Involvement of ERK pathway in albumin-induced MCP-1expression in mouse proximal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：KihoTakaya, DaisukeKoya, MotohideIsono, ToshiroSugimoto, TakeshiSugaya, AtsunoriKashiwagi,  MasakazuHaneda作者单位：1 Department of Medicine, Shiga University of MedicalScience, Shiga 520-2192; and Discovery ResearchLaboratory, Tanabe Seiyaku Company, Limited, Osaka, Japan 0 L( y7 a0 w+ X% }/ a. C
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      【摘要】2 W2 B; p/ _% D* D6 c2 t% o- Z
   Persistent proteinuria has been indicatedto be a major risk factor for the development of tubulointerstitialdamage through a process of proinflammatory molecule expression.Monocyte chemoattractant protein-1 (MCP-1) was shown to contribute torecruitment of immune cells into the renal interstitium in acute andchronic renal diseases. However, the molecular mechanisms by whichproteinuria causes MCP-1 expression in proximal tubular cells have notbeen fully clarified. In this study, we examined whether albuminoverload-induced MCP-1 expression was regulated by mitogen-activatedprotein kinase (MAPK) in mouse proximal tubular (mProx) cells. Exposureof mProx cells to delipidated bovine serum albumin (BSA) induced mRNAand protein expression of MCP-1 in a time- and dose-dependent manner. BSA activated extracellular signal-regulated kinase (ERK1/2) and p38MAPK. The MEK inhibitor U-0126 partially suppressed BSA-induced MCP-1expression and MCP-1 promoter/luciferase reporter activity. U-0126 alsoinhibited an increase in nuclear factor- B and activator protein-1DNA-binding activity of MCP-1 promoter by protein overload in mProxcells. In addition, we found that U-0126 inhibited BSA-induced nuclearfactor- B reporter activity and inhibitory protein degradation inmProx cells. In conclusion, these findings indicate that ERK signalingis involved in BSA-induced MCP-1 expression in mProx cells. 5 K, o( U5 O% g. o- |# T
      【关键词】 nuclear factor B tubulointerstitial damage/ i  v! X9 q" S* R3 m
   INTRODUCTION% {0 t4 d$ V+ t) V

THE IMPORTANT CORRELATION between the degree of proteinuria and the risk ofprogression of renal disease has been recognized ( 5, 18 ).It is widely observed that patients with significant proteinuria aremore likely to develop end-stage renal failure than those withoutproteinuria ( 21, 23, 25 ). It is also recognized that thedegree of renal dysfunction correlates with histological abnormalitiesin the renal tubulointerstitium, rather than in glomeruli, even inprimary glomerular disorders ( 4, 23, 27 ). Theseobservations suggest that the severity of proteinuria is a majordeterminant of tubulointerstitial injury ( 21 ).1 U- \2 X* l9 z/ R, j$ P$ p9 Z
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Recent reports have revealed that albumin, the major protein found inproteinuria, is able to induce phenotypic changes in proximal tubularcells and alter their function in a manner that producesproinflammatory chemokines in the renal tubulointerstitium ( 10 ). When proximal tubular cells are incubated withalbumin, they produce monocyte chemoattractant protein-1 (MCP-1) andRANTES (regulated upon activation, normal T cell expressed andsecreted) ( 31, 34 ). MCP-1 is an important chemoattractantfor macrophages and T lymphocytes ( 2, 6, 16 ), which arethe predominant inflammatory cells found in the interstitium in chronicglomerular disease ( 7, 17 ). Increasing evidence alsosuggests that MCP-1 may have an important role not only in theregulation of interstitial inflammation but also in other processesrelated to matrix deposition ( 17 ).

