Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells

Sule Sengul1,2, Craig Zwizinski1,2, and Vecihi Batuman1,2,3

1 Section of Nephrology, Department of Medicine, Tulane Medical Center, 3 Tulane Cancer Center, and 2 Veterans Administration Medical Center, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that light chain (LC) endocytosis by human proximal tubule cells (PTCs) leads to production of cytokines through activation of NF-kappa B. Here, we examined the role of MAPK pathways in these responses using four species of myeloma LCs (kappa 1, kappa 2, kappa 3, and lambda 1) previously shown to induce cytokine production by PTCs. Among these, kappa 1-LC, which yielded the strongest cytokine responses, was selected for detailed studies. Activation of MAPKs was probed by Western blot analysis for the active kinases, ERK 1/2, JNK 1/2, and p38 in kappa 1-LC-exposed human PTCs. To evaluate the functional role of MAPKs in LC-induced cytokine responses, we tested the effects of U-0126, an ERK inhibitor; SP-600125, an inhibitor of JNK; SB-203580, a p38 inhibitor; and curcumin, a JNK-AP-1 inhibitor, all added to media before 4-h exposure to 1.5 mg/ml kappa 1-LC. IL-6 and monocyte chemotactic protein-1 (MCP-1) were determined by ELISA. Both LC and human serum albumin (HSA) activated ERK, although the HSA effect was weaker. kappa 1-LC stimulated all three MAPKs, although phosphorylation of ERK was more pronounced and sustained than others. Inhibitors of ERK, JNK, and p38 reduced LC-induced IL-6 and MCP-1 production. These findings suggest that activation of MAPKs plays a role in LC-induced cytokine responses in PTCs. Activation of MAPKs may be involved in cytokine responses induced by other proteins as well as LCs and may be pivotal in the pathophysiology of tubulointerstitial injury in proteinuric diseases.

intracellular signaling mechanisms; interleukin-6; MCP-1; progression of renal diseases; myeloma; proteinuria


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INCREASED ENDOCYTOSIS of filtered proteins in the proximal tubule has been shown to induce cytokine production by proximal tubule cells (PTCs), possibly mediating tubulointerstitial injury (1, 33, 47, 48). This has been proposed as a major mechanism in the progression of kidney disease in proteinuric states. In multiple myeloma, excessive production and luminal delivery of light chains (LCs) to PTCs cause increased endocytosis of LCs via the endocytic receptors cubilin and megalin and result in eventual degradation within the lysosomes (3, 4). We previously demonstrated that increased endocytosis of myeloma LCs by human PTCs induces inflammatory cytokine production through activation of NF-kappa B (38). However, possible signaling mechanisms mediating these inflammatory responses and, specifically, the potential role of intracellular signaling pathways such as MAPKs have not been elucidated in response to LC endocytosis.

The present study was designed to investigate the cellular signaling mechanisms mediating these responses in cultured human PTCs. For this purpose, we first determined the phosphorylation of the three major MAPKs, ERK 1/2, JNK 1/2, and p38, in kappa 1-LC-exposed human PTC by immunoblotting using phospho-specific antibodies. To evaluate the functional role of phosphorylation of these MAPKs, we tested the effects of pharmacological inhibitors of MAPKs, U-0126, an inhibitor of ERK (11); SP-600125, an inhibitor of JNK (5, 16); SB-203580, an inhibitor of p38 (7, 46); and curcumin, an inhibitor of the JNK-AP1 pathway (23, 27), on kappa 1-LC-induced IL-6 and MCP-1 production. Our results suggest that MAPK pathways are involved in LC-induced cytokine responses.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and purification of myeloma LC. Myeloma LCs isolated from the urine of myeloma patients and previously shown to induce cytokine production through activation of NF-kappa B in human PTCs were used in these experiments. LCs were isolated and purified by a slight modification of the method previously reported from our laboratory (30, 38). Briefly, LCs were precipitated from urine with ammonium sulfate (55 to 90% saturation determined empirically), extensively dialyzed against distilled water and lyophilized. The precipitated LCs were purified by dissolving the lyophilized desalted crude protein in buffer at pH 6.0, followed by chromatography on carboxymethyl-Sephadex (C-50; Pharmacia, Piscataway, NJ). Under these conditions, the LCs were bound to the column, whereas the contaminants were not. Bound LC was eluted with 0.6 mol/l of NaCl, redialyzed against distilled water, and lyophilized. The purity of LCs was confirmed by SDS-PAGE (21) and the immunological identity reported from the clinical laboratory was confirmed by Western blotting using goat anti-human kappa  and lambda -LC antibodies.

