ERK and p38 MAP kinase are involved in arachidonic acid release induced by H2O2 and PDGF in mesangial cells

Misako Hayama, Risa Inoue, Satoshi Akiba, and Takashi Sato

Department of Pathological Biochemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Increased prostaglandin production is implicated in the pathogenesis of glomerular disease. With this consideration, we examined the combined effects of reactive oxygen species and platelet-derived growth factor (PDGF), which might initiate glomerular dysfunction, on arachidonic acid release and cytosolic phospholipase A2 (cPLA2) activation in rat mesangial cells. H2O2-induced release of arachidonic acid was enhanced by PDGF, which by itself had little effect on the release, and the enhancement was completely inhibited by a cPLA2 inhibitor. The phosphorylation of cPLA2, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein (MAP) kinase was upregulated by H2O2 or PDGF alone and except for ERK was enhanced further by the two in combination. The release of arachidonic acid induced by PDGF together with H2O2 was inhibited partially by an inhibitor of ERK or p38 MAP kinase and completely when the two inhibitors were combined; the inhibitory pattern was similar to that for the phosphorylation of cPLA2. These results suggest that the ERK and p38 MAP kinase pathways are involved in the increase in cPLA2 activation and arachidonic acid release induced by PDGF together with H2O2.

hydrogen peroxide; p38 mitogen-activated protein kinase; extracellular signal-regulated kinase; cytosolic phospholipase A2; platelet-derived growth factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MESANGIAL CELLS SYNTHESIZE vasodilatory prostaglandins (PGs) such as PGE2, which act as relaxing factors and regulate glomerular hemodynamics under physiological conditions. However, an increase in renal PGE2 production, which is observed in renal diseases such as glomerulonephritis and diabetic glomerulopathy, induces a functional disorder of the mesangium including abnormal glomerular filtration rate and is implicated in the pathogenesis of nephropathy (46, 52).

Previously, reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radical were recognized to cause cell injury and therefore be involved in the pathogenesis of various diseases. However, recent observations indicate that ROS cause physiological responses other than pathological responses, including protein phosphorylation, Ca2+ signaling, and the activation of transcription factors by stimulating intracellular signaling systems, thus suggesting that ROS function as second messengers in the signal transduction pathway stimulated by the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (21, 36, 38, 50).

In glomerular disorders, ROS are generated by several inflammatory cells including neutrophils, monocytes, and macrophages gathered in the inflamed glomeruli, and also by the mesangial cell itself (7, 9, 10). Previous studies revealed that the intracellular generation of ROS or exposure to H2O2 (a major species of ROS) leads to an increase in PGE2 production and arachidonic acid release in rat renal glomeruli and rat mesangial cells (2, 5, 48). In other cell systems including vascular smooth muscle cells, H2O2 potentiated arachidonic acid release and phospholipase A2 (PLA2) activation (11, 51). Therefore, ROS are involved in the mechanism underlying an increase in PGE2 production leading to glomerular dysfunction and renal injury.

Platelet-derived growth factor (PDGF) has been shown to induce the migration and proliferation of mesangial cells and therefore is considered to participate in the development and progression of glomerular diseases (1, 15, 26, 31). PDGF receptor-stimulated migration and proliferation are reported to require ROS production in vascular smooth muscle cells (49). In mesangial cells, it has been reported that ROS induce tyrosine phosphorylation of the PDGF receptor (24), and further, that PDGF-induced proliferation of the cells is enhanced by H2O2 via a mechanism involving the generation of PGs (20). Because PDGF is also generated by macrophages or glomerular cells in renal injury, it is possible that ROS and PGDF synergistically contribute to the initiation and progression of glomerular diseases.

In this context, evidence indicating that PDGF as well as H2O2 induce the activation of mitogen-activated protein (MAP) kinase, now defined as extracellular signal-regulated kinase (ERK), in mesangial cells (29, 30, 53) led us to suggest that the synergistic increase in PGE2 production is implicated in the pathogenesis of glomerular dysfunction, because ERK is known to increase cytosolic PLA2 (cPLA2) activity (35, 39). Previously, we showed (27) that cPLA2 activation and an increase in PGE2 generation are associated with increased MAP kinase activity in rat mesangial cells exposed to high-glucose medium. Thus in the present study, we investigated the synergistic effect of H2O2 and PGDF on the activation of MAP kinases, including ERK and p38 MAP kinase, which lead to cPLA2 activation in rat mesangial cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Materials. Recombinant human PDGF-BB was obtained from Pepro Tech (London, UK). Methyl arachidonyl fluorophosphonate (MAFP) was from Cayman Chemical (Ann Arbor, MI). PD-98059, SB-202190, and anisomycin were from Calbiochem (La Jolla, CA). Antibodies against phosphorylated ERK1/2 or phosphorylated p38 MAP kinase were from New England Biolabs (Beverly, MA). Antibodies against cPLA2 and phosphoserine were from Santa Cruz Biotechnology (Santa Cruz, CA) and nanoTools Antikörpertechnik (Germany), respectively. [3H]arachidonic acid (100 Ci/mmol) was from PerkinElmer Life Sciences (Boston, MA). Other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan) or Sigma (St. Louis, MO).