The intracellular mechanisms by which albumin upregulates MCP-1 inproximal tubular cells have not been fully characterized. The promoterregion of the mouse MCP-1/JE gene contains putative binding sites for anumber of transcription factors, including the ubiquitous nuclearfactor- B (NF- B)/Rel family and activator protein-1 (AP-1)( 20 ). NF- B proteins normally exist in the cytosol asdimers bound to inhibitory proteins (I B). After exposure to diversestimuli, I B undergoes proteolysis, allowing NF- B to enter thenucleus and activate the expression of genes encoding chemokines andother proteins ( 1, 13 ). The significance of NF- B inregulation of the MCP-1 gene has been reported in various types ofcells, including renal cells ( 19, 26, 30, 32 ). Forinstance, exposure of rat proximal tubular cells to albumin inducedNF- B activation ( 32, 34 ). Inhibition of NF- B with pharmacological agents ( N -tosyl-phenylalanine chloromethylketone and dexamethasone) or an antisense oligonucleotide to the rat p65 subunit of NF- B significantly reduced albumin-induced MCP-1 transcription in rat proximal tubular cells ( 32 ).
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On the other hand, recent evidence suggests that activation of AP-1 isalso required for induction of MCP-1. Shyy et al. ( 28 ) reported that AP-1 binding to12- O -tetradecanoylphorbol-13-acetate-responsive elements iscritical for shear stress-induced expression of the MCP-1 gene. It hasalso been reported that lipopolysaccharide, transforming growthfactor-, or interleukin-1 stimulates MCP-1 gene expression byactivating AP-1 in certain types of cells ( 19, 29, 33 ). Arole in the regulation of murine MCP-1/JE expression for the AP-1 siteexposed to tumor necrosis factor- and transforming growth factor- has been suggested by other studies, because MCP-1/JE expression wasdecreased when antisense DNAs targeting c-Jun and c-Fos or inhibitorsof c-Jun and c-Fos were used ( 12, 29 ). AP-1 activity isregulated 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 NH 2 -terminal kinase (JNK)have been identified to be involved in a wide range of cellularresponses to extracellular signals ( 24 ). Recently, Dixonand Brunskill ( 9 ) demonstrated that albumin was able tostimulate proliferation of proximal tubular cells via ERK, suggesting apossible link between albuminuria and the derangements of proximaltubular cell growth observed in progressive renal scarring. However, itremains to be clarified whether ERK is involved in the regulation ofMCP-1 expression./ f) c) ?" S$ Q' I3 `, }, C
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In the present study, we investigated the role of ERK activated by BSAin regulation of MCP-1 expression.

METHODS$ w: o& ?7 p5 b1 x9 i2 O2 l

Materials

BSA (fatty acid free, fraction V) was obtained from Bayel(Pittsburgh, PA). BSA did not contain a high level of endotoxin ( Japan).: D# P) ~% @- Q0 \" `& o8 ?8 x

Antiphospho-p42/p44 ERK, antiphospho-p38 MAPK, antiphospho-I B-,and anti-I B- antibodies were purchased from New England Biolabs(Beverly, MA). Antiphospho-JNK antibody, U-0126, SB-203580, andluciferase kit were purchased from Promega (Madison, WI). Anti-ERK2,-p38 MAPK, and -JNK2 antibodies were obtained from Santa CruzBiotechnology (Santa Cruz, CA). Anti- -actin antibody was obtainedfrom Sigma Chemical (St. Louis, MO). The 5×NF- B luciferase reportervector (NF- B-Luc) was obtained from Clontech (Palo Alto, CA).[ - 32 P]dCTP and [ - 32 P]ATP werepurchased from New England Nuclear (Boston, MA). All other reagentswere of chemical grade and were purchased from standard suppliers./ f$ O9 ]- I% E4 {
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Proximal Tubular Cell Culture) R/ Z8 u: u2 b
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Microdissection of the proximal tubule. Proximal tubular segments were microdissected from C57BL/6J adult mousekidney. The kidneys were perfused through the renal artery and excised,and coronal sections were cut with a surgical blade. The sections weretransferred to a flask containing 10 ml of ice-cold Hanks' balancedsalt solution containing 0.1% BSA and 0.1% type I collagenase, whichwas used to perfuse the kidneys. The flask was incubated for 10 min at37°C in a shaking water bath. The samples were suffused with 5%CO 2 -95% O 2 during the incubations. Tissueswere transferred to Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS) and placed on ice. Then theproximal tubular segments were microdissected under a stereoscopic microscope.
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Immortalization of proximal tubular cells. The microdissected proximal tubules were aspirated into 20 µl of K1medium composed of 1:1 DMEM-Ham's F-12 culture medium with 10% FCSand placed on type IV collagen-precoated 96-well plates. The proximaltubules were maintained in a minimum volume of the modified 1:1mesenchymal cultured K1 medium-fresh K1 medium for 24-48 h. Afterattachment to the culture plate, proximal tubules were cocultured withthe mouse mesenchymal feeder cells. At ~7-10 days aftermicrodissection, primary cell outgrowths were transfected with 100 µg/ml of the simian virus-40 large T antigen gene by Transfectamreagent (Promega) according to the manufacturer's protocol. The cellswere maintained in culture for 2 days after transfection. Then theywere dissociated with 0.25% trypsin-EDTA and reseeded onto the type IVcollagen-precoated 48-well plates with fresh feeder cells. After passages 2 and 3, the transfection procedure wasrepeated, and the cells were expanded in the presence of feeder cells.After passage 3, the immortalized cells were no longerdependent on the presence of feeder cells and were maintained in K1medium at 37°C in a 5% CO 2 -95% air humidified atmosphere.