We tested our LC preparation for endotoxin using the chromogenic Limulus amebocyte lysate (LAL) test (Charles River Labs, Charleston, SC). We found that our LCs used in these experiments were essentially endotoxin free (<3.7 EU/mg LC protein). The LCs used in these experiments did not contain measurable quantities of IL-6 and TNF-alpha (sensitivity of the ELISA kits 0.7 and 4.4 pg/ml, respectively), indicating that cytokine contamination does not occur during purification (38).

LCs were collected from myeloma patients with modest renal insufficiency without albuminuria and no evidence of glomerular involvement. Thus the LCs studied here are considered tubulopathic. Kidney biopsies were not performed in the patients.

Cell cultures. In all experiments, SV40 immortalized human PTCs were used. Grown as a monolayer, these cells show marker brush-border enzymes and have biochemical and morphological characteristics similar to other widely used PTC lines, including LLC-PK1, OK, HK-2, and human PTCs in stable culture (31). Brush-border enzymes and phloridzin-inhibitable glucose transport were similar to other established PTC models. This transformed cell line's response to inflammatory stimuli was extensively compared with parental PTCs and was found similar (14). Cells were routinely grown in DRM-23E medium supplemented with 0.5% (vol:vol) fetal bovine serum in T-75 flasks (Falcon, Becton Dickinson Labware) at 37°C in a humidified atmosphere of 95% air-5% CO2 and refed at intervals of 2 or 3 days. At confluence, the culture medium was aspirated, the cultures were rinsed with HBSS, and the cells were removed by trypsin/ethylene-diamine tetraacetic acid digestion, reseeded into T-75 flasks containing the complete medium, and cultured to confluence.

Preparation of whole cell lysates. Cells planted onto 35-mm sterile tissue culture dishes (Corning Glass Works, Corning, NY) or six-well tissue culture plates were grown at 37°C in serum-free medium in an incubator for 24 h. After each experiment, medium was removed and cells were washed with PBS. The following steps were done on ice: 200 µl of RIPA buffer consisting of 50 mM Tris · HCl (pH 7.4), 1% NP-40 (IGEPAL-CA630), 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA (EGTA) (pH 8.0), protease inhibitors (aprotinin, leupeptin, and pepstatin, 1 µg/ml each, and freshly added), 1 mM Na3VO4, and 1 mM PMSF (added immediately before use) were added to the dishes. After 10 min of incubation, dishes were scraped with a cell scraper and then lysates were transferred to 1.5-ml microcentrifuge tubes using syringes fitted with 21-gauge needles. Lysates were passed through a 21-gauge needle to shear DNA and centrifuged at 13,000 g for 10 min at 4°C. Finally, supernatants were harvested as whole cell lysates and used in immunoblotting studies.

Protein concentrations were determined in whole cell lysates prepared from PTCs by using Pierce BCA Protein Assay (Rockford, IL).

Western blot analysis for MAPKs. Equal amounts of proteins were separated by NuPAGE Bis-Tris gel by using precast gels (10% acrylamide) and a minigel apparatus (Novex). Electrophoresis was performed according to manufacturer's recommendations. Separated proteins were electrophoretically transferred to nitrocellulose membranes for 1 h at 30 V by using a semi-dry blotting apparatus (Novex). Proteins were probed with polyclonal phospho-specific antibodies against ERK 1/2, JNK 1/2, and p38 (1:5,000, 1:2,000, and 1:5,000 dilutions, respectively) by using a Western Breeze Chemiluminescent Kit according to the manufacturer's protocol (Novex). Integrity of phospho-specific antibodies against MAPKs was tested using sorbitol and nerve growth factor-treated PC12 cell extracts provided by the antibody supplier (Promega, Madison, WI). We determined total MAPKs using primary antibodies (1:1,000 dilution, Cell Signaling, Beverly, MA) to confirm equal protein loading in the gels for each experiment. Representative blots from one of at least three separate experiments were selected for illustration.

