Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells

Kiho Takaya1, Daisuke Koya1, Motohide Isono1, Toshiro Sugimoto1, Takeshi Sugaya2, Atsunori Kashiwagi1, and Masakazu Haneda1

1 Department of Medicine, Shiga University of Medical Science, Shiga 520-2192; and 2 Discovery Research Laboratory, Tanabe Seiyaku Company, Limited, Osaka, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Persistent proteinuria has been indicated to be a major risk factor for the development of tubulointerstitial damage through a process of proinflammatory molecule expression. Monocyte chemoattractant protein-1 (MCP-1) was shown to contribute to recruitment of immune cells into the renal interstitium in acute and chronic renal diseases. However, the molecular mechanisms by which proteinuria causes MCP-1 expression in proximal tubular cells have not been fully clarified. In this study, we examined whether albumin overload-induced MCP-1 expression was regulated by mitogen-activated protein kinase (MAPK) in mouse proximal tubular (mProx) cells. Exposure of mProx cells to delipidated bovine serum albumin (BSA) induced mRNA and protein expression of MCP-1 in a time- and dose-dependent manner. BSA activated extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. The MEK inhibitor U-0126 partially suppressed BSA-induced MCP-1 expression and MCP-1 promoter/luciferase reporter activity. U-0126 also inhibited an increase in nuclear factor-kappa B and activator protein-1 DNA-binding activity of MCP-1 promoter by protein overload in mProx cells. In addition, we found that U-0126 inhibited BSA-induced nuclear factor-kappa B reporter activity and inhibitory protein degradation in mProx cells. In conclusion, these findings indicate that ERK signaling is involved in BSA-induced MCP-1 expression in mProx cells.

nuclear factor-kappa B; tubulointerstitial damage


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE IMPORTANT CORRELATION between the degree of proteinuria and the risk of progression of renal disease has been recognized (5, 18). It is widely observed that patients with significant proteinuria are more likely to develop end-stage renal failure than those without proteinuria (21, 23, 25). It is also recognized that the degree of renal dysfunction correlates with histological abnormalities in the renal tubulointerstitium, rather than in glomeruli, even in primary glomerular disorders (4, 23, 27). These observations suggest that the severity of proteinuria is a major determinant of tubulointerstitial injury (21).

Recent reports have revealed that albumin, the major protein found in proteinuria, is able to induce phenotypic changes in proximal tubular cells and alter their function in a manner that produces proinflammatory chemokines in the renal tubulointerstitium (10). When proximal tubular cells are incubated with albumin, they produce monocyte chemoattractant protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cell expressed and secreted) (31, 34). MCP-1 is an important chemoattractant for macrophages and T lymphocytes (2, 6, 16), which are the predominant inflammatory cells found in the interstitium in chronic glomerular disease (7, 17). Increasing evidence also suggests that MCP-1 may have an important role not only in the regulation of interstitial inflammation but also in other processes related to matrix deposition (17).

The intracellular mechanisms by which albumin upregulates MCP-1 in proximal tubular cells have not been fully characterized. The promoter region of the mouse MCP-1/JE gene contains putative binding sites for a number of transcription factors, including the ubiquitous nuclear factor-kappa B (NF-kappa B)/Rel family and activator protein-1 (AP-1) (20). NF-kappa B proteins normally exist in the cytosol as dimers bound to inhibitory proteins (Ikappa B). After exposure to diverse stimuli, Ikappa B undergoes proteolysis, allowing NF-kappa B to enter the nucleus and activate the expression of genes encoding chemokines and other proteins (1, 13). The significance of NF-kappa B in regulation of the MCP-1 gene has been reported in various types of cells, including renal cells (19, 26, 30, 32). For instance, exposure of rat proximal tubular cells to albumin induced NF-kappa B activation (32, 34). Inhibition of NF-kappa B with pharmacological agents (N-tosyl-phenylalanine chloromethyl ketone and dexamethasone) or an antisense oligonucleotide to the rat p65 subunit of NF-kappa B significantly reduced albumin-induced MCP-1 transcription in rat proximal tubular cells (32).