Cell culture and preparation of [3H]arachidonic acid-labeled cells. Rat mesangial cells were prepared as described previously (27). Briefly, mesangial cells were obtained from a culture of glomeruli isolated by sieving from Sprague-Dawley rats (body wt 100-150 g) and grown in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenious acid. The cells, between passages 3 to 6, were made quiescent by incubation with RPMI 1640 containing 0.1 mg/ml BSA for 24 h and were subjected to experiments. For labeling with [3H]arachidonic acid, cells were incubated with the radioactive compound (0.5 µCi/ml) for 24 h in RPMI 1640 containing 0.4 mg/ml BSA. The labeled cells were washed with phosphate-buffered saline containing 0.1 mg/ml BSA and placed in RPMI 1640 containing 0.1 mg/ml BSA.

Arachidonic acid release. The labeled mesangial cells (in 35-mm dishes) were treated with various reagents and stimulated with 0.5 mM H2O2 and/or 50 ng/ml PDGF-BB for 60 min as described in the figure legends. The reaction was terminated by transferring the medium and the cell lysate, which was prepared by adding 0.1 N NaOH, into a 100:500:1.5 (vol/vol) ratio of ice-cold chloroform to methanol to HCl. Lipids in the medium and the lysate were extracted and separated by thin-layer chromatography on a silica gel G plate using an 80:80:2 (vol/vol) ratio of diethyl ether to petroleum ether to acetic acid as the developing system. The areas corresponding to free fatty acid and other lipids (diacylglycerol, triacylglycerol, and phospholipids) were scraped off. The radioactivity of each fraction was determined by liquid scintillation counting. The total radioactivity of the fractions recovered from a single lane of the plate was usually in the range of 3.8 × 105 to 4.3 × 105 disintegrations/min (dpm). The radioactivity of [3H]arachidonic acid released was corrected by adjusting the total radioactivity to 4.0 × 105 dpm.

Immunoblot analysis. The quiescent mesangial cells (in 35-mm dishes) were stimulated with 1 mM H2O2 and/or 100 ng/ml PDGF-BB for 10 min as described in the figure legends. After being washed, the cells were solubilized with a buffer (that contained 2% SDS, 20 mM beta -glycerophosphate, 2 mM Na3VO4, 3 mM EGTA, 2% beta -mercaptoethanol, 10% glycerol, 0.01% bromophenol blue, and 50 mM Tris · HCl at pH 6.8) and were subjected to SDS-PAGE on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, and antibodies against phosphorylated ERK1/2 or phosphorylated p38 MAP kinase were applied. The bound antibodies were visualized using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech).

Immunoprecipitation of cPLA2. The quiescent mesangial cells (in 35-mm dishes) were treated with various reagents and stimulated with 1 mM H2O2 and/or 100 ng/ml PDGF-BB for 15 min as described in the figure legends. After being washed, cells were lysed in an ice-cold lysis buffer [that contained 1% Triton X-100, 150 mM NaCl, 5 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 mM p-(amidinophenyl)methanesulfonyl fluoride, and 20 mM Tris · HCl at pH 7.4] and were subjected to immunoprecipitation of cPLA2 as described previously (28). Briefly, the lysate was incubated with anti-cPLA2 antibodies overnight at 4°C and further with protein A-agarose for 2 h at 4°C. After centrifugation, the pellet obtained was washed three times with the lysis buffer and was solubilized. The sample was subjected to SDS-PAGE on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane and antibodies against phosphoserine or cPLA2 were applied. The bound antibodies were visualized as described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Arachidonic acid release and cPLA2 activation. Because rat mesangial cells have been shown to respond to only the PDGF isoform that is a BB homodimer (40), we used PDGF-BB in the present study. When mesangial cells were exposed to H2O2, the release of arachidonic acid increased dose dependently, and the H2O2-induced release was enhanced further by PDGF-BB with the increasing concentration, although PDGF-BB by itself had little effect on the release as is shown in Fig. 1.