We examined the effects of four different myeloma LCs (kappa 1, kappa 2, kappa 3, and lambda 1) and human serum albumin (HSA) on ERK activation. For this experiment, myeloma LCs (1.5 mg/ml) and HSA (1.5 mg/ml, Sigma) were dissolved in serum-free regular media. Confluent monolayers of serum-deprived PTCs were treated with protein solutions for 10 min in six-well plates. At the end of exposure, cell lysates were prepared and blotted for both phosphorylated and total forms of ERK (Fig. 1). A kappa 1-LC (first LC on Fig. 1) that we previously demonstrated to yield the strongest cytokine responses (38) was selected for detailed studies.


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Fig. 1.   Effects of different light chains (LCs) and human serum albumin (HSA) on ERK activation. The effects of 4 myeloma LCs (kappa 1, lambda 1, kappa 2, and kappa 3, LC-1 to -4, respectively) and HSA on ERK activation were determined. Cells were treated with 4 different LCs and HSA for 10 min, cell lysates were then probed for phospho-specific (top) and total ERK (bottom). The effect of LCs on ERK activation was more marked than HSA. These LCs were the same proteins used in our previous reports (30, 38) and shown to induce strong cytokine responses in proximal tubule cells (PTCs), whereas HSA had no effect (38). (Representative figure from 3 different experiments is shown.)

For time course studies, kappa 1-LC (1.5 mg/ml, ~50 µM) dissolved in serum-free medium was added to confluent monolayers of cells for 10, 20, 40, 60, 120, and 240 min, and then cell lysates were prepared and used in immunoblotting studies for phosphorylated and total forms of MAPKs (Fig. 2, A-C).


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Fig. 2.   Effects of kappa 1-LC on MAPK activation. Human PTCs were exposed to 1.5 mg/ml (~50 µM) of kappa 1-LC for the indicated times. A: there was marked activation of ERK 1/2 in LC-exposed cells, detected as early as 10 min, decreasing but persisting over 4 h. Both JNK 1/2 and p38 were also activated. B: phospho-JNK 1/2 bands seemed to fade completely after 40 min of exposure to myeloma LC. C: phospho-p38 band started to fade after 40 min but was sustained up to 240 min. A-C, bottom: total MAPKs corresponding to each experiment. (Representative blots from 3 different experiments are shown.)

Because the most marked response appeared with the ERK pathway, we also performed dose-response experiments with kappa 1-LC (0.4-0.025 mg/ml) on ERK activation. In this experiment, cells were treated with decreasing concentrations of kappa 1-LC for 10 min and cell lysates were probed for ERK. For comparison, total ERK was also shown for each experiment (Fig. 3).


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Fig. 3.   Effects of varying doses of kappa 1-LC on ERK activation. Confluent monolayers of PTCs were exposed to decreasing concentrations of kappa 1-LC (0.4- 0.025 mg/ml) for 10 min. Top: effect of kappa 1-LC on ERK activation appeared to be dose dependent and was observed at concentrations as low as 0.025 mg/ml (~1 µM) at 10 min. Bottom: total ERK. (Representative blots from 3 different experiments are shown.)

Effects of pharmacological inhibitors on activation of MAPKs. To test the effects of MAPK inhibitors, after 24-h serum deprivation, confluent monolayers of cells were pretreated with the MAPK inhibitors (U-0126, SP-600125, and SB-203580) for 1 h at 1, 5, and 15 µM concentrations and then treated with 1.5 mg/ml (50 µM) of kappa 1-LC in the continued presence of inhibitors. These inhibitors are considered selective for their respective pathways by many investigators (5, 7, 11, 16, 23, 27, 46). After 10 min of exposure, cell lysates were prepared using the method described above. Blotting studies were performed using primary antibodies against phosphorylated and total forms of MAPK pathways (Fig. 4, A-C).


