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Volume 272, Number 16, Issue of April 18, 1997 pp. 10877-10881
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Interleukin-1 Induction of Mitogen-activated Protein Kinases in Human Mesangial Cells
ROLE OF OXIDATION^*

(Received for publication, September 16, 1996, and in revised form, December 31, 1996)

William A. Wilmer , Laura C. Tan , Jennifer A. Dickerson , Michele Danne and Brad H. Rovin

From the Department of Medicine, Ohio State University, Columbus, Ohio 43210

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

FOOTNOTES

REFERENCES

ABSTRACT

Interleukin-1 (IL-1) significantly influences renal cellular function through the induction of several gene products. The molecular mechanisms involved in gene regulation by IL-1 are poorly understood; however, the appearance of novel tyrosine phosphoproteins in IL-1-treated cells suggests that IL-1 may function through tyrosine phosphoprotein intermediates. The mitogen-activated protein (MAP) kinases are tyrosine phosphoproteins that could potentially mediate the effects of IL-1. Protein tyrosine phosphorylation following IL-1 treatment may be dependent on redox changes since the IL-1 receptor is not a protein-tyrosine kinase and oxidation has been shown to induce tyrosine phosphorylation. In this report we demonstrate that conditioning human glomerular mesangial cells with IL-1 results in the tyrosine phosphorylation and activation of two members of the MAP kinase family, extracellular signal-regulated protein kinase 2 (ERK2) and p54 Jun-NH₂-terminal kinase (JNK). This effect of IL-1 is abrogated by pretreating cells with the antioxidants N-acetyl-L-cysteine or dithiothreitol. Furthermore, the effects of IL-1 on ERK and JNK activation are reproduced by treating mesangial cells with membrane-permeable oxidants. IL-1 and oxidants also cause phosphorylation and activation of the upstream ERK regulatory element MAP kinase kinase. Interestingly, IL-1, but not exogenous oxidants, causes phosphorylation of the upstream JNK activator, JNK kinase. These data indicate that IL-1 activates ERK2 through an oxidation-dependent pathway. Exogenous oxidants and IL-1 activate JNK through different upstream mechanisms; however, antioxidant inhibition of JNK activation indicates that endogenous oxidants may play a role in IL-1-induced JNK activation. Thus IL-1 may affect mesangial cell function by activating MAP kinases, which can then regulate gene transcription. Furthermore, reactive oxygen species released during inflammatory glomerular injury may also affect mesangial function through a MAP kinase signal.

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INTRODUCTION

Interleukin-1 (IL-1)¹ has received considerable attention as a potential mediator of renal injury (reviewed in Refs. 1-3). IL-1 affects proliferation of cultured renal cells (4-6) and induces renal cell production of a variety of biologic modifiers (4, 7-10). IL-1 has been found in the kidneys of patients and experimental animals with glomerulonephritis (11-15), and blocking the IL-1 receptor was recently shown to attenuate injury in experimental glomerulonephritis (16, 17). Thus, IL-1 may be relevant to the pathogenesis of glomerular damage in vivo.

Despite a growing understanding of the effects of IL-1 on the kidney, the intracellular mechanisms that mediate the actions of IL-1 are poorly characterized. It is known, however, that IL-1 treatment of mesangial cells up-regulates tyrosine kinase activity and causes tyrosine phosphorylation of several mesangial cell proteins (10, 18-20). Therefore, IL-1 may activate gene transcription in human mesangial cells through the induction of tyrosine phosphoprotein intermediates.

In considering potential tyrosine phosphoprotein mediators of IL-1 activity, MAP kinases are of particular interest for several reasons. These kinases require tyrosine phosphorylation for activity, are cytokine-inducible, and have been implicated in regulating gene transcription (21-24). Furthermore, members of the ERK and JNK subgroups of the MAP kinase family have molecular masses similar in size (24, 25) to the tyrosine phosphoproteins that are induced by IL-1 treatment of human mesangial cells (18, 19).

Although IL-1 can induce protein tyrosine phosphorylation, its receptor is not a classic receptor tyrosine kinase (26). IL-1-induced tyrosine phosphorylation may be explained by the observation that protein tyrosine phosphorylation can be modulated by intracellular redox potential. Increasing intracellular oxidation can directly enhance protein tyrosine phosphorylation (27-29) and can also potentiate protein tyrosine phosphorylation induced by other stimuli (30, 31). Furthermore, cytokines such as IL-1 have been reported to increase oxygen radical release by mesangial cells (32).