On the other hand, recent evidence suggests that activation of AP-1 is also required for induction of MCP-1. Shyy et al. (28) reported that AP-1 binding to 12-O-tetradecanoylphorbol-13-acetate-responsive elements is critical for shear stress-induced expression of the MCP-1 gene. It has also been reported that lipopolysaccharide, transforming growth factor-beta , or interleukin-1beta stimulates MCP-1 gene expression by activating AP-1 in certain types of cells (19, 29, 33). A role in the regulation of murine MCP-1/JE expression for the AP-1 site exposed to tumor necrosis factor-alpha and transforming growth factor-beta has been suggested by other studies, because MCP-1/JE expression was decreased when antisense DNAs targeting c-Jun and c-Fos or inhibitors of c-Jun and c-Fos were used (12, 29). AP-1 activity is regulated by the mitogen-activated protein kinase (MAPK) superfamily. At least three members of MAPK, extracellular signal-regulated kinase (ERK), p38 MAPK, and the c-Jun NH2-terminal kinase (JNK) have been identified to be involved in a wide range of cellular responses to extracellular signals (24). Recently, Dixon and Brunskill (9) demonstrated that albumin was able to stimulate proliferation of proximal tubular cells via ERK, suggesting a possible link between albuminuria and the derangements of proximal tubular cell growth observed in progressive renal scarring. However, it remains to be clarified whether ERK is involved in the regulation of MCP-1 expression.

In the present study, we investigated the role of ERK activated by BSA in regulation of MCP-1 expression.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

BSA (fatty acid free, fraction V) was obtained from Bayel (Pittsburgh, PA). BSA did not contain a high level of endotoxin (<3.0 ng/ml), as detected by the Endospacy method (SRL, Tokyo, Japan).

Antiphospho-p42/p44 ERK, antiphospho-p38 MAPK, antiphospho-Ikappa B-alpha , and anti-Ikappa B-alpha antibodies were purchased from New England Biolabs (Beverly, MA). Antiphospho-JNK antibody, U-0126, SB-203580, and luciferase kit were purchased from Promega (Madison, WI). Anti-ERK2, -p38 MAPK, and -JNK2 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-beta -actin antibody was obtained from Sigma Chemical (St. Louis, MO). The 5×NF-kappa B luciferase reporter vector (NF-kappa B-Luc) was obtained from Clontech (Palo Alto, CA). [alpha -32P]dCTP and [gamma -32P]ATP were purchased from New England Nuclear (Boston, MA). All other reagents were of chemical grade and were purchased from standard suppliers.

Proximal Tubular Cell Culture

Microdissection of the proximal tubule. Proximal tubular segments were microdissected from C57BL/6J adult mouse kidney. The kidneys were perfused through the renal artery and excised, and coronal sections were cut with a surgical blade. The sections were transferred to a flask containing 10 ml of ice-cold Hanks' balanced salt solution containing 0.1% BSA and 0.1% type I collagenase, which was used to perfuse the kidneys. The flask was incubated for 10 min at 37°C in a shaking water bath. The samples were suffused with 5% CO2-95% O2 during the incubations. Tissues were transferred to Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and placed on ice. Then the proximal tubular segments were microdissected under a stereoscopic microscope.

Immortalization of proximal tubular cells. The microdissected proximal tubules were aspirated into 20 µl of K1 medium composed of 1:1 DMEM-Ham's F-12 culture medium with 10% FCS and placed on type IV collagen-precoated 96-well plates. The proximal tubules were maintained in a minimum volume of the modified 1:1 mesenchymal cultured K1 medium-fresh K1 medium for 24-48 h. After attachment to the culture plate, proximal tubules were cocultured with the mouse mesenchymal feeder cells. At ~7-10 days after microdissection, primary cell outgrowths were transfected with 100 µg/ml of the simian virus-40 large T antigen gene by Transfectam reagent (Promega) according to the manufacturer's protocol. The cells were maintained in culture for 2 days after transfection. Then they were dissociated with 0.25% trypsin-EDTA and reseeded onto the type IV collagen-precoated 48-well plates with fresh feeder cells. After passages 2 and 3, the transfection procedure was repeated, and the cells were expanded in the presence of feeder cells. After passage 3, the immortalized cells were no longer dependent on the presence of feeder cells and were maintained in K1 medium at 37°C in a 5% CO2-95% air humidified atmosphere.

The cells were stained by cytokeratin, but not by alpha -smooth muscle actin (data not shown). Thus this cell line expressed the proximal tubular phenotype.

Albumin Uptake Assay

Cells were exposed to serum-free DMEM containing BSA (5 mg/ml) at 37°C. Control cells were incubated on ice for measurement of nonspecific albumin uptake. After 90 min, the culture plates were placed on ice. Then the cells were scraped from the culture plates and lysed via sonication. After pH was adjusted to 7.4 with PBS (pH 6.0), insoluble material was removed by centrifugation. The supernatants were used for ELISA measurements (see BSA ELISA).