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Fig. 4.   Effects of pharmacological inhibitors on LC-induced activation of MAPKs. A: effects of U-0126 on kappa 1-LC-induced activation of ERK. Cells were pretreated with different concentrations (1, 5, and 15 µM) of U-0126, a pharmacological inhibitor of ERK, for 1 h and then treated with kappa 1-LC (1.5 mg/ml, ~50 µM) in the continued presence of U-0126 for 10 min. Top: U-0126 inhibited kappa 1-LC-induced activation of ERK. Bottom: total ERK was not affected. (Representative blots from 3 different experiments are shown.) B: effects of SP-600125 (SP) on kappa 1-LC-induced activation of JNK. Cells were pretreated with different concentrations (1, 5, and 15 µM) of SP, a pharmacological inhibitor of JNK, for 1 h and then exposed to kappa 1-LC (1.5 mg/ml, ~50 µM) for 10 min in the presence of SP. Top: activation of JNK was inhibited by SP. Bottom: total JNK was not affected. (Representative blots from 3 different experiments are shown.) C: effects of SB-203580 (SB) on kappa 1-LC-induced activation of p38. After 1-h pretreatment with SB (1, 5, and 15 µM), a pharmacological inhibitor of p38, cells were exposed to kappa 1-LC (1.5 mg/ml, 50 µM) for 10 min. Top: kappa 1-LC-induced activation of p38 was suppressed by SB. Bottom: total p38 was not affected. (Representative blots from 3 different experiments are shown.)

Effect of MAPK inhibitors on IL-6 and MCP-1 production. For MAPK inhibitor experiments, serum-deprived (24 h) cells were pretreated for 1 h with MAPK inhibitors (Figs. 5-8). Concentrations of the inhibitors (1-15 µM for U-0126, SP-600125, curcumin, and SB-203580) used in these experiments were determined from published literature (5, 7, 11, 16, 23, 27, 46) and pilot studies in our laboratory. These concentrations of inhibitors had negligible effects on basal cytokine production (see RESULTS). Cell viability was determined by trypan blue exclusion assays; in all experiments, at least 85% of cells remained viable (7). After pretreatment with inhibitors, cells were incubated with kappa 1-LC (1.5 mg/ml, ~50 µM) for 4 h in the presence and absence of the MAPK inhibitors (Figs. 5-8). After exposure, culture supernatants were harvested and stored at -70°C for ELISA assays.


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Fig. 5.   Effect of ERK inhibition on basal and kappa 1-LC-induced IL-6 and monocyte chemotactic protein (MCP-1) production. Cells were pretreated with ERK inhibitor U-0126 (1-15 µM) for 1 h before exposure to kappa 1-LC (1.5 mg/ml, ~50 µM) for 4 h. Basal secretion of both IL-6 (A) and MCP-1 (C) is normalized to 1. kappa 1-LC-stimulated IL-6 (B) and MCP-1 (D) production is normalized to 1 and results are expressed as fold-increase over basal or LC-stimulated cytokine production. A: kappa 1-LC-stimulated IL-6 production by nearly 4-fold compared with controls [from 121.01 ± 1.35 (normalized to 1) to 436.02 ± 45. 9 pg/106 cells]. U-0126 alone had no effect on IL-6 production. B: LC-induced IL-6 production was significantly inhibited by U-0126 (up to 75% with 15 µM U-0126). C: kappa 1-LC-stimulated MCP-1 production by 6-fold compared with controls [from 65.44 ± 1.08 (normalized to 1) to 406.69 ± 15.3 pg/106 cells]. D: U-0126 significantly inhibited MCP-1 production. Results are presented as means ± SE (n = 3, *P > 0.05 vs. control, ***P < 0.001 vs. control or kappa 1-LC).