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Fig. 1.   Arachidonic acid release induced by H2O2 and platelet-derived growth factor BB (PDGF-BB). A: [3H]arachidonic acid-labeled mesangial cells were stimulated with various concentrations of H2O2 for 60 min. B: labeled cells were stimulated with various concentrations of PDGF-BB in the presence () or absence (open circle ) of 0.5 mM H2O2 for 60 min. [3H]arachidonic acid released was measured as described in MATERIALS AND METHODS. Each point represents the mean ± SE of 3 separate experiments performed in duplicate; dpm, disintegrations/min.

It has been well characterized that receptor-stimulated activation of cPLA2 is associated with the immediate release of arachidonic acid and that the catalytic activity of cPLA2 is increased by phosphorylation at Ser505 of the enzyme via ERK (35, 39, 47). Therefore, we investigated the phosphorylation of cPLA2 using an antiphosphoserine antibody and the contribution of cPLA2 to the arachidonic acid release using the cPLA2 inhibitor MAFP upon stimulation with PDGF and H2O2. The results shown in Fig. 2 demonstrate that PDGF-BB and H2O2 alone induced an increase in phosphorylated cPLA2. When combined, they produced a synergistic increase in the phosphorylation, whereas the protein content of cPLA2 remained unchanged under the conditions. Furthermore, as shown in Fig. 3, the release of arachidonic acid stimulated by H2O2 alone or in combination with PDGF-BB was almost completely inhibited by MAFP.


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Fig. 2.   Phosphorylation of cytosolic phospholipase A2 (cPLA2) induced by H2O2 and PDGF-BB. Mesangial cells were stimulated with or without (control) 1 mM H2O2 and/or 100 ng/ml PDGF-BB for 15 min. Lysate prepared from the cells was subjected to immunoprecipitation of cPLA2 and the precipitate was analyzed by immunoblotting. A: antibodies against phosphoserine used. B: antibodies against cPLA2 used. Results are representative of 3 separate experiments.



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Fig. 3.   Effects of methyl arachidonyl fluorophosphonate (MAFP) on arachidonic acid release induced by H2O2 and PDGF-BB. [3H]arachidonic acid-labeled mesangial cells were treated with (+) or without (-) 10 µM MAFP for 30 min and stimulated with (+) or without (-) 0.5 mM H2O2 and/or 50 ng/ml PDGF-BB for 60 min. Each value represents the mean ± SE of 3 separate experiments performed in duplicate.

ERK activation and its effect on arachidonic acid release. The role of ERK1/2 as an upstream regulator for cPLA2 in arachidonic acid release after stimulation with PDGF-BB and H2O2 was investigated. The results in Fig. 4 indicate that PDGF-BB and H2O2 alone induced an apparent increase in the phosphorylation of ERK1/2 as detected with an antiphospho-ERK antibody but in combination did not evoke a further increase. Moreover, the synergistic increase in arachidonic acid release elicited by PDGF-BB together with H2O2 was suppressed by ~30% by PD-98059, a specific inhibitor of upstream kinase, MAP kinase kinase, or ERK kinase (MEK), although PD-98059 dose-dependently inhibited the release induced by H2O2 alone (as shown in Fig. 5). These results suggest that under the conditions used, ERK1/2 partially contributed to cPLA2 activation, which led to arachidonic acid release.


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Fig. 4.   Phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) induced by H2O2 and PDGF-BB. Mesangial cells were stimulated with or without (control) 1 mM H2O2 and/or 100 ng/ml PDGF-BB for 10 min. Phosphorylated ERK1/2 (phospho-ERK1/2) in the cells was detected as described in MATERIALS AND METHODS. Results are representative of 3 separate experiments.



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Fig. 5.   Effects of PD-98059 on arachidonic acid release induced by H2O2 and PDGF-BB. A: [3H]arachidonic acid-labeled mesangial cells were treated with various concentrations of PD-98059 for 30 min and stimulated with () or without (open circle ) 0.5 mM H2O2 for 60 min. B: labeled cells were treated with (+) or without (-) 30 µM PD-98059 for 30 min and stimulated with (+) or without (-) 0.5 mM H2O2 and 50 ng/ml PDGF-BB for 60 min. Each value represents the mean ± SE of 3 separate experiments performed in duplicate.