BSA ELISA

Polyvinyl ELISA plates were coated with 100 µl of rabbit anti-BSA diluted 1:500 in PBS (pH 7.4) at 4°C overnight before experiments. After the plates were washed twice with PBS containing 0.05% Tween 20 (PBS-T), they were incubated with 100 µl of cell lysates for 2 h at room temperature. Nonspecific peroxidation sites were blocked for 30 min at room temperature with 3% H2O2. After repeated washings, the plates were incubated with 100 µl of biotinylated anti-rabbit BSA diluted 1:250 in PBS for 2 h at room temperature. After repeated washings, the plates were incubated with 1:1 avidin-biotinylated horseradish peroxidase for 1 h at room temperature. After repeated washings, the plates were incubated with 100 µl of azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt peroxidase substrate (1 mg/ml), 0.1 M citric acid (pH 4.2), and 30% H2O2 (0.3 µl/ml) at room temperature for 30 min. The absorbance of each well was read at 405 nm using an automatic ELISA plate reader. A standard curve was plotted, and BSA concentration in each sample was calculated by comparison with a standard curve.

Proximal Tubular Cell Culture and Experimental Procedure

Murine proximal tubular (mProx) cells were cultured in DMEM containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultured cells from passages 4-30 were used for the experiments. Subconfluent cells were made quiescent by incubation with 0.1% FBS-DMEM for 24 h. Quiescent cells were incubated with various concentrations of fatty acid-free BSA in the experimental medium (DMEM with 0.1% BSA and 14 mM HEPES) for the indicated times at 37°C. For the experiments with inhibitors, cells were incubated with the indicated concentrations of U-0126 or SB-203580 for 60 min at 37°C before exposure to BSA. After incubation, cells were harvested for the determination listed below.

Northern Blot Analysis

Total RNA (12 µg) was isolated by guanidinium and phenol extraction (TRIzol Reagent, GIBCO BRL, Grand Island, NY), electrophoresed on 1% formaldehyde-agarose gels, and transferred to a nylon membrane (Nytran, Schleicher & Schuell, Dassel, Germany). Mouse MCP-1 was labeled with [alpha -32P]dCTP by a random primer labeling method (Bca BEST, Takara, Shiga, Japan). The membranes were hybridized with mouse MCP-1 cDNA in hybridization buffer (Perfect Hyb, Toyobo, Osaka, Japan) at 65°C for 16 h. The membranes were autoradiographed at -80°C overnight.

Immunoblot Analysis

Immunoblot analysis was performed as previously described (15). Briefly, proximal tubular cells were lysed in SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% bromphenol blue). The cell lysates containing 40 µg of proteins in SDS sample buffer were electrophoresed on 12% SDS-polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA). After the membranes were blocked, they were incubated with a 1:1,000 dilution of antiphospho-ERK, antiphospho-p38 MAPK, antiphospho-JNK, or antiphospho-Ikappa B-alpha antibodies at 4°C overnight. Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat antibody (Amersham, Buckinghamshire, UK) was used as a secondary antibody. The immunoreactive bands were detected with an enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA). The membranes were then reprobed with anti-ERK, anti-p38 MAPK, anti-JNK, anti-Ikappa B-alpha , or anti-beta -actin antibody as an internal control.

DNA Constructions and Transient Transfection

The pJECAT2.6 was a gift from Dr. J. M. Boss. The reporter construct contains DNA fragments for the six MCP-1/JE promoter fused to the coding region of the chloramphenicol acetyltransferase gene (20). To construct the luciferase reporter (MCP-1/JE-Luc), the MCP-1/JE promoter site constructions were subcloned into pGL3 vector (Promega) at MluI/XhoI sites. mProx cells (~1 × 106/assay) were transfected with 0.7 µg of the beta -galactosidase and luciferase reporter constructs by Lipofectamine (GIBCO BRL) for 6 h. The cells were incubated with BSA (30 mg/ml) for 12 h after transfection or treated with 10 µM U-0126 for 1 h before exposure to BSA. Luciferase activity was determined with a luciferase assay kit (Promega) and normalized by cotransfected beta -galactosidase activity (Promega).

MCP-1 Measurement by ELISA

The culture media of proximal tubular cells on a 24-well plate were collected and stored at -80°C until assay. Accumulation of MCP-1 protein in the media was measured with an MCP-1 ELISA kit (R & D System, Minneapolis, MN). The concentrations of MCP-1 were corrected for the total amount of cellular DNA, as previously reported (14).