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Fig. 6.   Effect of JNK inhibition on basal and kappa 1-LC-induced IL-6 and MCP-1 production. Cells were pretreated with JNK inhibitor SP (1-15 µM) for 1 h before exposure to myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basal secretion of both IL-6 (A) and MCP-1 (C) is normalized to 1. kappa 1-LC-stimulated IL-6 (B) and MCP-1 (D) production is normalized to 1 and results are expressed as fold-increase over basal or LC-stimulated cytokine production. A: in this experiment, LC induced ~3-fold increase in IL-6 production compared with control (from 133.05 ± 2.54 to 375.1 ± 1.00 pg/10 6 cells). Unlike previous experiments, SP alone also inhibited basal IL-6 production. B: LC-induced IL-6 production was significantly inhibited by SP. C: SP alone had no effect on MCP-1 production, but kappa 1-LC-induced MCP-1 production was significantly inhibited by SP (D) at all 3 concentrations. Results are presented as means ± SE (n = 3, *P > 0.05 vs. control, ***P < 0.001 vs. control or kappa 1-LC).



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Fig. 7.   Effect of curcumin (Curc) on basal and kappa 1-LC-induced IL-6 production. Cells were pretreated with Curc, an inhibitor of JNK-AP1 (1-15 µM), for 1 h before stimulation with myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basal secretion of both IL-6 (A) and MCP-1 (C) is normalized to 1. kappa 1-LC-stimulated IL-6 (B) and MCP-1 (D) production is normalized to 1 and results are expressed as fold-increase over basal or LC-stimulated cytokine production. A-C: Curc alone had no effect on cytokine production. B-D: Curc significantly inhibited kappa 1-LC-induced IL-6 and MCP-1. Results are presented as means ± SE (n = 3, *P > 0.05 vs. control, **P < 0.05 vs. kappa 1-LC, ***P < 0.001 vs. control or kappa 1-LC).



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Fig. 8.   Effect of p38 inhibition on basal and kappa 1-LC-induced IL-6 and MCP-1 production. Cells were pretreated with p38 inhibitor SB (1-15 µM) for 1 h before stimulation with myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basal secretion of both IL-6 (A) and MCP-1 (C) is normalized to 1. kappa 1-LC-stimulated IL-6 (B) and MCP-1 (D) production is normalized to 1 and results are expressed as fold-increase over basal or LC-stimulated cytokine production. SB alone had no effect on basal secretion of either IL-6 (A) or MCP-1 (C). LC-induced IL-6 production was significantly inhibited by SB at only 15-µM concentration (B). Similarly, LC-induced MCP-1 production was significantly inhibited by SB (D) at all 3 concentrations. Results are presented as means ± SE (n = 3, *P > 0.05 vs. control, ***P < 0.001 vs. control or kappa 1-LC).

Measurement of IL-6 and MCP-1 levels by ELISA. IL-6 and MCP-1 were measured in the supernatants using commercial human ELISA kits (Quantikine; R&D Systems) according to the manufacturer's protocol. The sensitivity of the ELISA is 0.7 pg/ml for IL-6 and 5 pg/ml for MCP-1 assays. Cytokine concentrations in the unknown samples were determined by comparison with a standard curve developed with known amounts of recombinant human cytokines provided with the kits. Experiments were conducted in triplicate using 96-well microplates, and results were read in a microplate reader. Cells were trypsinized and counted and expressed as picograms of cytokine per 106 cells. Results were presented as fold-increase over either basal or LC-stimulated responses normalized to 1.

Reagents and antibodies. Cell culture products and all other reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified. Antibodies against active (phosphorylated) ERK 1/2, JNK 1/2, and p38 were purchased from Promega. MAPK inhibitors (U-0126 and SB-203580) were purchased from Calbiochem (San Diego, CA). SP-600125 was purchased from Biomol (Plymouth Meeting, PA). Antibodies against total MAPKs were purchased from Cell Signaling.