p38 MAP kinase activation and its effect on arachidonic acid release. p38 MAP kinase, which plays a role in the stress-stimulated signal transduction pathway, has been reported to serve as an activator for cPLA2 via its phosphorylation in thrombin- or collagen-stimulated platelets (9, 32). Therefore, we examined the contribution of p38 MAP kinase to cPLA2 activation and to arachidonic acid release under the experimental conditions used here. As shown in Fig. 6, p38 MAP kinase was apparently phosphorylated by PDGF-BB and to a lesser extent by H2O2, and the phosphorylation of the kinase was synergistically increased by PDGF and H2O2 as detected using antiphospho-p38 MAP kinase. To examine whether the increase in phosphorylated p38 MAP kinase is implicated in the enhancement of arachidonic acid release, we used anisomycin, which is known to activate MAP kinases responsible for stress stimuli such as p38 MAP kinase but not ERK (12, 23, 37). As shown by the results in Fig. 7A, anisomycin alone did not induce any release of arachidonic acid, but it potentiated the H2O2-stimulated release in a dose-dependent fashion. Under these conditions, phosphorylation of p38 MAP kinase was elicited by anisomycin and H2O2 alone and was further increased by the two in combination (Fig. 7B).


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Fig. 6.   Phosphorylation of p38 MAP kinase induced by H2O2 and PDGF-BB. Mesangial cells were stimulated with or without (control) 1 mM H2O2 and/or 100 ng/ml PDGF-BB for 10 min. Phosphorylated p38 MAP kinase (phospho-p38 MAPK) in the cells was detected as described in MATERIALS AND METHODS. Results are representative of 3 separate experiments.



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Fig. 7.   Effects of anisomycin on arachidonic acid release and p38 MAP kinase phosphorylation induced by H2O2. A: [3H]arachidonic acid-labeled mesangial cells were treated with various concentrations of anisomycin for 30 min and stimulated with () or without (open circle ) 0.5 mM H2O2 for 60 min. Each value represents the mean ± SE of 3 separate experiments performed in duplicate. B: mesangial cells were treated with or without (control) 10 µM anisomycin for 30 min and stimulated with or without 0.5 mM H2O2 for 10 min. Results are representative of 3 separate experiments.

Effect of MAP kinase inhibitors on cPLA2 phosphorylation and arachidonic acid release. The results shown in Fig. 7 suggest that activation of p38 MAP kinase is involved in the enhancement of H2O2-induced arachidonic acid release. Therefore, we tried to confirm the contribution of p38 MAP kinase to the synergistically increased release of arachidonic acid stimulated by the combination of PDGF and H2O2 using the specific p38 MAP kinase inhibitor SB-202190. As shown in Fig. 8A, SB-202190 inhibited the release by 50%. However, SB-202190 together with the ERK inhibitor PD-98059, which by itself suppresses the release by only 30%, almost completely inhibited the release. These effects of the inhibitors paralleled their inhibitory effects on the phosphorylation of cPLA2: the increased phosphorylation of cPLA2 elicited by the combination of PDGF-BB and H2O2 was suppressed partially by either SB-202190 or PD-98059 alone and completely by two inhibitors combined (as seen in Fig. 8, B and C).


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Fig. 8.   Effects of PD-98059 on arachidonic acid release and cPLA2 phosphorylation induced by H2O2 and PDGF-BB. A: [3H]arachidonic acid-labeled mesangial cells were treated with (+) or without (-) 30 µM PD-98059, 50 µM SB-202190, or both for 30 min and stimulated with (+) or without (-) 0.5 mM H2O2 and 50 ng/ml PDGF-BB for 60 min. Each value represents the mean ± SE of 3 separate experiments performed in duplicate. B: mesangial cells were treated with (+) or without (-) 30 µM PD-98059, 50 µM SB-202190, or both for 30 min and stimulated with (+) or without (-) 1 mM H2O2 and 100 ng/ml PDGF-BB for 15 min. Lysate prepared from the cells was subjected to immunoprecipitation of cPLA2, and the precipitate obtained was analyzed by immunoblotting with antibodies against phosphoserine. Results are representative of 3 separate experiments. C: amounts of phosphorylated cPLA2 were estimated by measuring the density of the cPLA2 band shown in B and expressed as the percent of control (untreated and unstimulated cells). Each value represents the mean ± SE of 3 separate experiments.