This investigation was thus undertaken to determine whether IL-1-induced tyrosine phosphorylation in human mesangial cells activates ERK and/or JNK and to establish the role of intracellular oxidation in this pathway.

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EXPERIMENTAL PROCEDURES

Cell Culture and Treatment

Human mesangial cells were cultured from kidneys not suitable for transplantation and characterized as we have previously described (8). In this investigation, cells from four different donors were used between passages 5 and 10. Cells were grown to confluence in RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Inc.). Before conditioning, monolayers were washed to remove serum; all subsequent incubations were carried out in medium consisting of RPMI 1640 plus 0.25% bovine serum albumin.

In these experiments, mesangial cells were treated with medium alone (control) or with 1.1 ng/ml human recombinant IL-1 (specific activity, 5 × 10⁸ units/mg; Genzyme Corp., Cambridge, MA). This concentration of IL-1 was previously determined to cause a significant induction of tyrosine phosphoproteins in mesangial cells (18). The redox status of the cells was manipulated by treatment with the antioxidants DTT or NAC (Sigma) or the cell-permeable oxidizing agents diamide (33) or hydrogen peroxide (Sigma). In some experiments, mesangial cells were conditioned with PD 098059 (34, 35), a specific inhibitor of MEK1 and MEK2 activation (a gift from Dr. Alan Saltiel, Parke-Davis, Ann Arbor, MI). All reagents were diluted in RPMI 1640 plus 0.25% bovine serum albumin to achieve the indicated concentrations.

Tissue culture medium and cytokines were found to be virtually endotoxin free (<0.1 ng/ml) using the quantitative Limulus amebocyte lysate assay (M. A. Bioproducts, Walkersville, MD).

Immunoblotting and Immunoprecipitation of Mesangial Cell Lysates

Confluent mesangial cells grown in 100-mm plates were appropriately conditioned and, at specified times after treatment, lysed in buffer containing 1% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM Na₃VO₄, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 0.15 unit/ml aprotinin. For immunoblotting, 50-100 µg of cell protein were separated on a 10% SDS-polyacrylamide gel under reducing conditions unless otherwise indicated and then transferred to 0.45 µM nitrocellulose membranes (Hoefer Biotech, Inc., San Francisco, CA) using the electroblotting technique described by Kyhse-Andersen (36). Membranes were probed with the following primary antibodies: mouse monoclonal anti-phosphotyrosine (clone 4G10, Upstate Biotechnology, Inc., Lake Placid, NY), mouse monoclonal anti-pan ERK, which recognizes the 42- and 44-kDa isoforms (Transduction Laboratories, Lexington, KY), polyclonal rabbit anti-phosphorylated-ERK, which recognizes the tyrosine-phosphorylated 42- and 44-kDa ERK isoforms (New England Biolabs Inc., Beverly, MA), polyclonal rabbit anti-phosphorylated-SEK, which recognizes threonine-phosphorylated SEK1 (New England Biolabs), polyclonal rabbit anti-phosphorylated-MEK, which recognizes serine-phosphorylated MEK1/2 (New England Biolabs), and polyclonal rabbit anti-JNK, which is specific for the FLG epitope of JNK and thus recognizes the 46- and 54-kDa isoforms (Santa Cruz Biotechnology, Santa Cruz, CA). Non-immune rabbit IgG or mouse isotype-specific IgG served as a control for the primary antibodies. Appropriate biotinylated secondary antibodies were obtained from Zymed Laboratories, Inc. (San Francisco, CA). After incubation with streptavidin-horseradish peroxidase, blots were developed using enhanced chemiluminescence (Amersham Life Science, Inc.).

ERK was immunoprecipitated from mesangial lysates (500 µg of protein) with 5 µg of the anti-pan ERK antibody, followed by protein G-Sepharose. The immunoprecipitated material was run on a polyacrylamide gel, blotted to nitrocellulose, and probed with the anti-phosphotyrosine antibody as described above. To determine whether IL-1 increased JNK tyrosine phosphorylation, 500 µg of protein from mesangial lysates were immunoprecipitated with 5 µg of anti-phosphotyrosine, followed by protein A-agarose. After electrophoresis under nonreducing conditions (to avoid interference with the detection of p54 JNK from IgG heavy chains), the immunoprecipitated material was blotted and probed with the anti-JNK antibody, as described above.

