5) prompted us to investigate a potential role of these kinases in O2.-induced hypertrophy of culture proximal tubular cells.
Treatment of two different proximal tubular cell lines with AngII in a dose previously shown to stimulate O2. generation and induction of hypertrophy readily phosphorylates p44/42 MAP kinase without influencing protein expression of these enzymes. As predicted, this phosphorylation was associated with activation of p44/42 MAP kinases using Elk-1 as a substrate. AngII-mediated phosphorylation of MAP kinases has been described previously in cultured proximal tubular cells derived from opossum kidney, but potential consequences on cell growth have been not investigated in this study (18). AngII-stimulated activation of p44/42 MAP kinase appears to be specific for proximal tubular cells, as Terada et al. convincingly demonstrated on intact microdissected nephron segments that this vasopeptide-induced phosphorylation does not occur in other tubular segments (19).
Interestingly, we observed a biphasic AngII-induced phosphorylation of p44/42 MAP kinase: an early rapid transient activation (2 to 10 min) and a second peak after 12 h of stimulation. This observation is in agreement with a report by Huwiler and coworkers demonstrating in mesangial cells an AngII-induced rapid and transient early increase of MAP kinase activation that was associated with hypertrophy, whereas a more sustained phosphorylation (up to 60 min) was caused by mitogens (20). Later time points beyond 90 min were not investigated in this study (20). However, an interesting recent study demonstrates a biphasic induction pattern of p44/42 MAP kinase phosphorylation in cultured mesangial cells challenged with inflammatory cytokines that is reminiscent of our observation (21). In this system (21), the late activation was mediated by endogenous nitric oxide (NO) generation, a response that is unlikely in the AngII-stimulated p44/42 MAP kinase phosphorylation because AngII attenuates NO production in proximal tubular cells (22).
Inhibition of membrane-bound NAD(P)H oxidase with DIP or scavenging O2. with NAC attenuates the AngII-stimulated p44/42 MAP kinase phosphorylation. Furthermore, interference with O2. generation appears to suppress basal MAP kinase activity in the absence of AngII. We have demonstrated previously that AngII treatment of MCT and LLC-PK1 cells resulted in intracellular generation of reactive oxygen species (4). In addition, exogenous H2O2 also stimulates phosphorylation of p44/42 MAP kinase with a biphasic induction pattern similar to AngII-mediated activation, although the later phosphorylation occurs slightly earlier at 4 h. These results collectively suggest that AngII-induced phosphorylation and activation of p44/42 MAP kinase is mediated by O2. as an intermediate signal transduction molecule. Indeed, several recent studies performed in nonrenal cells provide ample evidence that oxidative stress can activate MAP kinases (5,6,7,23,24). For example, Wang and coworkers observed in cardiac fibroblasts that AngII-induced p44/42 MAP kinase activation was suppressed by NAC and NO, suggesting a role for free oxygen radicals (23). Addition of H2O2 to rat pheochromocytoma PC 12 cells stimulates p44/42 MAP kinase phosphorylation (24). Although the molecular mechanism(s) that cause oxidative stress to stimulate p44/42 MAP kinase phosphorylation are not entirely clear, a recent study provides evidence that H2O2 inactivates specific phosphatases, an effect that could contribute to enhanced phosphorylation of target proteins (25).
We have demonstrated previously that p27Kip 1 expression is a necessary prerequisite for AngII-induced G1 phase arrest of proximal tubular cells with subsequent hypertrophy (3). This AngII-mediated p27Kip 1 expression was due to posttranscriptional mechanisms, and the increased level of p27Kip 1 binds to cyclin D1-CdK4 complexes and inhibits their activity (3). Therefore, we propose the following signal transduction pathway: AngII, after binding to AT1-receptors, activates membrane-bound NAD(P)H oxidase, leading to an increase in O2. generation. This intracellular accumulation of reactive oxygen species subsequently leads to phosphorylation and activation of p44/42 MAP kinases. Activation of this enzyme then leads to an increase in p27Kip 1 expression, G1 phase arrest, and ultimately cell hypertrophy. One may argue that inhibition of p44/42 MAP kinase expression prevents G1 phase arrest and hypertrophy by interfering with more upstream events of the cell cycle, such as G0-G1 phase transit in which expression of immediate early genes such as c-fos and c-myc are necessary (26). AngII-induced expression of such immediate early genes occurs in proximal tubular cells before G1 phase arrest and hypertrophy (1). However, several recent investigations in nonrenal cells generated evidence that the relationship between p44/42 MAP kinase activation, expression of CdK inhibitors, and hypertrophy is a more direct one. Activation of p44/42 MAP kinase has been associated with cell cycle arrest and hypertrophy rather than with proliferation under distinct conditions (27,28,29,30). Bornfeldt and colleagues recently found that PD98059 inhibits mitogen-induced proliferation of human VSMC that do not secrete prostanoids (27). In contrast, VSMC that could synthesize prostaglandin E2 through the inducible form of cyclooxygenase (COX-2) p44/42 MAP kinase activation leads to cell cycle arrest, suggesting that opposing effects of p44/42 MAP kinase activation depend on the availability of specific downstream targets (27). Nerve growth factor (NGF)-induced growth arrest in NIH 3T3 cells depends on p44/42 MAP kinase, because PD98059 blocked the ability of NGF to inhibit the activities of CdK4 and CdK2 by preventing NGF-mediated induction of the CdK inhibitor p21Cip 1 (28). Raf-1, an upstream serine-threonine protein kinase of p44/42 MAP kinase that can associate with Ras, induces p21Cip 1 in these cells in a p53-independent manner (29).