Statistical analysis. Results were expressed as means ± SE. Multiple comparisons were made by ANOVA and Bonferroni's multiple-comparison tests. Statistical analyses, curve fitting, and calculations were done using GraphPad Prism, version 3 for Windows NT (1999, GraphPad Software, San Diego, CA). Statistical significance was defined as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LCs induce activation of ERK in PTCs. Four different LCs (kappa 1, kappa 2, kappa 3, and lambda 1) at 1.5-mg/ml concentration induced activation of ERK 1/2 compared with control (Fig. 1). There was a weaker response to HSA (1.5 mg/ml) also, although this protein did not elicit cytokine responses in human PTCs (38).

kappa 1-LC induces activation of ERK, JNK, and p38 in PTCs. Human PTCs exposed to kappa 1-LC (1.5 mg/ml, ~50 µM) for 10-240 min resulted in marked activation of ERK 1/2, JNK 1/2, and p38. ERK activation was detected as early as 10 min, decreasing but persisting over 4 h (Fig. 2A). The kappa 1-LC-induced phosphorylation of both JNK 1/2 and p38 was again discernible at 10 min and sustained for up to 4 h, although phospho-JNK 1/2 bands seemed to fade after 40 min (Fig. 2, B and C). Blots for total MAPKs remained constant throughout the duration of the experiments.

Because the most marked response appeared with ERK 1/2, we determined the effect of varying doses of kappa 1-LC on ERK phosphorylation. We found the effect of this LC to be dose dependent and effective at concentrations as low as 0.025 mg/ml (~1 µM) (Fig. 3).

Effects of pharmacological inhibitors on activation of MAPKs. We tested the effects of MAPK inhibitors at the concentrations (1, 5, and 15 µM) used in these experiments on respective MAPK pathways. These agents inhibited phospho-MAPKs at the same concentrations demonstrated to inhibit IL-6 and MCP-1 production, although the degree of apparent MAPK inhibition did not always seem proportionate to the degree of inhibition in the cytokine response. These inhibitors had no effect on total MAPKs (Fig. 4, A-C).

MAPK inhibitors suppress kappa 1-LC-induced IL-6 and MCP-1 production. We tested the effect of the pharmacological inhibitors of ERK, JNK, JNK-AP-1, and p38 pathways on production of IL-6 and MCP-1 in control cells and in cells exposed to kappa 1-LC, 1.5 mg/ml for 4 h.

Myeloma kappa 1-LC increased IL-6 production by about fourfold from baseline compared with control cells (from 121.01 ± 1.35 to 436.02 ± 45.9 pg/106 cells, P < 0.001; Fig. 5A). This same LC induced an approximately sixfold increase in MCP-1 production compared with control cells (from 65.44 ± 1.08 to 406.69 ± 15.3 pg/106 cells, P < 0.001; Fig. 5C). The pharmacological inhibitor of ERK, U-0126, showed a marked and dose-dependent inhibition of IL-6 in LC-exposed cells but had no effect on basal IL-6 and MCP-1 secretion in control cells not exposed to LC (Fig. 5, A-C). U-0126 also inhibited MCP-1 production (Fig. 5D). However, the inhibitory effect of this inhibitor of ERK on MCP-1 production in LC-exposed cells was not as marked as its effect on IL-6 (Fig. 5).

SP-600125, an inhibitor of JNK, and curcumin, an inhibitor of JNK-AP1, also inhibited both IL-6 and MCP-1 in kappa 1-LC-exposed cells (Figs. 6 and 7). kappa 1-LC again induced marked stimulation of IL-6 (~3- to 4-fold; Figs. 6A and 7A). These inhibitors did not have significant effects on basal secretion of cytokines in control cells, except for the experiment with SP-600125, which showed a significant inhibition of IL-6 (Fig. 6A). kappa 1-LC-induced IL-6 and MCP-1 production was also significantly inhibited by curcumin (Fig. 7, B and D).

Finally, we examined the effect of SB-203580, an inhibitor of p38. These experiments showed that p38 blockade caused a significant inhibition of both IL-6 and MCP-1 production in LC-exposed cells (Fig. 8, B and D). However, the effect of this inhibitor on IL-6 production was evident only at 15 µM and was not seen at lower concentrations (Fig. 8B). SB-203580 did not have a significant effect on basal secretion of either IL-6 or MCP-1 (Fig. 8, A and C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cytokine production in response to filtered serum proteins has recently attracted increasing attention as a major mechanism of progression of kidney diseases in proteinuric states (1, 32, 33, 43, 47, 48). We recently showed that excessive LC endocytosis causes IL-6, MCP-1, and IL-8 production through activation of NF-kappa B in cultured human PTCs and proposed that this may be an important mechanism of tubulointerstitial disease commonly seen in multiple myeloma (38). However, the potential role of signaling pathways such as MAPKs in these responses is unknown.