The cells were stained by cytokeratin, but not by -smooth muscleactin (data not shown). Thus this cell line expressed the proximaltubular 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 ofnonspecific albumin uptake. After 90 min, the culture plates wereplaced on ice. Then the cells were scraped from the culture plates andlysed via sonication. After pH was adjusted to 7.4 with PBS (pH 6.0),insoluble material was removed by centrifugation. The supernatants wereused for ELISA measurements (see BSA ELISA ).

BSA ELISA! z9 q$ p& o7 H$ o- i& N- t
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Polyvinyl ELISA plates were coated with 100 µl of rabbitanti-BSA diluted 1:500 in PBS (pH 7.4) at 4°C overnight beforeexperiments. After the plates were washed twice with PBS containing0.05% Tween 20 (PBS-T), they were incubated with 100 µl of celllysates for 2 h at room temperature. Nonspecific peroxidationsites were blocked for 30 min at room temperature with 3%H 2 O 2. After repeated washings, the plates wereincubated with 100 µl of biotinylated anti-rabbit BSA diluted 1:250in PBS for 2 h at room temperature. After repeated washings, theplates were incubated with 1:1 avidin-biotinylated horseradishperoxidase for 1 h at room temperature. After repeated washings,the plates were incubated with 100 µl ofazinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium saltperoxidase substrate (1 mg/ml), 0.1 M citric acid (pH 4.2), and 30%H 2 O 2 (0.3 µl/ml) at room temperature for 30 min. The absorbance of each well was read at 405 nm using an automaticELISA plate reader. A standard curve was plotted, and BSA concentrationin each sample was calculated by comparison with a standard curve.( k# W) {! m+ F8 {6 @- |4 @
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Proximal Tubular Cell Culture and Experimental Procedure

Murine proximal tubular (mProx) cells were cultured in DMEMcontaining 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 theexperimental medium (DMEM with 0.1% BSA and 14 mM HEPES) for theindicated times at 37°C. For the experiments with inhibitors, cellswere incubated with the indicated concentrations of U-0126 or SB-203580for 60 min at 37°C before exposure to BSA. After incubation, cellswere harvested for the determination listed below.( m5 N7 }7 S$ @1 `1 A