The duration of MAP kinase activation appears to play a pivotal role whether a cell is committed to proliferation or arrested in the G1 phase of the cell cycle. In agreement with our findings, Tombes et al. recently found that transient activation of p44/42 MAP kinase in hepatocytes by NGF leads to proliferation (30). In contrast, NGF caused a sustained activation of p44/42 MAP kinases in PC 12 cells, resulting in p21Cip 1-dependent G1 phase arrest (30). Hence, a growth factor may have totally different effects on growth behavior of distinct cells depending on the duration of MAP kinase activation.
We have reported previously that the AngII-induced hypertrophy depends to some extent on endogenous activation of transforming growth factor-ß (TGF-ß), because a neutralizing anti-TGF-ß antibody partly attenuated cellular enlargement (31). This previous finding does not contradict our present results, and the relationship between AngII, TGF-ß, MAP kinases, and oxygen radicals is complex. For example, AngII stimulates TGF-ß synthesis in VSMC through MAP kinase activation (32). On the other hand, p44/42 MAP kinases are activated by TGF-ß in some cell systems (33). Thus, AngII-mediated oxygen radical formation and TGF-ß synthesis may work in concert in inducing MAP kinase activation with a potential positive feedback on TGF-ß formation.
AngII stimulates phosphorylation of stress-activated p38 MAP kinase in proximal tubular cells in a biphasic pattern, similar to p44/42 MAP kinase. However, apparent differences in the total amount of phosphorylated enzyme may be due to differences in antibody affinity rather than being true physiologic effects. Induction of p38 MAP kinase by vasoactive factors such as AngII as well as by H2O2 has been described in other cell types (reviewed in reference (34). However, inhibition of p38 MAP kinase with SB203580 failed to attenuate AngII as well as H2O2-induced p27Kip 1 expression. Furthermore, this specific inhibitor of p36 MAP kinase did not influence AngII-stimulated de novo protein synthesis in proximal tubular cells. These data suggest that AngII-stimulated p38 MAP kinase activation may not be an essential part of the hypertrophic response of proximal tubular cells induced by this vasoactive peptide. This finding is somewhat at variance with the observations of Ushio-Fukai et al. showing that p38 MAP kinase is a critical component of oxidant stress-mediated hypertrophy of VSMC (35). Although the discrepancy between our study and that of Ushio-Fukai is currently unclear, it is likely that cell type-specific effects may play a role. Furthermore, a negative effect of p38 MAP kinase on the regulation of p44/42 MAP kinase has also been described in VSMC (36), indicating that even in the same cell type, activation of the p38 MAP kinase pathway may have opposite effects depending on the specific growth conditions and time frame of stimulation.
We found in preliminary in vitro experiments that MAP kinase can directly phosphorylate recombinant p27Kip 1. Others have also previously reported this findings (37). However, phosphorylation of p27Kip 1 is assumed to initiate degradation by the ubiquitin-proteasome complex (38). It remains unclear how p27Kip 1 phosphorylation could contribute to an increase in expression of this CdK inhibitor as observed in our system. One possibility may be the phosphorylation at sites other than those necessary for degradation.
Phosphorylation of various MAP kinases is not restricted to the cell culture environment, and activation of these enzymes has been reported in the kidney in vivo by diverse stimuli such as inflammatory responses, hyperosmolarity, and proliferation (reviewed in references (33,) (39,) and (40). It is likely that activation of p44/42 MAP kinase in proximal tubular cells plays an integrative response in adaptive processes occurring after acute and chronic loss of renal tissue.
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M. E. Ullian, A. K. Gelasco, W. R. Fitzgibbon, C. N. Beck, and T. A. Morinelli N-Acetylcysteine Decreases Angiotensin II Receptor Binding in Vascular Smooth Muscle Cells J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2346 - 2353. [Abstract] [Full Text] [PDF]
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 J. Dong, J. I. Everitt, S. S. Lau, and T. J. Monks Induction of ERK1/2 and Histone H3 Phosphorylation within the Outer Stripe of the Outer Medulla of the Eker Rat by 2,3,5-Tris-(Glutathion-S-yl)hydroquinone Toxicol. Sci., August 1, 2004; 80(2): 350 - 357. [Abstract] [Full Text] [PDF]
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L. Zeng, H. Xu, T.-L. Chew, R. Chisholm, M. M. Sadeghi, Y. S. Kanwar, and F. R. Danesh Simvastatin Modulates Angiotensin II Signaling Pathway by Preventing Rac1-Mediated Upregulation of p27 J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1711 - 1720. [Abstract] [Full Text] [PDF]
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, controls; 
, controls +
PD98059; [UNK], AngII; 
, AngII + PD98059. This tracing is representative
of five independent experiments with qualitatively similar results.