MAPKs are important mediators of cellular stress responses phosphorylating selected intracellular proteins, including transcription factors when challenged with a wide variety of stimuli (12, 37). Three major MAPK families have been identified in mammalian cells: ERK 1 and 2, also known as p42/44 MAP kinase; JNK, also known as SAPK 1; and p38 MAP kinase, also known as SAPK 2 (28, 34). They have been shown to be activated as early responses to a variety of stimuli, including peptide mitogens, growth factors, oxidants, proinflammatory-inflammatory cytokines, lipid mediators, complement, albumin, and physical stressors in many cultured cell systems (8-10, 13, 15, 18, 19, 22, 26, 35, 36, 41, 45). Increased production of inflammatory cytokines through activation of different MAPKs has been described in response to IL-1beta , TNF-alpha , and physical pressure in both cultured mesangial and PTCs (22, 35, 40). Many transcription factors, such as c-Jun, ELK-1, ATF1, ATF2, NF-kappa B, and cAMP response element binding protein, have been shown to be regulated by MAPK pathways (20, 44). MAPKs could act via the transcription factors and they could directly or indirectly regulate cytokine gene transcription (17, 41).

We recently observed that inflammatory cytokine production in response to excessive LC endocytosis in cultured human PTCs is mediated via activation of NF-kappa B (38). In the present study, we demonstrated that LC-induced cytokine responses involve activation of MAPKs. Among these MAPKs, ERK 1/2 appeared to play the dominant role in LC-exposed cells. However, all three MAPKs appeared to participate, suggesting that LC-induced cytokine responses involve complex and multiple mechanisms.

Our studies provide two lines of evidence supporting these conclusions. First, we found that phosphorylated MAPKs are upregulated in LC-exposed cells. Immunoblotting experiments showed activation of all three MAPKs studied here. The most robust and sustained responses were seen with ERK at exposures as early as 10 min and decreasing over time but persisting as long as 4 h (Fig. 2A). Second, the inhibitors of the three major MAPKs as well as the JNK-AP-1 inhibitor curcumin all resulted in significant inhibition of LC-induced IL-6 and MCP-1 production (Figs. 5-8). Taken together, these two lines of evidence show that MAPKs are involved in LC-induced cytokine production in human PTCs.

Although the ERK pathway appeared to be more prominent than other kinases, all three MAPKs tested here were clearly involved. That all three signaling cascades might participate in cytokine responses is not surprising, because it is well documented that there may be considerable overlap and perhaps "cross talk" among these MAPKs (41). Furthermore, cell stress responses are well known to use multiple effector pathways.

The effects of pharmacological inhibitors of MAPKs should be interpreted with caution, however. Although many investigators consider these inhibitors specific for their respective pathways (5, 7, 11, 16, 23, 27, 46), it has been shown that some of these agents may have either variable or additional effects that may be relevant to the responses observed here. For example, SB-203580 can directly inhibit thromboxane synthase and cyclooxygenase-1 and -2 (6, 25). Curcumin, considered a selective AP-1 inhibitor, at higher concentrations (40-60 µM) has been shown to inhibit TNF-, phorbol ester-, and hydrogen peroxide-mediated NF-kappa B activation at a step before I-kappa B phosphorylation in a myeloid cell line (39). In another study, curcumin was shown to inhibit AP-1, NF-kappa B, as well as Egr-1 in cultured endothelial cells (29). At lower concentrations, i.e., 10 µM, curcumin was demonstrated to inhibit endogenous VCAM-1 expression in human microvascular endothelial cells independent of and without an effect on NF-kappa B. In this study, antibodies against c-Jun and c-Fos inhibited NF-kappa B activation, suggesting an interaction between AP-1 and NF-kappa B (2). Although these studies were conducted in different cell lines and used inhibitors at higher concentrations than used in our studies, they raise the possibility that some of the cytokine-blocking effects of these pharmacological inhibitors of MAPKs may be mediated through other pathways and may involve overlapping mechanisms. In our experiments, the apparent effects of the MAPK inhibitors on their respective MAPKs were not always proportionate to the degree of cytokine inhibition, and this is also consistent with the possibility that the effects of MAPK inhibitors on cytokine responses may involve additional mechanisms.

