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作者：XianghongZhang,  YouhuaLiu作者单位：1 Department of Pathology, University of PittsburghSchool of Medicine, Pittsburgh, Pennsylvania 15261; and Department of Cell Biology, Peking Union MedicalCollege, Beijing 10000 China 2 V3 a% ^7 v% T# l) A+ }6 N9 A
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      【摘要】8 U$ p5 L0 O+ z7 e# r0 Q$ I( W( a" f
   Hepatocytegrowth factor (HGF) receptor, the product of the c-met protooncogene, is transcriptionally regulated by a wide variety ofcytokines as well as extracellular environmental cues. In this report,we demonstrate that c-met expression was significantly suppressed byoxidative stress. Treatment of mouse renal inner medullary collectingduct epithelial cells with 0.5 mM H 2 O 2 inhibited c-met mRNA and protein expression, which was concomitant with induction of Egr-1 transcription factor. Ectopic expression of Egr-1 in renal epithelial cells markedly inhibited endogenous c-metexpression in a dose-dependent fashion, suggesting a causative effectof Egr-1 in mediating c-met suppression. The cis -acting element responsible for H 2 O 2 -induced c-metinhibition was localized at nucleotide position 223 to 68 of c-metpromoter, in which reside an imperfect Egr-1 and three Sp1-bindingsites. Egr-1 markedly suppressed c-met promoter activity but did notdirectly bind to its cis -acting element in the c-met gene. Induction of Egr-1 by oxidative stressattenuated the binding of Sp1 to its cognate sites, but it did notaffect Sp1 abundance in renal epithelial cells. Immunoprecipitationuncovered that Egr-1 physically interacted with Sp1 by forming theSp1/Egr-1 complex, which presumably resulted in a decreasedavailability of unbound Sp1 as a transcriptional activator for the c-met gene. Thus it appears that inhibition of c-metexpression by oxidative stress is mediated by the interplay between Sp1and Egr-1 transcription factors. Our findings reveal a noveltranscriptional regulatory mechanism by which Egr-1 sequesters Sp1 as atranscriptional activator of c-met via physical interaction. 5 C2 _3 U* H! u5 G1 ?) A
      【关键词】 hepatocyte growth factor cmet receptor gene transcription tubular epithelial cells H O4 ]5 e4 w7 Z9 r/ Y+ U
   INTRODUCTION4 U( \. F0 G7 X& V8 N

HEPATOCYTE GROWTH FACTOR (HGF) receptor is a member of the receptor tyrosine kinasesuperfamily and is encoded by the c-met protooncogene( 3, 29 ). Mature c-met receptor is a heterodimeric proteinconsisting of an extracellular -subunit and a -subunit harboringan extracellular portion, a transmembrane segment and the cytoplasmictyrosine kinase domain ( 32 ). On binding to its specificligand, c-met receptor undergoes autophosphorylation of multipletyrosine residues in its cytoplasmic region and triggers cascades ofsignal transduction events that lead to such diverse cellular responsesas cell survival, proliferation, migration, and differentiation( 5, 6, 17, 24-26 ). Because all biological activitiesof HGF are mediated by a single receptor, the expression and levels ofc-met protein not only determine the cell-type specificity of HGFactions but also dictate the overall activities of this pairedsignaling system. Consistent with this view, earlier studies indicatethat it is the abundance and activity of c-met receptor, rather thanHGF itself, that best correlate with the biological function of HGF indiverse organs under physiological and pathological conditions( 8, 28, 39 ).) A* r1 ~3 m- B: \, t+ }7 [
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The expression of the c-met receptor is primarily regulated at thetranscriptional level ( 21 ). Unlike its ligand, which isexpressed mainly in mesenchyme-derived cells in normal tissues ( 19, 26 ), c-met expression is relatively ubiquitous with a widespread pattern. For instance, in normal adult kidney and cultured kidney cells, c-met protein is detected in all types of renal cellsincluding glomerular mesangial cells, podocytes, proximal tubularcells, collecting duct epithelial cells, and interstitial fibroblasts,although high level of c-met is only observed in distal tubules andcollecting duct epithelia under normal circumstances ( 46 ).This prevalent pattern of c-met constitutive expression is closelyoverlapped with, and predominantly dictated by, the ubiquitousspecificity protein (Sp) family of transcription factors ( 18, 37, 46 ). Both Sp1 and Sp3 avidly bind to the GC boxes (Sp1-bindingsites) in the c-met promoter region and transcriptionally activate itsexpression; and together, they interact with each other, formingheterodimeric complexes and elicit synergistic actions on c-met gene transcription ( 46 ). Thebroad expression pattern of the c-met gene likely highlightsa wide implication of HGF in normal cell physiology in diverse organs.1 |; @# G& B* m8 r" {" M0 S
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A wide variety of cytokines, growth factors, and hormones has beenreported to induce c-met expression in renal epithelial cells and othertypes of cells ( 21, 23, 27, 36 ). Expression of the c-met gene is also induced rapidly in the injured organs after diverse types of tissue-injurious insults ( 20, 21 ). Contrary to HGF whose expression is often stimulated in both injured and distal intact organs, c-met induction exclusively occurs at thesite of injury and is principally correlated with the injury andsubsequent repair process in a site-specific manner ( 14, 21, 34 ). In addition, c-met expression is induced in renal epithelial cells by altered extracellular environmental cues such as ahigh concentration of glucose in vitro and in diabetic states in vivo( 22 ). Hence, in response to a vast diversity of stimuli, the c-met gene is commonly activated to induce itsexpression, a process that may be necessary and important for injuryrepair and regeneration. However, little is documented as to c-mettranscriptional suppression in response to certain conditions, and italso remains largely unknown about the molecular mechanism governingc-met inhibition.
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In this report, we demonstrate that oxidative stress inhibits c-met gene expression at both mRNA and protein levels. Thesuppression of c-met expression is accompanied by induction of Egr-1transcription factor in renal epithelial cells afterH 2 O 2 treatment. Our results suggestthat Egr-1 sequesters Sp1 by physical interaction, which leads to adecreased availability of free Sp1 for c-met transcriptional activation.1 h4 G+ a1 A6 D# i/ F4 s