Northern Blot Analysis
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Total RNA (12 µg) was isolated by guanidinium and phenolextraction (TRIzol Reagent, GIBCO BRL, Grand Island, NY),electrophoresed on 1% formaldehyde-agarose gels, and transferred to anylon membrane (Nytran, Schleicher & Schuell, Dassel, Germany). MouseMCP-1 was labeled with [ - 32 P]dCTP by a random primerlabeling method (Bca BEST, Takara, Shiga, Japan). The membranes werehybridized with mouse MCP-1 cDNA in hybridization buffer (Perfect Hyb,Toyobo, Osaka, Japan) at 65°C for 16 h. The membranes wereautoradiographed at 80°C overnight.9 Q, }9 E3 N8 K% R3 \
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Immunoblot Analysis/ ^; k# Z/ {" w; E7 y! I5 x
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Immunoblot analysis was performed as previously described( 15 ). Briefly, proximal tubular cells were lysed in SDSsample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10%glycerol, 50 mM dithiothreitol, and 0.01% bromphenol blue). The celllysates containing 40 µg of proteins in SDS sample buffer wereelectrophoresed on 12% SDS-polyacrylamide gels and electrotransferredto polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford,MA). After the membranes were blocked, they were incubated with a1:1,000 dilution of antiphospho-ERK, antiphospho-p38 MAPK,antiphospho-JNK, or antiphospho-I B- antibodies at 4°Covernight. Horseradish peroxidase-conjugated anti-rabbit, anti-mouse,or anti-goat antibody (Amersham, Buckinghamshire, UK) was used as asecondary antibody. The immunoreactive bands were detected with anenhanced chemiluminescence detection system (NEN Life Science Products,Boston, MA). The membranes were then reprobed with anti-ERK, anti-p38MAPK, anti-JNK, anti-I B-, or anti- -actin antibody as aninternal control.' _( U" X/ W% B0 E0 Y, k

DNA Constructions and Transient Transfection9 c4 `: y% m# r1 K

The pJECAT2.6 was a gift from Dr. J. M. Boss. The reporterconstruct contains DNA fragments for the six MCP-1/JE promoter fused tothe 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 Mlu I/ Xho I sites. mProx cells(~1 × 10 6 /assay) were transfected with 0.7 µg ofthe -galactosidase and luciferase reporter constructs byLipofectamine (GIBCO BRL) for 6 h. The cells were incubated withBSA (30 mg/ml) for 12 h after transfection or treated with 10 µMU-0126 for 1 h before exposure to BSA. Luciferase activity wasdetermined with a luciferase assay kit (Promega) and normalized bycotransfected -galactosidase activity (Promega).+ W# h6 K* P. O: K
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MCP-1 Measurement by ELISA
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The culture media of proximal tubular cells on a 24-well platewere collected and stored at 80°C until assay. Accumulation ofMCP-1 protein in the media was measured with an MCP-1 ELISA kit (R & DSystem, Minneapolis, MN). The concentrations of MCP-1 were correctedfor the total amount of cellular DNA, as previously reported( 14 ).

Nuclear Extraction and Electrophoretic Mobility Shift Assay8 S- o% z- p! e
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Nuclear protein was prepared from proximal tubular cells aspreviously described ( 15 ). Electrophoretic mobility shiftassays (EMSAs) were performed by incubation of 5 µg of nuclearproteins with 1 µg of poly(dI-dC) in binding buffer (20 mM HEPES, pH7.9, 1.8 mM MgCl 2, 2 mM dithiothreitol, 0.5 mM EDTA, and0.5 mg/ml BSA) at room temperature for 20 min and reaction with[ - 32 P]ATP-labeled AP-1/GC or B-1/ B-2oligonucleotide at room temperature for an additional 30 min. Thereaction mixtures were electrophoresed on a 5% polyacrylamide gel and autoradiographed.: F; y' @: \1 C; e( S; u. q6 K0 s3 b' \
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The coding strand sequences of the DNAs of interest were asfollows: 5'-GCACCCTGCCTGACTCCACCCCCCTGGCTTACAA-3' (AP-1/GC) and 5'-CCCGAAGGGTCTGGGAACTTCCAATACTGCCTCAGAATGGGAATTTCCACGCTCTTATCC-3' ( B-1/ B-2).
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Statistical Analysis# z+ ?7 h+ `( m