Nuclear Extraction and Electrophoretic Mobility Shift Assay

Nuclear protein was prepared from proximal tubular cells as previously described (15). Electrophoretic mobility shift assays (EMSAs) were performed by incubation of 5 µg of nuclear proteins with 1 µg of poly(dI-dC) in binding buffer (20 mM HEPES, pH 7.9, 1.8 mM MgCl2, 2 mM dithiothreitol, 0.5 mM EDTA, and 0.5 mg/ml BSA) at room temperature for 20 min and reaction with [gamma -32P]ATP-labeled AP-1/GC or kappa B-1/kappa B-2 oligonucleotide at room temperature for an additional 30 min. The reaction mixtures were electrophoresed on a 5% polyacrylamide gel and autoradiographed.

The coding strand sequences of the DNAs of interest were as follows: 5'-GCACCCTGCCTGACTCCACCCCCCTGGCTTACAA-3' (AP-1/GC) and 5'-CCCGAAGGGTCTGGGAACTTCCAATACTGCCTCAGAATGGGAATTTCCACGCTCTTATCC-3' (kappa B-1/kappa B-2).

Statistical Analysis

Values are means ± SD. Analysis of variance followed by Scheffé's test was used to determine significant differences in multiple comparisons. P < 0.05 was defined as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of Immortalized mProx Cells

Albumin uptake into mProx cells. To ensure that our mProx cells would reabsorb albumin, as occurs in the normal kidney, the cells were incubated with 5 mg/ml BSA for 90 min at 37°C, and then cellular BSA was measured by ELISA (see METHODS). The cells showed about fivefold enhanced albumin reabsorption compared with control (0.247 µg albumin/mg protein at 37°C vs. 0.042 µg albumin/mg protein at 4°C). Approximately 75% of uptake was blocked by cold incubation, indicating that the majority of cellular albumin uptake was specific. These results also indicate that this cell line expresses a proximal tubular phenotype and is functionally similar to primary cultures.

Effect of BSA on MCP-1 mRNA Expression

We examined whether BSA stimulated the expression of MCP-1 mRNA in mProx cells. By Northern blotting, BSA (30 mg/ml) induced MCP-1 mRNA in a time-dependent manner from 1 h to a maximal stimulation at 6 h (Fig. 1A). The induction of MCP-1 mRNA by BSA was dose dependent, with a maximal stimulation at 30 mg/ml (Fig. 1B).


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Fig. 1.   Effect of BSA on monocyte chemoattractant protein-1 (MCP-1) mRNA expression. After serum deprivation of mouse proximal tubular (mProx) cells for 24 h, cells were exposed to BSA (30 mg/ml) for indicated times (A) or to BSA at 5-30 mg/ml for 6 h (B). RNA was extracted, and MCP-1 mRNA was analyzed by Northern blot, with 28S and 18S mRNA as internal standards (left). Right: quantitative results; n = 4. *P < 0.05 vs. control.

Activation of MAPKs and NF-kappa B by BSA

Activation of MAPKs was assessed by immunoblotting using antibodies that recognize only the phosphorylated form of ERK, p38 MAPK, or JNK. In mProx cells, BSA stimulated the activities of ERK and p38 MAPK. ERK was significantly activated at 5 min after stimulation and returned to basal levels at 30 min (Fig. 2A). Activation of p38 MAPK was also seen at 1 min after stimulation (Fig. 2B). However, JNK was not activated by BSA (data not shown).


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Fig. 2.   Activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) by BSA. mProx cells were incubated with BSA (30 mg/ml) for indicated times. Top: equal amounts of cell lysate were subjected to immunoblotting using antibodies against phospho-ERK (pERK1/2, A) or phospho-p38 MAPK (pp38, B). Blots were developed with enhanced chemiluminescence. Membranes were reprobed with anti-ERK or anti-p38 MAPK antibodies. Blots are representative of 4 independent experiments. Bottom: quantitative results. *P < 0.05 vs. control.

To define the kinetics of Ikappa B-alpha proteins involved in NF-kappa B activation, we examined the phosphorylation and degradation of Ikappa B-alpha . Exposure to BSA led to transient phosphorylation of Ikappa B-alpha in 1 h. In parallel, Ikappa B-alpha is degraded and then returned to control levels within 2.5 h. After 2.5 h, phosphorylation and expression of Ikappa B-alpha seemed to be increased (Fig. 3).