MBP Kinase Assay

ERK activity in mesangial cell lysates was measured as the ability of these lysates to phosphorylate MBP, a substrate for ERK (37). Briefly, 10 µl of mesangial protein were incubated with 10 µl of a 2 mg/ml MBP solution in buffer containing 20 mM MOPS, pH 7.2, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM Na₃VO₄, 1 mM DTT (Upstate Biotechnology Inc.). A solution containing inhibitors to protein kinase C, protein kinase A, and calmodulin-dependent kinase was added to reduce interference from other serine/threonine kinases (Upstate Biotechnology Inc.). The reaction was started by the addition of 10 µCi of [-³²P]ATP (6000 Ci/mmol, DuPont NEN) in buffer containing 75 mM MgCl₂ and 500 µM ATP. After 10 min at 30 °C, an aliquot of the reaction mixture was removed and blotted onto phosphocellulose paper. The papers were washed several times in 0.75% phosphoric acid before counting in a scintillation counter. After accounting for nonspecific binding and binding of endogenous proteins that were phosphorylated, the results were expressed as pmol of phosphate incorporated into MBP/min/µg of mesangial cell protein.

c-Jun NH₂-terminal Kinase Assay

Mesangial cell proteins were harvested and JNK activity was assayed (as described by Cano et al. (38)) after immunoprecipitation of JNK isoforms. Briefly, cells were lysed in buffer containing 20 mM Hepes, pH 7.9, 0.3 M NaCl, 2 mM EGTA, 20 mM -glycerophosphate, 1 mM DTT, 0.1% Triton X-100, 1 mM Na₃VO₄, 400 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 18 µg/ml aprotinin. After centrifugation and normalization of the protein content, extracts were mixed with 1.0 µg of polyclonal anti-JNK antibody (Santa Cruz Biotechnology) overnight at 4 °C. The JNK·anti-JNK immune complexes were precipitated with protein G-Sepharose for 1 h, washed several times in kinase buffer (20 mM Hepes, pH 7.9, 2 mM EGTA, 10 mM MgCl₂, 1 mM DTT, 100 µM Na₃VO₄, and 25 mM -glycerophosphate), and resuspended in 40 µl of kinase buffer containing 2 µg of the fusion protein GST-c-Jun_(1-79) (Santa Cruz Biotechnology), 100 µM ATP, and 3-5 µCi of [-³²P]ATP. The reaction was allowed to proceed for 20 min at room temperature and was terminated by the addition of SDS-loading buffer. Phosphorylated GST-c-Jun_1-79 was resolved on a 12% SDS-polyacrylamide gel and then autoradiographed.

Statistical Analysis

Data are presented as mean ± S. E. Multiple comparisons were examined for significant differences using ANOVA. Individual comparisons were subsequently made using the Bonferroni post-test. A p < 0.05 was considered significant.

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RESULTS AND DISCUSSION

Intracellular Redox Potential Affects Protein Tyrosine Phosphorylation in Human Mesangial Cells

The addition of IL-1 to mesangial monolayers resulted in a time-dependent increase in tyrosine phosphorylation of several proteins. Two proteins with apparent molecular masses of 41.2 and 52.6 kDa (p41.2 and p52.6, respectively) were the dominant proteins phosphorylated after incubation with IL-1 (Fig. 1A). Proteins of the same molecular mass were tyrosine-phosphorylated when mesangial cells were treated with diamide, which directly oxidizes intracellular thiols, or with the membrane-permeable reactive oxygen intermediate, hydrogen peroxide (Fig. 1B). Because increasing intracellular oxidation mimicked the effects of IL-1 on protein tyrosine phosphorylation, we examined whether antioxidants could inhibit IL-1-induced tyrosine phosphorylation. As shown in Fig. 2, when mesangial cells were treated with the antioxidants NAC or DTT before incubation with IL-1, the tyrosine phosphorylation of p41.2 and p52.6 was attenuated. Similarly, diamide-induced tyrosine phosphorylation of p41.2 and p52.6 was blocked in NAC-treated cells (Fig. 2). These data demonstrate that oxidation stimulates protein tyrosine phosphorylation in human mesangial cells and suggest that IL-1-mediated protein tyrosine phosphorylation may occur through an oxidation-dependent mechanism.