Values are means ± SD. Analysis of variance followed byScheffé's test was used to determine significant differences inmultiple comparisons. P significant.
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RESULTS$ p! u: J0 m1 @2 X
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Characterization of Immortalized mProx Cells
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Albumin uptake into mProx cells. To ensure that our mProx cells would reabsorb albumin, as occurs in thenormal kidney, the cells were incubated with 5 mg/ml BSA for 90 min at37°C, and then cellular BSA was measured by ELISA (see METHODS ). The cells showed about fivefold enhanced albuminreabsorption compared with control (0.247 µg albumin/mg protein at37°C vs. 0.042 µg albumin/mg protein at 4°C). Approximately 75%of uptake was blocked by cold incubation, indicating that the majorityof cellular albumin uptake was specific. These results also indicatethat this cell line expresses a proximal tubular phenotype and isfunctionally similar to primary cultures.
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Effect of BSA on MCP-1 mRNA Expression7 ~% H3 |4 `( L6 V/ ?2 {
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We examined whether BSA stimulated the expression of MCP-1 mRNA inmProx cells. By Northern blotting, BSA (30 mg/ml) induced MCP-1 mRNA ina time-dependent manner from 1 h to a maximal stimulation at6 h (Fig. 1 A ). Theinduction of MCP-1 mRNA by BSA was dose dependent, with a maximalstimulation at 30 mg/ml (Fig. 1 B ).+ J/ R4 X, w# x9 x& ~3 U
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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) forindicated times ( A ) or to BSA at 5-30 mg/ml for 6 h ( B ). RNA was extracted, and MCP-1 mRNA was analyzed byNorthern blot, with 28S and 18S mRNA as internal standards( left ). Right : quantitative results; n = 4. * P

Activation of MAPKs and NF- B by BSA

Activation of MAPKs was assessed by immunoblotting usingantibodies that recognize only the phosphorylated form of ERK, p38 MAPK, or JNK. In mProx cells, BSA stimulated the activities of ERK andp38 MAPK. ERK was significantly activated at 5 min after stimulationand returned to basal levels at 30 min (Fig. 2 A ). Activation of p38 MAPKwas also seen at 1 min after stimulation (Fig. 2 B ). However,JNK was not activated by BSA (data not shown).
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Fig. 2. Activation of extracellular signal-regulated kinase (ERK) and p38mitogen-activated protein kinase (MAPK) by BSA. mProx cells wereincubated with BSA (30 mg/ml) for indicated times. Top :equal amounts of cell lysate were subjected to immunoblottingusing antibodies against phospho-ERK (pERK1/2, A )or phospho-p38 MAPK (pp38, B ). Blots were developed withenhanced chemiluminescence. Membranes were reprobed with anti-ERK oranti-p38 MAPK antibodies. Blots are representative of 4 independentexperiments. Bottom : quantitative results. * P

To define the kinetics of I B- proteins involved in NF- Bactivation, we examined the phosphorylation and degradation of I B-. Exposure to BSA led to transient phosphorylation ofI B- in 1 h. In parallel, I B- is degraded and thenreturned to control levels within 2.5 h. After 2.5 h,phosphorylation and expression of I B- seemed to be increased(Fig. 3 ).% f/ F9 Z8 \% F
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Fig. 3. Phosphorylation and degradation of I B- by BSA.mProx cells were exposed to BSA (30 mg/ml) for indicated times. Cellextracts (40 µg of protein) were subjected to Western blotting withanti-phospho-I B- antibody (pI B), anti-I B- antibody, andanti- -actin antibody. Blot is representative of 5 experiments.
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Effect of MAPK Pathway on BSA-Induced MCP-1 Expression% Z  s: G1 c! A; J3 l6 s
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We next investigated the intracellular signaling pathways involvedin BSA-induced MCP-1 expression. Because we clearly demonstrated thatBSA 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 pretreatmentwith U-0126 (data not shown). Treatment with U-0126 partiallysuppressed BSA-induced MCP-1 mRNA expression. On the other hand,SB-203580 tended to inhibit BSA-induced MCP-1 mRNA, but this effect wasnot statistically significant (Fig. 4 A ). To further confirm theroles of ERK and p38 MAPK in MCP-1 expression, accumulation of MCP-1protein in the medium was analyzed by ELISA. Consistent with theresult of Fig. 4 A, treatment with U-0126 suppressed BSA-induced MCP-1 protein in a dose-dependent manner. SB-203580 tendedto suppress BSA-induced MCP-1 protein, but this effect was notstatistically significant. The combination of U-0126 and SB-203580suppressed BSA-induced MCP-1 protein, but these effects were notadditive or synergistic (Fig. 4 B ). To elucidate themechanism of transcriptional regulation of MCP-1 in mProx cells, thesix 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 cellstransfected with MCP-1/JE-Luc demonstrated an increase in luciferaseactivity on exposure to BSA (Fig. 4 C ). When mProx cells weretreated with 10 µM U-0126 before exposure to BSA, luciferase activitywas partially suppressed (Fig. 4 C ). These results suggest that the ERK pathway is involved in mediating the BSA-induced increaseof 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 µMSB-203580 for 1 h and then exposed to BSA (30 mg/ml) for 2 h. Left : Northern blot of mRNA. Right : quantitativeresults; n = 4. * P # P B : cellswere treated with 5 or 10 µM U-0126 or/and SB-203580 for 1 h andthen exposed to BSA (30 mg/ml) for 24 h. Accumulation of MCP-1protein in medium was analyzed by ELISA on duplicate samples; n = 3. * P P C : 6 MCP-1/JE promoter fusion reporterconstructs linked to the luciferase gene were transfected into mProxcells. Transfected cells were treated with 10 µM U-0126 for 1 hbefore exposure to BSA (30 mg/ml) for 8 h. Luciferase activity wasmeasured by a luciferase assay kit. Values are normalized bycotransfected -galactosidase activity. * P P