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Fig. 3.   Phosphorylation and degradation of Ikappa B-alpha by BSA. mProx cells were exposed to BSA (30 mg/ml) for indicated times. Cell extracts (40 µg of protein) were subjected to Western blotting with anti-phospho-Ikappa B-alpha antibody (pIkappa B), anti-Ikappa B-alpha antibody, and anti-beta -actin antibody. Blot is representative of 5 experiments.

Effect of MAPK Pathway on BSA-Induced MCP-1 Expression

We next investigated the intracellular signaling pathways involved in BSA-induced MCP-1 expression. Because we clearly demonstrated that BSA activated ERK and p38 MAPK (Fig. 2), the effect of inhibitor of MEK (U-0126) or p38 MAPK (SB-203580) on expression of MCP-1 was examined. We first confirmed that albumin uptake was not affected by pretreatment with U-0126 (data not shown). Treatment with U-0126 partially suppressed BSA-induced MCP-1 mRNA expression. On the other hand, SB-203580 tended to inhibit BSA-induced MCP-1 mRNA, but this effect was not statistically significant (Fig. 4A). To further confirm the roles of ERK and p38 MAPK in MCP-1 expression, accumulation of MCP-1 protein in the medium was analyzed by ELISA. Consistent with the result of Fig. 4A, treatment with U-0126 suppressed BSA-induced MCP-1 protein in a dose-dependent manner. SB-203580 tended to suppress BSA-induced MCP-1 protein, but this effect was not statistically significant. The combination of U-0126 and SB-203580 suppressed BSA-induced MCP-1 protein, but these effects were not additive or synergistic (Fig. 4B). To elucidate the mechanism of transcriptional regulation of MCP-1 in mProx cells, the six MCP-1/JE promoter fusion reporter constructs, 2,724-bp fragment (-2,642 to +82 with respect to the MCP-1/JE start of transcription), fused to the luciferase gene (MCP-1/JE-Luc), were used. mProx cells transfected with MCP-1/JE-Luc demonstrated an increase in luciferase activity on exposure to BSA (Fig. 4C). When mProx cells were treated with 10 µM U-0126 before exposure to BSA, luciferase activity was partially suppressed (Fig. 4C). These results suggest that the ERK pathway is involved in mediating the BSA-induced increase of MCP-1 expression.


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Fig. 4.   Effect of inhibitors of MAPK on BSA-induced MCP-1 mRNA and protein. A: mProx cells were treated with 10 µM U-0126 or 10 µM SB-203580 for 1 h and then exposed to BSA (30 mg/ml) for 2 h. Left: Northern blot of mRNA. Right: quantitative results; n = 4. *P < 0.05 vs. control; #P < 0.05 vs. BSA. B: cells were treated with 5 or 10 µM U-0126 or/and SB-203580 for 1 h and then exposed to BSA (30 mg/ml) for 24 h. Accumulation of MCP-1 protein in medium was analyzed by ELISA on duplicate samples; n = 3. *P < 0.05 vs. control; # P < 0.05 vs. BSA. C: 6 MCP-1/JE promoter fusion reporter constructs linked to the luciferase gene were transfected into mProx cells. Transfected cells were treated with 10 µM U-0126 for 1 h before exposure to BSA (30 mg/ml) for 8 h. Luciferase activity was measured by a luciferase assay kit. Values are normalized by cotransfected beta -galactosidase activity. * P < 0.05 vs. control; # P < 0.05 vs. BSA.

Effect of the MAPK Pathway on BSA-Induced DNA Binding of NF-kappa B and AP-1

Previous studies revealed that the distal regulatory region of the murine gene contains two kappa B sites: kappa B-1 and kappa B-2. The proximal regulatory region contains a third kappa B site (kappa B-3), an AP-1-binding site, a GC box, and a TATA box (Fig. 5A) (20). To elucidate the role of these binding sites in response to BSA, we performed EMSA using a labeled AP-1/GC box site as a probe. BSA stimulated DNA binding to AP-1 at 1 h (Fig. 5B). Addition of excess unlabeled AP-1/GC oligonucleotide resulted in complete absence of the band in autoradiographs, confirming the specificity of the reaction. In contrast, the binding was not affected by incubation with an irrelevant oligonucleotide, i.e., NF-kappa B consensus oligonucleotide (Fig. 5B). Similarly, increased DNA binding of NF-kappa B was maximal 1 h after exposure to BSA in mProx cells. Addition of excess unlabeled kappa B-1/kappa B-2 oligonucleotide competitively blocked the specific binding of NF-kappa B, whereas AP-1 consensus oligonucleotide did not affect this binding (Fig. 5C).