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Fig. 1. Induction of tyrosine phosphoproteins in human mesangial cells. Lysates were prepared from human mesangial cells as described under "Experimental Procedures" and immunoblotted with an anti-phosphotyrosine antibody. A, cells were treated with IL-1 (1.1 ng/ml) for the indicated lengths of time. The relative positions of molecular mass markers (kDa) are shown. The arrows indicate two prominent tyrosine phosphoproteins with apparent molecular masses of 41.2 and 52.6 kDa. B, cells were treated with 1.1 ng/ml IL-1 for 30 min or 2 mM diamide (DIA) for 10 min and then washed and incubated in fresh medium for 20 min or 250 µM hydrogen peroxide for 30 min. The arrows indicate the 41.2- and 52.6-kDa tyrosine phosphoproteins. Results are representative of four experiments. C, control.
[View Larger Version of this Image (33K GIF file)]

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Fig. 2. Induction of tyrosine phosphoproteins is attenuated in cells pretreated with antioxidants. Cells were untreated (C), treated with 1.1 ng/ml IL-1 for 30 min or 2 mM diamide (DIA) for 10 min, washed, and incubated in fresh medium for 20 min. In some experiments, the cells were pretreated with 20 mM NAC or 2 mM DTT for 30 min followed by IL-1 or diamide. Whole cell lysates were then immunoblotted with anti-phosphotyrosine. The arrows indicate the positions of the 41.2- and 52.6-kDa tyrosine phosphoproteins. Results are representative of four experiments.
[View Larger Version of this Image (48K GIF file)]

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The p41.2 and p52.6 tyrosine phosphoproteins have molecular masses similar to those reported for the MAP kinases ERK2 and p54 JNK, respectively (24, 25). Furthermore, the predominant isoforms of ERK and JNK expressed in human mesangial cells were found to have molecular masses of 42.1 and 52.8 kDa, respectively (Fig. 3). These data suggested the possibility that IL-1 treatment of mesangial cells results in oxidation-dependent phosphorylation and activation of ERK2 and p54 JNK. This was tested in the following series of experiments.

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Fig. 3. JNK and ERK isoforms expressed in human mesangial cells. Lysates from untreated human mesangial cells were immunoblotted for JNK or ERK. The arrowhead indicates that the dominant JNK isoform has a molecular mass of approximately 52.8 kDa. The higher molecular mass doublet seen in this blot is a nonspecific band that occurs with the secondary antibody. The dominant ERK isoform (arrowhead) has an apparent molecular mass of 42.1 kDa. Other isoforms with molecular masses greater than 50 kDa are apparent and have been described with the anti-ERK antibody used for immunoblotting.
[View Larger Version of this Image (40K GIF file)]

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ERK2 Is Tyrosine-phosphorylated and Activated in Mesangial Cells by IL-1 or Oxidizing Agents

To determine whether IL-1 induced the tyrosine phosphorylation of ERK2, whole cell lysates were probed with an antibody specific for tyrosine-phosphorylated ERK. As shown in Fig. 4A, this antibody detected tyrosine-phosphorylated ERK in lysates from IL-1-treated mesangial cells but not from control cells. In most experiments, a single 41.6-kDa isoform of tyrosine-phosphorylated ERK was detected. Occasionally a minor band with an apparent molecular mass of 44.2 kDa (probably ERK1) was also seen. These results were confirmed by immunoprecipitating ERK from IL-1-treated mesangial lysates and staining the immunoprecipitates with an anti-phosphotyrosine antibody. This demonstrated a tyrosine-phosphorylated ERK isoform with an apparent molecular mass of 41.1 kDa (data not shown).

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Fig. 4. Tyrosine phosphorylation of ERK in human mesangial cells. Mesangial cell lysates were probed for tyrosine-phosphorylated ERK after the indicated treatments. A, cells were untreated (C), treated with IL-1 (1.1 ng/ml) for 30 min, or treated with 20 mM NAC or 2 mM DTT for 30 min followed by IL-1. The tyrosine-phosphorylated ERK isoform detected had an apparent molecular mass of 41.6 kDa. Results are representative of three experiments. B, cells were treated with 2 mM diamide (DIA) (as in Fig. 1) or with 250 µM hydrogen peroxide for 15 min. The ERK isoform detected had a molecular mass of 41.6 kDa. Results are representative of four experiments.
[View Larger Version of this Image (28K GIF file)]

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The role of redox status in ERK2 induction by IL-1 was evaluated by incubating cells with NAC or DTT before IL-1. These antioxidants blocked IL-1-induced tyrosine phosphorylation of ERK2 (Fig. 4A). Furthermore, the oxidizing agents diamide and hydrogen peroxide reproduced the effects of IL-1 and caused tyrosine phosphorylation of ERK2 (Fig. 4B). This pattern of ERK phosphorylation in response to IL-1, oxidants, or antioxidants was identical to the pattern of induction of the p41.2 tyrosine phosphoprotein (Fig. 1).