Effect of the MAPK Pathway on BSA-Induced DNA Binding of NF- Band AP-1
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Previous studies revealed that the distal regulatory region of themurine gene contains two B sites: B-1 and B-2. The proximal regulatory region contains a third B site ( B-3), an AP-1-binding site, a GC box, and a TATA box (Fig. 5 A ) ( 20 ). Toelucidate the role of these binding sites in response to BSA, weperformed EMSA using a labeled AP-1/GC box site as a probe. BSAstimulated DNA binding to AP-1 at 1 h (Fig. 5 B ).Addition of excess unlabeled AP-1/GC oligonucleotide resulted incomplete absence of the band in autoradiographs, confirming thespecificity of the reaction. In contrast, the binding was not affectedby incubation with an irrelevant oligonucleotide, i.e., NF- Bconsensus oligonucleotide (Fig. 5 B ). Similarly, increasedDNA binding of NF- B was maximal 1 h after exposure to BSA inmProx cells. Addition of excess unlabeled B-1/ B-2 oligonucleotidecompetitively blocked the specific binding of NF- B, whereas AP-1consensus oligonucleotide did not affect this binding (Fig. 5 C ).9 c! @6 h+ X# n1 D2 z+ {
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Fig. 5. Activation of DNA binding to nuclear factor- B(NF- B) and activator protein-1 (AP-1) by BSA and effect of inhibitorof MEK on BSA-induced NF- B and AP-1 DNA binding. A :distal regulatory region of the murine MCP-1/JE gene contains 2 Bsites, and proximal regulatory region contains an AP-1-binding site.mProx cells were treated with 10 µM U-0126 for 1 h and thenexposed to BSA (30 mg/ml) for 1 h. Electrophoretic mobility shiftassays were performed by incubating nuclear proteins (5 µg) with[ - 32 P]ATP end-labeled AP-1/GC oligonucleotide( B ) or B-1/ B-2 oligonucleotide containing MCP-1 Bconsensus sequences ( C ). Reaction mixtures wereelectrophoresed on a 5% polyacrylamide gel and autoradiographed.

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

Involvement of MAPK in BSA-Induced I B- Degradation andNF- B Activation4 g( D7 P; F! ^