    DISCUSSION
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MATERIALS AND METHODS
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Previously it was reported that H2O2 induces an increase in arachidonic acid release that is suppressed by mepacrine in rat mesangial cells (5), which suggests the involvement of PLA2 in the mechanism although the molecular species of the PLA2 is unknown. It is recognized that cPLA2 is responsible for the immediate arachidonic acid release and PGE2 production upon stimulation. In fact, H2O2 is known to activate cPLA2 to release arachidonic acid in other cell systems including vascular smooth muscle cells, striatal neurons, and fibroblasts (13, 42, 44). In the present study, we showed that the increase in arachidonic acid release stimulated by H2O2 and the synergistic effect of H2O2 and PDGF (see Fig. 1A) were completely inhibited by MAFP, a cPLA2 inhibitor (see Figs. 1 and 3). Concomitantly, the phosphorylation of cPLA2 was accelerated on stimulation with H2O2 and synergistically increased by H2O2 and PDGF (see Fig. 2). These results suggest that the activation of cPLA2 fully contributes to the synergistic release of arachidonic acid on stimulation with the combination of H2O2 and PGDF as well as the release evoked with H2O2 by itself. Our results indicate that PDGF-BB alone induces sufficient phosphorylation of cPLA2 (see Fig. 2) but it exerts little effect on arachidonic acid release (see Figs. 1 and 3). It has been reported for mesangial cells that PDGF-BB at the concentration used here induces an increase in intracellular Ca2+ concentration to 0.2 µM or less (8), whereas H2O2 has this effect to ~1.0 µM (35). In addition, it was reported that 0.3-1.0 µM of intracellular Ca2+ concentration is required for the translocation of cPLA2 from the cytosol to the membrane to hydrolyze membrane phospholipids (14, 16). Therefore, we suggest that PDGF-BB has little effect on arachidonic acid release, probably due to insufficient increase in the intracellular Ca2+ concentration.

Rat mesangial cells are reported to possess Ca2+-independent PLA2 (iPLA2) and secretory PLA2 (such as group IIA PLA2) in addition to cPLA2, all of which are activated upon stimulation with inflammatory cytokines or H2O2. We demonstrated recently an activation of iPLA2 in mesangial cells after stimulation with interleukin-1beta (4). It is documented that in mesangial cells, interleukin-1beta -induced PGE2 generation is dependent on the expression of group II PLA2 (45), and that interleukin-1beta and tumor necrosis factor-alpha also stimulate the expression of group IIA PLA2 via ROS generation (19). Furthermore, in uterine stromal cells, the contribution of iPLA2 to H2O2-induced arachidonic acid release was shown (7). These findings suggest the involvement of iPLA2 or secretory PLA2 in the arachidonic acid release observed in the present study. However, bromoenol lactone, a specific inhibitor of iPLA2, did not have any effect on the release (data not shown). Manoalide, an inhibitor of secretory PLA2, had no effect on the release, and also the activity of the PLA2 was not detected in the supernatant of mesangial cells stimulated with PDGF-BB and H2O2 for 60 min (data not shown). Therefore, we suggest that H2O2 and PDGF stimulate mesangial cells to activate cPLA2 and to release arachidonic acid.

cPLA2 activity is known to be upregulated by phosphorylation via ERK (35, 39). It was reported in mesangial cells that H2O2 phosphorylates and activates ERK and that PDGF induces the same events (29, 30, 53). We confirmed in the present study that PDGF and H2O2 each stimulated the phosphorylation of ERK1/2 (see Fig. 4). However, in combination, PDGF and H2O2 did not elicit a synergistic effect on the phosphorylation. Similarly, the synergistic release of arachidonic acid induced by this combination was inhibited partially by PD-98059 (an MEK inhibitor), whereas the release by H2O2 was completely inhibited by PD-98059 (see Fig. 5). Therefore, we suggest that another mechanism contributes to the synergistic activation of cPLA2 caused by the combination of PDGF and H2O2.