We considered tyrosine phosphorylation of ERK to be an index of enzyme activation. To verify that IL-1 treatment of mesangial cells resulted in a functional increase in ERK activity, cell lysates were tested in a MBP phosphorylation assay. As shown in Table I, lysates from IL-1-treated mesangial cells caused a significantly greater phosphorylation of MBP than lysates from untreated cells. Diamide and hydrogen peroxide increased MBP kinase activity similar to IL-1 (Table I). Pretreatment with 2 mM DTT for 30 min completely blocked IL-1-induced MBP kinase activity (1.5 ± 0.3 versus 3.8 ± 0.9 pmol of phosphate incorporated/min/µg of protein; activity from the control cells was 1.5 ± 0.3, n = 4). Similarly, MBP kinase activity from cells treated with 20 mM NAC for 30 min followed by IL-1 (2.9 ± 0.5 pmol of phosphate incorporated/min/µg of protein, n = 6) was not different from control cell activity (2.2 ± 0.4, n = 5) but was significantly less than the activity from cells treated with IL-1 alone (4.98 ± 0.4, n = 5).

Table I. ERK activity in human mesangial cells Cells were treated as follows before lysis: control, medium alone; IL-1, 1.1 ng/ml for 30 min; diamide, 2 mM for 10 min then fresh medium for 20 min; H2O2, 150 µM for 30 min. ERK activity is measured as pmol of phosphate incorporated into MBP/min/µg of cell protein. Condition Control IL-1 Diamide H2O2 n 11 10 4 7 Activity 1.8 ± 0.2 3.9 ± 0.5a 5.0 ± 0.6a 3.5 ± 0.6b a p < 0.01 versus control. b p < 0.05 versus control.

Taken together, these data conclusively demonstrate that IL-1 and oxidizing agents activate ERK2 in human mesangial cells. ERK activation in response to IL-1 or oxidants has been reported for other cell types (23, 37, 39-41), consistent with the data presented here. A link between IL-1 and oxidant-induced ERK activation has not, however, been established previously. The observation that IL-1-induced ERK2 tyrosine phosphorylation and activation are inhibitable by antioxidants provides evidence that reactive oxygen intermediates act as intracellular signals for IL-1.

JNK Is Tyrosine-phosphorylated and Activated in Mesangial Cells by IL-1 or Oxidants

To determine if IL-1 caused tyrosine phosphorylation of JNK, tyrosine phosphoproteins were immunoprecipitated from mesangial lysates and probed with anti-JNK antibody. As shown in Fig. 5, a single isoform of tyrosine-phosphorylated JNK (molecular mass 56 kDa, nonreducing conditions) was detected in immunoprecipitates from IL-1-treated cells. Phosphorylated JNK was not found in control lysates.

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Fig. 5. Immunoprecipitation of JNK from mesangial cells. Cells were untreated (C) or treated with IL-1 (1.1 ng/ml) for 30 min. Cells were lysed and proteins were immunoprecipitated with anti-phosphotyrosine (C, IL) or with an isotype control antibody (IL/C). These immunoprecipitates were electrophoresed under nonreducing conditions and immunoblotted for JNK. The arrowhead indicates the position of JNK (approximate molecular mass 56 kDa) that was tyrosine-phosphorylated in cells treated with IL-1. Results are representative of two experiments.
[View Larger Version of this Image (68K GIF file)]

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Subsequent experiments were undertaken to assess the role of oxidation in IL-1-induced JNK activation. JNK was immunoprecipitated from mesangial lysates, and these immunoprecipitates were used to phosphorylate the fusion protein GST-c-Jun, a JNK substrate. Minimal c-Jun phosphorylation was detected using control cell immunoprecipitates, but immunoprecipitates from IL-1-treated cells caused significant c-Jun phosphorylation (Fig. 6A). Diamide and hydrogen peroxide also induced c-Jun kinase activity (Fig. 6B). Immunoprecipitates from cells exposed to DTT or NAC before IL-1 showed significantly decreased c-Jun kinase activity compared with cells incubated with IL-1 alone (Fig. 6A).

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Fig. 6. Phosphorylation of GST-c-Jun by mesangial cell lysates. Mesangial cells were treated with the indicated stimuli and lysed, and JNK was immunoprecipitated. These immunoprecipitates were tested for phosphorylation of a GST-c-Jun fusion protein. A, cells were untreated (C) or treated with 1.1 ng/ml IL-1 for 30 min. Some monolayers were incubated with 20 mM NAC or 2 mM DTT 60 min before IL-1 treatment. Results are representative of three experiments. B, cells were treated with 2 mM diamide (DIA) as in Fig. 1 or with 250 µM hydrogen peroxide for 15 min. Results are representative of five experiments.
[View Larger Version of this Image (47K GIF file)]

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These data clearly demonstrate that IL-1 and oxidizing agents activate JNK in human mesangial cells. IL-1 and oxidant activation of JNK has also been described in other cells (24, 42-44). The observation that IL-1-induced JNK activity can be attenuated by antioxidants provides evidence that reactive oxygen species may mediate the effects of IL-1 on JNK activation in human mesangial cells. This conclusion is supported by a recent study that showed antioxidant inhibition of IL-1-induced JNK activation in bovine chondrocytes (43).