Because U-0126 was able to inhibit BSA-stimulated DNA-bindingactivity of NF- B (Fig. 5 C ), we hypothesized that ERK isinvolved in the activation of NF- B. To learn how the ERK and NF- Bpathways interact, we examined the effect of U-0126 on BSA-inducedI B- degradation and NF- B activation. Pretreatment of mProxcells with U-0126 attenuated the I B- degradation by BSA (Fig. 6 A ).
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Fig. 6. Effect of inhibitor of MEK on I B- degradation andNF- B luciferase activity. A : mProx cells were treatedwith 10 µM U-0126 for 1 h and then exposed to BSA (30 mg/ml) for1.5 h. Cell extracts (40 µg of protein) were subjected toWestern blotting with anti-I B- antibody. Blot is representativeof 4 experiments. B : 5×NF- B luciferase gene(NF- B-Luc) was transfected into mProx cells. Transfected cells weretreated with 10 µM U-0126 for 1 h before exposure to BSA (30 mg/ml) for 8 h. Luciferase activity of duplicate samples wasmeasured by a luciferase assay kit. Values are normalized bycotransfected -galactosidase activity; n = 5. * P P
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To further confirm the involvement of ERK in the activation of NF- B,we performed reporter assay using 5×NF- B luciferase vector(NF- B-Luc). Mouse proximal tubule cells transfected with NF- B-Lucdemonstrated an increase in luciferase activity on exposure to BSA(Fig. 6 B ). When mProx cells were treated with 10 µM U-0126 before exposure to BSA, the luciferase activity was partially suppressed.
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These results suggest that the ERK pathway is involved in theactivation of NF- B by BSA.1 u2 B" t# d5 G) i( k# k& S/ F. R+ n- O
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DISCUSSION
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Increasing evidence suggests that proteinuria itselfmay 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 andtranslation of MCP-1 in rat proximal tubular cells atpathophysiologically relevant concentrations ( 31 ), signaltransduction pathways involved in the effect of albumin on MCP-1 havenot been fully characterized. In the present study, we demonstratedthat 1 ) albumin induced the expression of MCP-1 in mProxcells, a newly established mouse proximal tubular cell line; 2 ) albumin induced the activation of ERK and thephosphorylation and degradation of I B-; 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- B, the binding sites of which existed in the promoter region ofMCP-1; and 5 ) the transcriptional activity of NF- B andI B- degradation were inhibited by U-0126.

In the present study, ERK was significantly activated 5 min afterstimulation with BSA and returned to basal levels by 30 min in mProxcells. A similar effect of albumin was observed in opossum kidneyproximal tubular cells, in which ERK was activated 1 min afterincubation with recombinant human serum albumin, reached maximalactivation 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- -inducedexpression of MCP-1 in human umbilical vein endothelial cells( 11 ). In the present study, the inhibitor for p38 MAPKtended to suppress BSA-induced MCP-1 expression, but this effect wasnot statistically significant. Thus further studies are needed toclarify the involvement of p38 MAPK in the regulation of MCP-1.

We found that U-0126, an inhibitor of MEK, was able to inhibitBSA-induced mRNA and protein expression of MCP-1 and MCP-1 reporteractivity in mProx cells. In addition, phosphorylation and degradationof I B were also induced by BSA in mProx cells. Although albuminoverload induced MCP-1 expression through activation of NF- B in ratproximal tubular cells ( 32 ), we hypothesized that theERK-AP-1 and I B-NF- B pathways could be responsible foralbumin-induced expression of MCP-1. Indeed, the cooperative action ofNF- B and AP-1 in interleukin-1 -induced MCP-1 gene expression wassuggested in human endothelial cells ( 19 ). The murineMCP-1/JE gene contains two B sites in the distal regulatory regionand one AP-1-binding site in the proximal regulatory region ( 20 ). We found that BSA could stimulate DNA binding ofAP-1 and NF- 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- B-binding activity and NF- B-dependent transcription as well as the degradation of I B in mProx cells. Theseresults indicate that the ERK pathway interacts with the I B-NF- Bpathway after stimulation with BSA in mProx cells. This hypothesis issupported by the recent findings in melanoma cells, in whichNF- B-induced kinase (NIK) was found to regulate NF- B activationthrough a novel NIK-MEK-ERK-NF- B signaling pathway in addition tothe classical NIK-IKK-I B-NF- B pathway ( 8 ).' o6 n) `  J3 Q
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Although further investigation is needed to elucidate the precise rolesof ERK, the present findings support the hypothesis that the ERKpathway is involved in BSA-induced MCP-1 expression and suggest apossible interaction between NF- B and ERK. This information could beuseful in the design of anti-inflammatory strategies to suppress notonly cell proliferation but also transcriptional activation ofcytokines in renal diseases.
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