MATERIALS AND METHODS
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Cell culture and treatment. Mouse inner medullary collecting duct epithelial cell line 3 (mIMCD-3)was obtained from American Type Culture Collection (ATCC, Manassas,VA). Human kidney proximal tubular cells (HKC) were provided by Dr. L. Racusen of Johns Hopkins University (Baltimore, MD). Cells weremaintained in DMEM and Ham's F-12 medium (1:1 of DMEM/F12; LifeTechnologies, Grand Island, NY) supplemented with 10% FBS. Cells wereseeded in plastic petri dishes (100 mm; Falcon) at 60-70%confluence. After being cultivated for 16 h in the complete mediumcontaining 10% FBS, cells were serum-starved for 24 h andincubated for various periods of time as indicated in the absence(control) or presence of 0.5 mM H 2 O 2 (Sigma-Aldrich, St. Louis, MO), except when otherwise indicated. Thecells were then collected for analysis of gene expression at both mRNAand protein levels by Northern and Western blot analyses, respectively.

RNA preparation and Northern blot analysis. Total RNA was extracted from the cells using Ultraspec RNA solution(Biotecx, Houston, TX) according to the instructions specified by themanufacturer. Samples of 20 µg total RNA were electrophoresed on1.0% formadehyde-agarose gels and then transferred to GeneScreen plusnylon membrane (DuPont, Boston, MA) by capillary blotting followed byultraviolet cross-linking. Membranes were prehybridized for 4 h at65°C in a buffer containing 6× SSC, 5× Denhardt's solution, 1%SDS, 10% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. 32 P-labeled DNA probes were prepared by the random primerlabeling kit (Stratagene, La Jolla, CA) using[ - 32 P]dCTP. The rat c-met cDNA probe wasgenerated in our laboratory as described previously ( 23 ).The human egr-1 cDNA was obtained from the ATCC. Denatured probes wereadded to the same hybridization buffer at a concentration of1-2 × 10 6 cpm/ml, and hybridization was allowedto proceed at 65°C for 16 h. Membranes were washed and exposedto X-ray film (Eastman Kodak, Rochester, NY) at 80°C with the aidof an intensifying screen as described elsewhere ( 21, 44 ).Quantitation was performed by determination of the intensity of thehybridization signals using the National Institutes of Health Image program.

Western blot analysis. The mIMCD-3 cells were solubilized with RIPA lysis buffer (1% NonidetP-40, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 0.5%sodium deoxycholate, 1 mM sodium orthovanadate, 2 µg/ml aprotin, 2 µg/ml leupeptin in PBS) at 4°C for 20 min, and the supernatants were collected after centrifugation at 13,000 g at 4°C for10 min. Protein concentration was determined using a bicinchoninic acid(BCA) protein assay kit with bovine serum albumin as a standard (Sigma), and cell lysates were mixed with an equal amount of 2× loading buffer (100 mM Tris · HCl, 4% SDS, 20%glycerol, and 0.2% bromophenol blue). Samples were heated at 100°Cfor 10 min before loading and separated on precasted 10%SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) under nonreducingconditions. The proteins were transferred to a nitrocellulose membrane(Amersham, Arlington Heights, IL) in transfer buffer containing 48 mMTris · HCl, 39 mM glycine, 0.037% SDS, and 20%methanol at 4°C for 2 h. Nonspecific binding to the membrane wasblocked for 1 h at room temperature with 5% nonfat milk in TBSbuffer (20 mM Tris · HCl, 150 mM NaCl, and 0.1%Tween 20), and then the membrane was incubated for 1 h withvarious primary antibodies followed by incubation for 1 h with asecondary horseradish peroxidase-conjugated IgG in 5% nonfat milk. Thespecific antibodies against c-met, Egr-1, Sp1, Sp3, and actin, werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA). The signalswere visualized by the enhanced chemiluminescence system (ECL,Amersham) as described elsewhere ( 43 ).