Differential Phosphorylation of MEK and SEK by IL-1 and Oxidizing Agents

The activation of ERK is dependent on an upstream Tyr/Thr kinase designated as MEK (45). In order for MEK to phosphorylate and activate ERK, MEK must itself be serine-phosphorylated (46, 47). This portion of the ERK activation cascade was analyzed by immunoblotting mesangial lysates with an antibody specific for serine-phosphorylated (and presumably activated) MEK. As shown in Fig. 7, IL-1, hydrogen peroxide, and diamide each induced MEK phosphorylation. The phosphorylation of MEK correlated with its activation because PD 098059, a specific inhibitor of MEK activation (35), prevented IL-1- and oxidant-induced ERK tyrosine phosphorylation (data not shown). This is consistent with a recent study that showed oxidant induction of ERK was mediated in part through MEK activation (40). These data support the conclusion that IL-1 activates ERK through an oxidation-dependent pathway. Furthermore, the results show that an IL-1-induced redox signal acts upstream of ERK tyrosine phosphorylation, at least at the level of MEK activation, if not earlier in the cascade. These data also suggest that redox signals may regulate phosphorylation events other than protein tyrosine phosphorylation.

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Fig. 7. Differential phosphorylation of MEK and SEK by IL-1 and oxidants. Mesangial cells were treated with IL-1 (1.1 ng/ml for 30 min), hydrogen peroxide (250 µM for 15 min), or diamide (DIA) (2 mM for 10 min, then washed and incubated in fresh medium for 20 min). The cells were lysed and proteins were immunoblotted for serine-phosphorylated MEK1/2 or threonine-phosphorylated SEK1, as indicated. Results are representative of three experiments.
[View Larger Version of this Image (35K GIF file)]

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Like ERK, JNK phosphorylation and activation are dependent on an upstream Tyr/Thr kinase. One potential upstream JNK kinase is SEK1, which itself must be serine/threonine-phosphorylated for activity (48, 49). Immunoblotting mesangial lysates for the threonine-phosphorylated form of SEK1 demonstrated phosphorylated SEK1 in lysates of IL-1-treated cells but not in control cells (Fig. 7). In contrast, hydrogen peroxide and diamide did not induce phosphorylation of SEK1 (Fig. 7). This suggests that IL-1 and exogenous oxidants activate JNK by different mechanisms. A similar discrepancy was seen in neutrophils in which exogenous oxidants activated MAP kinase, but endogenously derived oxidants did not mediate MAP kinase activation by N-formyl-methionyl-leucyl phenylalanine (40). Although exogenous oxidants may conceivably activate JNK directly, a recent report suggests that hydrogen peroxide could not directly activate JNK in vitro (43). Alternatively, exogenous oxidants may act through a different upstream regulatory element than SEK1 (50). The observation that exogenous oxidants do not mimic the ability of IL-1 to phosphorylate (and presumably activate) SEK1 challenges the idea of a redox signal mediating IL-1-induced JNK activation. This, however, is difficult to reconcile with the antioxidant inhibition of IL-1-induced JNK activation (this study and Ref. 43). A potential explanation of these results is that in contrast to exogenous oxidants, endogenously derived reactive oxygen species generated during IL-1 treatment do contribute to the activation of SEK1. This scenario is supported by the observation that IL-1-induced SEK1 phosphorylation was prevented in mesangial cells pretreated with DTT (data not shown).