p38 MAP kinase has been shown to be activated by cytokines, endotoxic lipopolysaccharide, and stress caused by osmotic shock, heat shock, UV irradiation, and chemical agents (18, 22, 33, 43). Interestingly, it has been reported that, in human platelets, p38 MAP kinase could phosphorylate and activate cPLA2 after stimulation with thrombin and collagen (9, 32). Furthermore, stress-inducing stimuli such as H2O2 increased the phosphorylation and activation of cPLA2 and enhanced Ca2+ ionophore-induced arachidonic acid release, both of which were prevented by p38 MAP kinase inhibitor but not by ERK inhibitor in human platelets (10). Oxidative stress is also known to induce the activation of p38 MAP kinase in other cell systems (3, 6, 17, 41). These observations led us to suggest the participation of p38 MAP kinase in the synergistic increase in phosphorylation of cPLA2 and in the release of arachidonic acid induced by the combination of PDGF and H2O2. Our results showed that PDGF and H2O2 each stimulated the phosphorylation of p38 MAP kinase and in combination had a synergistic effect (see Fig. 6). Anisomycin, which activates MAP kinases responsive to stress stimuli such as p38 MAP kinase but not ERK (12, 23, 37), potentiated the H2O2-induced release of arachidonic acid and phosphorylation of p38 MAP kinase (see Fig. 7). Furthermore, whereas the ERK inhibitor PD-98059 and p38 MAP kinase inhibitor SB-202190 alone only partially inhibited the arachidonic acid release evoked by stimulation with PDGF and H2O2, combination of the two completely inhibited the release (see Fig. 8A). The synergistic increase in the phosphorylation of cPLA2 induced by PDGF and H2O2 was also attenuated by the combination of those inhibitors (Fig. 8, B and C). These observations suggest that both ERK and p38 MAP kinase are implicated in the increase in cPLA2 phosphorylation and arachidonic acid release after stimulation with PDGF together with H2O2. Recently, similar inhibitory effects of ERK and p38 MAP kinase inhibitors administered in combination on zymosan-stimulated cPLA2 phosphorylation and arachidonic acid release were reported in macrophages (23).

The mechanism by which PDGF and H2O2 synergistically stimulated phosphorylation of cPLA2 and release of arachidonic acid was not elucidated in the present study. It has been reported, however, that the PDGF receptor-operated signaling pathway, leading to ERK1/2 activation, DNA synthesis, and chemotaxis, is dependent on the generation of H2O2 in vascular smooth muscle cells (49). Furthermore, H2O2 has been shown to stimulate tyrosine phosphorylation of the PDGF receptor to induce a mitogenic response and an increase in the number of mesangial cells (24). Likewise, H2O2-induced tyrosine phosphorylation of PDGF receptors was reported recently for oligodendrocytes (6). Therefore, we suggest that one possible mechanism by which PDGF and H2O2 elicited synergistic effects on arachidonic acid release and phosphorylation of cPLA2 and p38 MAP kinase involves enhanced activation of the PDGF receptor.

In conclusion, we observed that PDGF and H2O2 induce a synergistic enhancement of cPLA2 activation to release arachidonic acid via a cooperative activation of ERK1/2 and p38 MAP kinase. Our results suggest that PDGF and oxygen radicals, which are generated in inflamed glomeruli or mesangial cells, cooperatively induce an increase in the production of PGE2 that is implicated in the pathogenesis of glomerular dysfunction, thus contributing to the initiation and progression of renal injury.


    ACKNOWLEDGEMENTS

This work was supported by the Frontier Research Program of the Ministry of Education, Science, Sports, and Culture of Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Sato, Dept. of Pathological Biochemistry, Kyoto Pharmaceutical Univ., Misasagi, Yamashina-ku, Kyoto 607-8414, Japan (E-mail: sato{at}mb.kyoto-phu.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00210.2001

Received 6 July 2001; accepted in final form 10 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abboud, HE. Platelet-derived growth factor and mesangial cells. Kidney Int 41: 581-583, 1992[ISI][Medline].

2.   Adler, S, Stahl RAK, Baker PJ, Chen YP, Pritzl PM, and Couser WG. Biphasic effect of oxygen radicals on prostaglandin production by rat mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 252: F743-F749, 1987[Abstract/Free Full Text].

3.   Aikawa, R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, Shiojima I, Hiroi Y, and Yazaki Y. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest 100: 1813-1821, 1997[Abstract/Free Full Text].

4.   Akiba, S, Hayama M, and Sato T. Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1beta and dibutyryl cAMP in rat mesangial cells. FEBS Lett 437: 225-228, 1998[ISI][Medline].

5.   Baud, L, Nivez MP, Chansel D, and Ardaillou R. Stimulation by oxygen radicals of prostaglandin production by rat renal glomeruli. Kidney Int 20: 332-339, 1981[ISI][Medline].

6.   Bhat, NR, and Zhang P. Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death. J Neurochem 72: 112-119, 1999[ISI][Medline].

7.   Birbes, H, Gothié E, Pageaux JF, Lagarde M, and Laugier C. Hydrogen peroxide activation of Ca2+-independent phospholipase A2 in uterine stromal cells. Biochem Biophys Res Commun 276: 613-618, 2000[ISI][Medline].

8.   Bonventre, JV, Weber PC, and Gronich JH. PAF and PDGF increase cytosolic [Ca2+] and phospholipase activity in mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 254: F87-F94, 1988[Abstract/Free Full Text].