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Fig. 5.   Activation of DNA binding to nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1) by BSA and effect of inhibitor of MEK on BSA-induced NF-kappa B and AP-1 DNA binding. A: distal regulatory region of the murine MCP-1/JE gene contains 2 kappa B sites, and proximal regulatory region contains an AP-1-binding site. mProx cells were treated with 10 µM U-0126 for 1 h and then exposed to BSA (30 mg/ml) for 1 h. Electrophoretic mobility shift assays were performed by incubating nuclear proteins (5 µg) with [gamma -32P]ATP end-labeled AP-1/GC oligonucleotide (B) or kappa B-1/kappa B-2 oligonucleotide containing MCP-1 kappa B consensus sequences (C). Reaction mixtures were electrophoresed on a 5% polyacrylamide gel and autoradiographed.

Pretreatment of mProx cells with U-0126 attenuated the DNA binding activity of AP-1 by BSA (Fig. 5B). DNA-binding activity of NF-kappa B was also inhibited by U-0126 (Fig. 5C).

Involvement of MAPK in BSA-Induced Ikappa B-alpha Degradation and NF-kappa B Activation

Because U-0126 was able to inhibit BSA-stimulated DNA-binding activity of NF-kappa B (Fig. 5C), we hypothesized that ERK is involved in the activation of NF-kappa B. To learn how the ERK and NF-kappa B pathways interact, we examined the effect of U-0126 on BSA-induced Ikappa B-alpha degradation and NF-kappa B activation. Pretreatment of mProx cells with U-0126 attenuated the Ikappa B-alpha degradation by BSA (Fig. 6A).


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Fig. 6.   Effect of inhibitor of MEK on Ikappa B-alpha degradation and NF-kappa B luciferase activity. A: mProx cells were treated with 10 µM U-0126 for 1 h and then exposed to BSA (30 mg/ml) for 1.5 h. Cell extracts (40 µg of protein) were subjected to Western blotting with anti-Ikappa B-alpha antibody. Blot is representative of 4 experiments. B: 5×NF-kappa B luciferase gene (NF-kappa B-Luc) was transfected into mProx cells. Transfected cells were treated with 10 µM U-0126 for 1 h before exposure to BSA (30 mg/ml) for 8 h. Luciferase activity of duplicate samples was measured by a luciferase assay kit. Values are normalized by cotransfected beta -galactosidase activity; n = 5. *P < 0.05 vs. control; # P < 0.05 vs. BSA.

To further confirm the involvement of ERK in the activation of NF-kappa B, we performed reporter assay using 5×NF-kappa B luciferase vector (NF-kappa B-Luc). Mouse proximal tubule cells transfected with NF-kappa B-Luc demonstrated an increase in luciferase activity on exposure to BSA (Fig. 6B). When mProx cells were treated with 10 µM U-0126 before exposure to BSA, the luciferase activity was partially suppressed.

These results suggest that the ERK pathway is involved in the activation of NF-kappa B by BSA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Increasing evidence suggests that proteinuria itself may mediate disease progression in chronic glomerulopathy, rather than simply reflect the severity of glomerular injury (3, 22). Although albumin was found to induce the transcription and translation of MCP-1 in rat proximal tubular cells at pathophysiologically relevant concentrations (31), signal transduction pathways involved in the effect of albumin on MCP-1 have not been fully characterized. In the present study, we demonstrated that 1) albumin induced the expression of MCP-1 in mProx cells, a newly established mouse proximal tubular cell line; 2) albumin induced the activation of ERK and the phosphorylation and degradation of Ikappa B-alpha ; 3) the inhibition of MEK with U-0126 reduced MCP-1 expression; 4) U-0126 inhibited albumin-induced activation of DNA binding of AP-1 and NF-kappa B, the binding sites of which existed in the promoter region of MCP-1; and 5) the transcriptional activity of NF-kappa B and Ikappa B-alpha degradation were inhibited by U-0126.

In the present study, ERK was significantly activated 5 min after stimulation with BSA and returned to basal levels by 30 min in mProx cells. A similar effect of albumin was observed in opossum kidney proximal tubular cells, in which ERK was activated 1 min after incubation with recombinant human serum albumin, reached maximal activation at 5 min, and returned to basal levels by 30 min (9). These results suggest that albumin is able to rapidly activate ERK in proximal tubular cells. Although BSA could also activate p38 MAPK in mProx cells, the MKK6-p38 stress kinase cascade was found to be critical for tumor necrosis factor-alpha -induced expression of MCP-1 in human umbilical vein endothelial cells (11). In the present study, the inhibitor for p38 MAPK tended to suppress BSA-induced MCP-1 expression, but this effect was not statistically significant. Thus further studies are needed to clarify the involvement of p38 MAPK in the regulation of MCP-1.