In summary, this report demonstrates that IL-1 and exogenous oxidizing agents activate ERK2 in human glomerular mesangial cells. For both IL-1 and oxidants, activation of ERK2 proceeds through MEK. These data, along with the finding that IL-1-induced ERK2 activation is attenuated by antioxidants, provide strong evidence that IL-1 activates ERK through an oxidation-dependent pathway. IL-1 and exogenous oxidizing agents also activate p54 JNK but appear to signal through different upstream pathways. Exogenous oxidants act independently of SEK1, whereas IL-1 treatment results in SEK1 phosphorylation and presumably activation. Thus, IL-1 may activate JNK through an oxygen radical independent pathway. Alternatively, since antioxidants block JNK activation and SEK1 phosphorylation in response to IL-1, endogenously derived oxygen radicals may serve as signal transduction elements for IL-1 in the JNK cascade. The implications of these results for human renal disease remain to be elucidated, but the data clearly suggest that at the molecular level MAP kinases may mediate the effects of IL-1 on glomerular cells. Additionally, this report illustrates a potential mechanism through which exogenous oxygen radicals, released during glomerular inflammation, can influence renal cellular function. Finally, the fact that endogenous reactive oxygen species may act as second messengers in the MAP kinase activation pathway identifies a potential target for therapeutic intervention in glomerular disease.