We recently demonstrated that LC-induced cytokine production was through the activation of NF-kappa B, similar to the observations reported by investigators attributing such responses to increased protein endocytosis (1, 32, 33, 48). It is of interest that LC effects were seen at concentrations as low as 25 µg/ml, concentrations that can easily occur in typical patients with myeloma and even in proteinuric patients without myeloma (38).

LCs are well documented to undergo vigorous endocytosis in PTCs via the endocytic receptors cubilin-megalin (3, 4). Other investigators who demonstrated albumin endocytosis as a proinflammatory event also proposed that albumin endocytosis is accomplished through the tandem cubilin-megalin system (42). The interaction between LC ligand with the endocytic receptors cubilin-megalin suggests an attractive speculation into how endocytosis can result in activation of MAPKs and the subsequent or simultaneous activation of nuclear transcription factors causing increased production of inflammatory cytokines in PTCs. Whether LC preferentially binds to cubilin and is endocytosed by its "chaperone" megalin or binds to megalin directly, the cytoplasmic domain of megalin, which contains NPXY motifs (42), could easily initiate the required phosphorylation cascade through tyrosine phosphorylation, progressing via ERK 1/2 and eventually resulting in phosphorylation of I-kappa B, the penultimate step in the activation of NF-kappa B. Our experiments suggested a significant role for MAPKs in LC-mediated cytokine responses. It is relevant that MAPKs have been demonstrated to have a key role in endocytosis (24), lending credence to our thesis that LC endocytosis activates MAPKs.

Previous studies on the endocytosis-induced cytokines in PTC have focused almost exclusively on albumin (1, 33, 47, 48). These studies demonstrated that albumin exposure in various PTC lines resulted in production of cytokines through activation of NF-kappa B. Albumin has also been demonstrated by Dixon and Brunskill (10) to stimulate p44/42 in opossum kidney PTCs; however, whether this results in cytokine production has not been examined. Our observations with myeloma LCs have similarities to these findings but also differ in fundamental ways. With the use of human PTCs and HSA even at concentrations exceeding those reported in the literature, we failed to elicit cytokine responses (38). In the present study, we did observe activation of ERK by HSA, although this effect was much weaker compared with LCs. Taken together, our observations on LCs and HSA and the previously published studies on albumin suggest that endocytosis of all filtered proteins may have inflammatory consequences. Our results suggest that LCs may be more potent than albumin in inducing cytokines in PTCs. Furthermore, LC effects were observed at very low concentrations (~1 µM) that can occur even in patients without myeloma, suggesting a potential role of LCs in the progression of kidney disease in other proteinuric diseases.

This study reveals clues that may improve our understanding of how proteinuria and excessive endocytosis of serum proteins can cause inflammatory responses in PTCs. A comprehensive description of the key steps that start with endocytosis resulting in production of cytokines will require additional experiments. Elucidation of the precise mechanisms of cytokine responses involved in protein endocytosis may open up novel areas probing therapeutic interventions that may be helpful in intervening with the progression of kidney disease in proteinuria.


    ACKNOWLEDGEMENTS

These studies were supported by a Merit Review Grant from the Department of Veterans Affairs. Partial support was also provided from developmental funds of the Tulane Cancer Center (research fellowship award to S. Sengul). Parts of this study are presented as an abstract at the 2002 annual meeting of the American Society of Nephrology in Philadelphia, PA.


    FOOTNOTES

Present address of S. Sengul: Ankara University Medical School, Division of Nephrology, Ibni Sina Hospital, Ankara, Turkey.

Address for reprint requests and other correspondence: V. Batuman, Nephrology Section, SL-45, Tulane Medical School, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: vbatuma{at}tulane.edu).

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.

First published February 11, 2003;10.1152/ajprenal.00350.2002

Received 27 September 2002; accepted in final form 4 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 284(6):F1245-F1254