We found that U-0126, an inhibitor of MEK, was able to inhibit BSA-induced mRNA and protein expression of MCP-1 and MCP-1 reporter activity in mProx cells. In addition, phosphorylation and degradation of Ikappa B were also induced by BSA in mProx cells. Although albumin overload induced MCP-1 expression through activation of NF-kappa B in rat proximal tubular cells (32), we hypothesized that the ERK-AP-1 and Ikappa B-NF-kappa B pathways could be responsible for albumin-induced expression of MCP-1. Indeed, the cooperative action of NF-kappa B and AP-1 in interleukin-1beta -induced MCP-1 gene expression was suggested in human endothelial cells (19). The murine MCP-1/JE gene contains two kappa B sites in the distal regulatory region and one AP-1-binding site in the proximal regulatory region (20). We found that BSA could stimulate DNA binding of AP-1 and NF-kappa B in the MCP-1 gene in mProx cells. As expected, U-0126 inhibited AP-1 induced by BSA. Interestingly, U-0126 also partially inhibited BSA-induced NF-kappa B-binding activity and NF-kappa B-dependent transcription as well as the degradation of Ikappa B in mProx cells. These results indicate that the ERK pathway interacts with the Ikappa B-NF-kappa B pathway after stimulation with BSA in mProx cells. This hypothesis is supported by the recent findings in melanoma cells, in which NF-kappa B-induced kinase (NIK) was found to regulate NF-kappa B activation through a novel NIK-MEK-ERK-NF-kappa B signaling pathway in addition to the classical NIK-IKK-Ikappa B-NF-kappa B pathway (8).

Although further investigation is needed to elucidate the precise roles of ERK, the present findings support the hypothesis that the ERK pathway is involved in BSA-induced MCP-1 expression and suggest a possible interaction between NF-kappa B and ERK. This information could be useful in the design of anti-inflammatory strategies to suppress not only cell proliferation but also transcriptional activation of cytokines in renal diseases.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Haneda, Dept. of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan (E-mail: haneda{at}belle.shiga-med.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.

First published January 7, 2003;10.1152/ajprenal.00230.2002

Received 18 June 2002; accepted in final form 30 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barnes, PJ, and Karin M. Nuclear factor kappa B---a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066-1071, 1997[Free Full Text].

2.   Boucher, A, Droz D, Adafer E, and Noel LH. Characterization of mononuclear cell subsets in renal cellular interstitial infiltrates. Kidney Int 29: 1043-1049, 1986[ISI][Medline].

3.   Burton, C, and Walls J. Proximal tubular cell, proteinuria and tubulo-interstitial scarring. Nephron 68: 287-293, 1994[ISI][Medline].

4.   Cameron, JS. Immunologically mediated interstitial nephritis: primary and secondary. Adv Nephrol 18: 207-248, 1989.

5.   Cameron, JS. Proteinuria and progression in human glomerular disease. Am J Nephrol 10: 81-87, 1990[ISI][Medline].

6.   Carr, MW, Roth SJ, Luther E, Rose SS, and Springer TA. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci USA 91: 3652-3656, 1994[Abstract].

7.   Danoff, TM. Chemokines in interstitial injury. Kidney Int 53: 1807-1808, 1998[ISI][Medline].

8.   Dhawan, P, and Richmond A. A novel NF-kappa B-inducing kinase-MAPK signaling pathway up-regulates NF-kappa B activity in melanoma cells. J Biol Chem 277: 7920-7928, 2002[Abstract/Free Full Text].

9.   Dixon, R, and Brunskill NJ. Albumin stimulates p44/p42 extracellular signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells. Clin Sci Lond 98: 295-301, 2000[ISI][Medline].

10.   Eddy, AA, and Giachelli CM. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 47: 1546-1557, 1995[ISI][Medline].

11.   Goebeler, M, Karin K, Gillitzer R, Kunz M, Yoshimura T, Brocker EB, Rapp UR, and Ludwig S. The MKK6/p38 stress kinase cascade is critical for tumor necrosis factor-alpha -induced expression of monocyte-chemoattractant protein-1 in endothelial cells. Blood 93: 857-865, 1999[Abstract/Free Full Text].

12.   Hanazawa, S, Takeshita A, Amano S, Semba T, Nirazuka T, Katoh H, and Kitano S. Tumor necrosis factor-alpha induces expression of monocyte chemoattractant JE via fos and jun genes in clonal osteoblastic MC3T3-E1 cells. J Biol Chem 268: 9526-9532, 1993[Abstract/Free Full Text].