FOOTNOTES

REFERENCES

1. Rovin, B. H., and Schreiner, G. F. (1991) Annu. Rev. Med. 42, 25-33 [CrossRef][Medline] [Order article via Infotrieve]
2. Sedor, J. R., Nakazato, Y., and Konieczkowski, M. (1992) Kidney Int. 41, 595-599 [Medline] [Order article via Infotrieve]
3. Atkins, R. C. (1995) Kidney Int. 48, 576-586 [Medline] [Order article via Infotrieve]
4. Chen, W. P., Chen, A., and Lin, C. Y. (1995) J. Pathol. 175, 327-337 [CrossRef][Medline] [Order article via Infotrieve]
5. Moutabarrik, A., Nakanishi, I., Zaid, K., Namiki, M., Kawaguchi, N., Onishi, S., Ishibashi, M., and Okuyama, A. (1994) Exp. Nephrol. 2, 196-204 [Medline] [Order article via Infotrieve]
6. Lonnemann, G., Shapiro, L., Engler-Blum, G., Muller, G. A., Koch, K. M., and Dinarello, C. A. (1995) Kidney Int. 47, 837-844 [Medline] [Order article via Infotrieve]
7. Muhl, H., and Pfeilschifter, J. (1995) J. Clin. Invest. 95, 1941-1946
8. Rovin, B. H., Yoshiumura, T., and Tan, L. (1992) J. Immunol. 148, 2148-2153 [Abstract]
9. Zoja, C., Wang, J. M., Bettoni, S., Sironi, M., Renzi, D., Chiaffarino, F., Abboud, H. E., Van Damme, J., Mantovani, A., Remuzzi, G., and Rambaldi, A. (1991) Am. J. Pathol. 138, 991-1003 [Abstract]
10. Daphna-Iken, D., and Morrison, A. R. (1995) Am. J. Physiol. 269, F831-F837 [Abstract/Free Full Text]
11. Matsumoto, K., Dowling, J., and Atkins, R. C. (1988) Am. J. Nephrol. 8, 463-470 [Medline] [Order article via Infotrieve]
12. Jenkins, D. A., Wojtacha, D. R., Swan, P., Fleming, S., and Cumming, A. D. (1994) Nephrol. Dial. Transplant. 9, 1228-1233 [Abstract/Free Full Text]
13. Noronha, I. L., Kruger, C., Andrassy, K., Ritz, E., and Waldherr, R. (1993) Kidney Int. 43, 682-692 [Medline] [Order article via Infotrieve]
14. Matsumoto, K., and Hatano, M. (1989) Clin. Exp. Immunol. 75, 123-128 [Medline] [Order article via Infotrieve]
15. Werber, H. I., Emancipator, S. N., Tykocinski, M. L., and Sedor, J. R. (1987) J. Immunol. 138, 3207-3212 [Abstract]
16. Lan, H. Y., Nikolic-Paterson, D. J., Mu, W., Vannice, J. L., and Atkins, R. C. (1995) Kidney Int. 47, 1303-1309 [Medline] [Order article via Infotrieve]
17. Tang, W. W., Feng, L., Vannice, J. L., and Wilson, C. B. (1994) J. Clin. Invest. 93, 273-279
18. Rovin, B. H., and Tan, L. C. (1994) Kidney Int. 46, 1059-1068 [Medline] [Order article via Infotrieve]
19. Lovett, D. H., Martin, M., Bursten, S., Szamel, M., Gemsa, D., and Resch, K. (1988) Kidney Int. 34, 26-35 [Medline] [Order article via Infotrieve]
20. Tetsuka, T., and Morrison, A. R. (1995) Am. J. Physiol. 269, C55-C59 [Abstract/Free Full Text]
21. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
22. Kyriakis, J. M., Banerjee, P., Nikolakai, E., Dai, T., Ruble, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
23. Huwiler, A., and Pfeilschifter, J. (1994) FEBS Lett. 350, 135-138 [CrossRef][Medline] [Order article via Infotrieve]
24. Kyriakis, J. M., Woodgett, J. R., and Avruch, J. (1995) Ann. N. Y. Acad. Sci. 766, 303-319 [Medline] [Order article via Infotrieve]
25. Pelech, S. L., and Sanghera, J. S. (1992) Trends Biochem. Sci. 17, 233-238 [Medline] [Order article via Infotrieve]
26. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., McMahan, C. J., Grubin, C. E., Wignall, J. M., Jackson, J. L., Call, S. M., Friend, D., Alpert, A. R., Gillis, S., Urdal, D. L., and Dower, S. K. (1988) Science 241, 585-589 [Abstract/Free Full Text]
27. Bauskin, A. R., Alkalay, I., and Ben-Neriah, Y. (1991) Cell 66, 685-696 [CrossRef][Medline] [Order article via Infotrieve]
28. Nakamura, K., Hori, T., Sato, N., Sugie, K., Kawakami, T., and Yodoi, J. (1993) Oncogene 8, 3133-3139 [Medline] [Order article via Infotrieve]
29. Schieven, G. L., Kirihara, J. M., Myers, D. E., Ledbetter, J. A., and Uckun, F. M. (1993) Blood 82, 1212-1220 [Abstract/Free Full Text]
30. Zor, U., Ferber, E., Gergely, P., Szucs, K., Dombradi, V., and Goldman, R. (1993) Biochem. J. 295, 879-888
31. Heffetz, D., and Zick, Y. (1989) J. Biol. Chem. 264, 10126-10132 [Abstract/Free Full Text]
32. Radeke, H. H., Meier, B., Topley, N., Floge, J., Habermehl, G. G., and Resch, K. (1990) Kidney Int. 37, 767-775 [Medline] [Order article via Infotrieve]
33. Kosower, N. S., and Kosower, E. M. (1995) Methods Enzymol. 251, 123-133 [Medline] [Order article via Infotrieve]
34. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585-13588 [Abstract/Free Full Text]
35. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489-27494 [Abstract/Free Full Text]
36. Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209 [CrossRef][Medline] [Order article via Infotrieve]
37. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J. (1996) J. Biol. Chem. 271, 4138-4142 [Abstract/Free Full Text]
38. Cano, E., Hazzalin, C. A., Kardalinou, E., Buckle, R. S., and Mahadevan, L. C. (1995) J. Cell Sci. 108, 3599-3609 [Abstract]
39. Bird, T. A., Schule, H. D., Delaney, P. B., Sims, J. E., Thoma, B., and Dower, S. K. (1992) Cytokine 4, 429-440 [CrossRef][Medline] [Order article via Infotrieve]
40. Fialkow, L., Chan, C. K., Rotin, D., Grinstein, S., and Downey, G. P. (1994) J. Biol. Chem. 269, 31234-31242 [Abstract/Free Full Text]
41. Knight, R. J., and Buxton, D. B. (1996) Biochem. Biophys. Res. Commun. 218, 83-88 [CrossRef][Medline] [Order article via Infotrieve]
42. Guan, Z., Tetsuka, T., Baier, L. D., and Morrison, A. R. (1996) Am. J. Physiol. 270, F634-F641 [Abstract/Free Full Text]
43. Lo, Y. Y. C., Wong, J. M. S., and Cruz, T. F. (1996) J. Biol. Chem. 271, 15703-15707 [Abstract/Free Full Text]
44. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79 [CrossRef][Medline] [Order article via Infotrieve]
45. Cobb, M., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846 [Free Full Text]
46. Alessi, D. R., Saito, Y., Campbell, D. G., Cohen, P., Sithanandam, G., Rapp, U., Ashworth, A., Marshall, C. J., and Cowley, S. (1994) EMBO J. 13, 1610-1619 [Medline] [Order article via Infotrieve]
47. Zheng, C. F., and Guan, K. L. (1994) EMBO J. 13, 1123-1131 [Medline] [Order article via Infotrieve]
48. Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800 [Medline] [Order article via Infotrieve]
49. Lin, A., Minden, A., Martinetto, H., Claret, F. X., Lange-Carter, C., Mercurio, F., Johnson, G. L., and Karin, M. (1995) Science 268, 286-290 [Abstract/Free Full Text]
50. Meier, R., Rouse, J., Cuenda, A., Nebreda, A. R., and Cohen, P. (1996) Eur. J. Biochem. 236, 796-805 [Medline] [Order article via Infotrieve]

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