9.   Börsch-Haubolds, AG, Kramer RM, and Watson SP. Phosphorylation and activation of cytosolic phospholipase A2 by 38-kDa mitogen-activated protein kinase in collagen-stimulated human platelets. Eur J Biochem 245: 751-759, 1997[Abstract].

10.   Buschbeck, M, Ghomashchi F, Gelb MH, Watson SP, and Börsch-Haubold AG. Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2. Biochem J 344: 359-366, 1999[ISI][Medline].

11.   Cane, A, Breton M, Koumanov K, Béréziat G, and Colard O. Oxidant-induced arachidonic acid release and impairment of fatty acid acylation in vascular smooth muscle cells. Am J Physiol Cell Physiol 274: C1040-C1046, 1998[Abstract/Free Full Text].

12.   Cano, E, Hazzalin CA, and Mahadevan LC. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol 14: 7352-7362, 1994[Abstract].

13.   Chakraborti, S, and Chakraborti T. Down-regulation of protein kinase C attenuates the oxidant hydrogen peroxide-mediated activation of phospholipase A2 in pulmonary vascular smooth muscle cells. Cell Signal 7: 75-83, 1995[ISI][Medline].

14.   Channon, J, and Leslie CC. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. J Biol Chem 265: 5409-5413, 1990[Abstract/Free Full Text].

15.   Choudhury, GG, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, and Abboud HE. PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF. Am J Physiol Renal Physiol 273: F931-F938, 1997[ISI][Medline].

16.   Clark, JD, Lin L-L, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, and Knopf JL. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043-1051, 1991[ISI][Medline].

17.   Clerk, A, Fuller SJ, Michael A, and Sugden PH. Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses. J Biol Chem 273: 7228-7234, 1998[Abstract/Free Full Text].

18.   Cohen, P. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol 7: 353-361, 1997[ISI].

19.   Dorsam, G, Taher MM, Valerie KC, Kuemmerle NB, Chan JC, and Franson RC. Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A2 in rat mesangial cells. J Pharmacol Exp Ther 292: 271-279, 2000[Abstract/Free Full Text].

20.   D'Souza, RJ, Phillips HM, Radeke HH, Aber GM, and Strange RC. Mesangial cell DNA synthesis induced by hydrogen peroxide, interleukin-6, and platelet-derived growth factor: effects of indomethacin and dazmegrel. Nephron 72: 263-268, 1996[ISI][Medline].

21.   Finkel, T, and Holbrook NJ. Oxidants, oxidative stress, and the biology of ageing. Nature 408: 239-247, 2000[ISI][Medline].

22.   Galcheva-Gargova, Z, Dérijard B, Wu IH, and Davis RJ. An osmosensing signal transduction pathway in mammalian cells. Science 265: 806-808, 1994[ISI][Medline].

23.   Gijón, MA, Spencer DM, Siddiqi AR, Bonventre JV, and Leslie CC. Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium. J Biol Chem 275: 20146-20156, 2000[Abstract/Free Full Text].

24.   González-Rubio, M, Voit S, Rodríguez-Puyol D, Weber M, and Marx M. Oxidative stress induces tyrosine phosphorylation of PDGF alpha - and beta -receptors and pp60c-src in mesangial cells. Kidney Int 50: 164-173, 1996[ISI][Medline].

25.   Ha, H, and Lee HB. Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Kidney Int 58 Suppl77: S19-S25, 2000[ISI].

26.   Hänsch, GM, Wagner C, Bürger A, Dong W, Staehler G, and Stoeck M. Matrix protein synthesis by glomerular mesangial cells in culture: effects of transforming growth factor beta  (TGF beta ) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA. J Cell Physiol 163: 451-457, 1995[ISI][Medline].

27.   Hayama, M, Akiba S, Fukuzumi M, and Sato T. High glucose-induced cytosolic phospholipase A2 activation responsible for eicosanoid production in rat mesangial cells. J Biochem 122: 1196-1201, 1997[Abstract].

28.   Hayama, M, Inoue R, Akiba S, and Sato T. Inhibitory effect of cepharanthine on fibronectin production in growth factor-stimulated rat mesangial cells. Eur J Pharmacol 390: 37-42, 2000[ISI][Medline].

29.   Huwiler, A, Fabbro D, and Pfeilschifter J. Platelet-derived growth factor stimulates de novo synthesis of mitogen-activated protein kinase in renal mesangial cells. Eur J Biochem 227: 209-213, 1995[Abstract].