13.   Henkel, T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah Y, and Baeuerle PA. Rapid proteolysis of Ikappa B-alpha is necessary for activation of the transcription factor NF-kappa B. Nature 365: 182-185, 1993[ISI][Medline].

14.   Ishida, T, Haneda M, Maeda S, Koya D, and Kikkawa R. Stretch-induced overproduction of fibronectin in mesangial cells is mediated by the activation of mitogen-activated protein kinase. Diabetes 48: 595-602, 1999[Abstract].

15.   Isono, M, Haneda M, Maeda S, Omatsu-Kanbe M, and Kikkawa R. Atrial natriuretic peptide inhibits endothelin-1-induced activation of JNK in glomerular mesangial cells. Kidney Int 53: 1133-1142, 1998[ISI][Medline].

16.   Leonard, EJ, and Yoshimura T. Human monocyte chemoattractant protein-1 (MCP-1). Immunol Today 11: 97-101, 1990[ISI][Medline].

17.   Lloyd, CM, Minto AW, Dorf ME, Proudfoot A, Wells TNC, Salant DJ, and Gutierrez-Ramos JC. RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis. J Exp Med 185: 1371-1380, 1997[Abstract/Free Full Text].

18.   Mallick, NP, Short CD, and Hunt LP. How far since Ellis? Nephron 46: 113-124, 1987[ISI][Medline].

19.   Martin, T, Cardarelli PM, Parry GCN, Felts KA, and Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1. Eur J Immunol 27: 1091-1097, 1997[ISI][Medline].

20.   Ping, D, Jones PL, and Boss JM. TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene. Immunity 4: 455-469, 1996[ISI][Medline].

21.   Remuzzi, G, and Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 339: 1448-1456, 1998[Free Full Text].

22.   Remuzzi, G, Ruggenenti P, and Benigni A. Understanding the nature of renal disease progression. Kidney Int 51: 2-15, 1997[ISI][Medline].

23.   Risdon, RA, Sloper JC, and De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 1: 363-366, 1968.

24.   Robinson, MJ, and Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9: 180-186, 1997[ISI][Medline].

25.   Rossing, P, Hommel E, Smidt UM, and Parving HH. Impact of arterial blood pressure and albuminuria on the progression of diabetic nephropathy in IDDM patients. Diabetes 42: 715-719, 1993[Abstract].

26.   Rovin, BH, Dickerson JA, Tan LC, and Hebert CA. Activation of nuclear factor-kappa B correlates with MCP-1 expression by human mesangial cells. Kidney Int 48: 1263-1271, 1995[ISI][Medline].

27.   Schainuck, LI, Striker GE, Cutler RE, and Benditt EP. Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1: 631-640, 1970[Medline].

28.   Shyy, JYJ, Lin MC, Han J, Lu Y, Petrime M, and Chien S. The cis-acting phorbol ester 12-O-tetradecanoyl phorbol 13-acetate-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci USA 92: 8069-8073, 1995[Abstract].

29.   Takeshita, A, Chen Y, Watanabe A, Kitano S, and Hanazawa S. TGF-beta induces expression of monocyte chemoattractant JE/monocyte chemoattractant protein 1 via transcriptional factor AP-1 induced by protein kinase in osteoblastic cells. J Immunol 155: 419-426, 1995[Abstract].

30.   Ueda, A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, and Okubo T. NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 153: 2052-2063, 1994[Abstract/Free Full Text].

31.   Wang, Y, Chen J, Chen L, Tay YC, Rangan GK, and Harris DCH Induction of monocyte chemoattractant protein-1 in proximal tubule cells by urinary protein. J Am Soc Nephrol 8: 1537-1545, 1997[Abstract].

32.   Wang, Y, Rangan GK, Goodwin B, Tay YC, Wang Y, and Harris DCH Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor-kappa B in proximal tubule cells. J Am Soc Nephrol 10: 1204-1213, 1999[Abstract/Free Full Text].

33.   Wang, Y, Rangan GK, Goodwin B, Tay YC, Wang Y, and Harris DCH Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kappa B dependent. Kidney Int 57: 2011-2022, 2000[ISI][Medline].

34.   Zoja, C, Donadelli R, Colleoni S, Figliuzzi M, Bonazzola S, Morigi M, and Remuzzi G. Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation. Kidney Int 53: 1608-1615, 1998[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 284(5):F1037-F1045
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