30.   Ishikawa, Y, and Kitamura M. Dual potential of extracellular signal-regulated kinase for the control of cell survival. Biochem Biophys Res Commun 264: 696-701, 1999[ISI][Medline].

31.   Kashgarian, M, and Sterzel RB. The pathobiology of the mesangium. Kidney Int 41: 524-529, 1992[ISI][Medline].

32.   Kramer, RM, Roberts EF, Um SL, Börsch-Haubolds AG, Watson SP, Fisher MJ, and Jakubowski JA. p38 Mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. J Biol Chem 271: 27723-27729, 1996[Abstract/Free Full Text].

33.   Kyriakis, JM, and Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18: 567-577, 1996[ISI][Medline].

34.   Laurent, B, and Ardaillou R. Reactive oxygen species: production and role in the kidney. Am J Physiol Renal Fluid Electrolyte Physiol 251: F765-F776, 1986[ISI][Medline].

35.   Lin, L-L, Wartmann M, Lin AY, Knopf JL, Seth A, and Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269-278, 1993[ISI][Medline].

36.   Lounsbury, KM, Hu Q, and Ziegelstein RC. Calcium signaling and oxidant stress in the vasculature. Free Radic Biol Med 28: 1362-1369, 2000[ISI][Medline].

37.   Matsumoto, T, Yokote K, Tamura K, Takemoto M, Ueno H, Saito Y, and Mori S. Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration. J Biol Chem 274: 13954-13960, 1999[Abstract/Free Full Text].

38.   Meyer, TN, Gloy J, Hug MJ, Greger R, Schollmeyer P, and Pavenstädt H. Hydrogen peroxide increases the intracellular calcium activity in rat mesangial cells in primary culture. Kidney Int 49: 388-395, 1996[ISI][Medline].

39.   Nemenoff, RA, Winitz S, Qian NX, van Putten V, Johnson GL, and Heasley LE. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem 268: 1960-1964, 1993[Abstract/Free Full Text].

40.   Pfeilschifter, J, and Hosang M. Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells. Cell Signal 3: 413-424, 1991[ISI][Medline].

41.   Raingeaud, J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, and Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270: 7420-7426, 1995[Abstract/Free Full Text].

42.   Rao, GN, Runge MS, and Alexander RW. Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells. Biochim Biophys Acta 1265: 67-72, 1995[ISI][Medline].

43.   Rouse, J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, and Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027-1037, 1994[ISI][Medline].

44.   Samanta, S, Perkinton MS, Morgan M, and Williams RJ. Hydrogen peroxide enhances signal-responsive arachidonic acid release from neurons: role of mitogen-activated protein kinase. J Neurochem 70: 2082-2090, 1998[ISI][Medline].

45.   Schalkwijk, C, Pfeilschifter J, Märki F, and van den Bosch H. Interleukin-1beta - and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2. J Biol Chem 267: 8846-8851, 1992[Abstract/Free Full Text].

46.   Schambelan, M, Blake S, and Sraer J. Increased prostaglandin production by glomeruli isolated from rats with streptozotocin-induced diabetes mellitus. J Clin Invest 75: 404-412, 1985[ISI][Medline].

47.   Schievella, AR, Regier MK, Smith WL, and Lin L-L. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem 270: 30749-30754, 1995[Abstract/Free Full Text].

48.   Shaw, S, Naegeli P, Etter JD, and Weidmann P. Role of intracellular signalling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells. Clin Exp Pharmacol Physiol 22: 924-933, 1995[ISI][Medline].

49.   Sundaresan, M, Yu ZY, Ferrans VJ, Irani K, and Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296-299, 1995[Abstract].

50.   Suzuki, YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997[ISI][Medline].

51.   van Rossum, GS, de Wit R, Bunt G, Verkleij AJ, van den Bosch H, Post JA, and Boonstra J. Activation of mitogen-activated protein kinase and cytosolic phospholipase A2 by hydrogen peroxide in fibroblasts. Lipids 34 Suppl: S65, 1999[ISI][Medline].

52.   Williams, B, and Schrier RW. Glucose-induced protein kinase C activity regulates arachidonic acid release and eicosanoid production by cultured glomerular mesangial cells. J Clin Invest 92: 2889-2896, 1993[ISI][Medline].

53.   Wilmer, WA, Tan LC, Dickerson JA, Danne M, and Rovin BH. Interleukin-1beta induction of mitogen-activated protein kinases in human mesangial cells. J Biol Chem 272: 10877-10881, 1997[Abstract/Free Full Text].


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