Construction of plasmid, DNA transfection, and luciferase assay. The various chimeric plasmids containing different lengths of the5'-flanking region of the human c-met gene linked to the coding sequence for chloramphenicol acetyltransferase (CAT) had beendescribed previously ( 18 ). In this study, the highlysensitive firefly luciferase reporter system was used. For thatpurpose, various DNA fragments containing different lengths of thec-met promoter region were excised with Sst I and Bgl II from different c-met-CAT reporter plasmid vectors. TheDNA fragments were gel purified and then subcloned into the Sst I/ Bgl II site of the promoterless pGL3-Basicluciferase expression vector (Promega, Madison, WI). The resultantchimeric reporter plasmids (designated as pGL3-0.7met, pGL3-0.2met, or pGL3-0.1met, etc.) were verified by detailedrestriction mapping. For transient transfection, the mIMCD-3 cells wereseeded in six-well plates at 2 × 10 5 cells/well. Thecells were then transfected with various luciferase reporter plasmidsusing the Lipofectamine 2000 reagent according to the instructionsspecified by the manufacturer (Life Technologies). A fixed amount ofinternal control reporter Renilla reniformis luciferasedriven under thymidine kinase (TK) promoter (pRL-TK, Promega) wascotransfected for normalizing the transfection efficiency andcorrecting firefly luciferase activity. After transfection, the cellswere incubated for an additional 24 h in the absence or presenceof 0.5 mM H 2 O 2 before being harvested forluciferase assay. Luciferase assay was performed using the DualLuciferase Assay System kit essentially according to the protocolspecified by the manufacturer (Promega). Relative luciferase activityof each construct (arbitrary unit) was reported as fold-induction overpGL3-0.1met after normalizing for transfection efficiency. Allexperiments were repeated in a minimum of three separate experiments (triple wells per experiment) to assume reproducibility. The values from these experiments were combined and subjected to analysis ofvariance using SigmaStat statistical software (Jandel Scientific, SanRafael, CA). P significant.1 M; K7 _  _5 Z# Q! A
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Preparation of nuclear protein extract. The control and H 2 O 2 -treated mIMCD-3 cells werecultured in the conditions described above. For preparation of nuclearprotein extracts, cells were washed twice with cold PBS and scraped off the plate with a rubber policeman. Cells were collected and the nucleiwere isolated according to the method described elsewhere ( 9, 46 ). Briefly, the pelleted cells were resuspended in 4 vol of buffer A containing protease inhibitors (10 mM HEPES, pH7.9, 1.5 mM MgCl 2, 0.5% Nonidet P-40, 10 mM KCl, 0.2 mMPMSF, and 0.5 mM dithiothreitol plus 1% protease inhibitor cocktail from Sigma-Aldrich). The cell suspension was transferred into a Douncehomogenizer immediately after incubation on ice for 10 min to allow thecells to swell. The cells were then lysed by 10 strokes. Cell nucleiwere collected by centrifugation at 3,500 g for 15 min at4°C and suspended in the same volume of buffer A withoutNonidet P-40. The nuclei were resuspended in 150 µl of bufferB (20 mM HEPES, pH 7.9, 10% glycerol, 1.5 mM MgCl 2, 10 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM dithiothreitol plusprotease inhibitor cocktail). Fifty-microliters of buffer B containing 1.6 M KCl were added into the nuclei suspension in adropwise fashion, followed by incubation on ice for 1 h. The nuclear extracts were collected by centrifugation at 4°C at 25,000 g for 30 min. Aliquots of protein extract were quicklyfrozen and stored at 80°C after the protein concentration had beendetermined using a BCA protein assay kit (Sigma-Aldrich).
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EMSA. The double-strand Sp1/Egr-1 oligonucleotide (oligo) corresponding tothe nucleotide sequence of 86~ 61 in the c-met promoter whichcontains a perfect Sp1 site overlapped with an imperfect Egr-1 site waslabeled with [ - 32 P]ATP using T 4 kinase(Life Technologies). An oligo containing two Egr-1-binding sites waspurchased from Santa Cruz Biotechnology and used as a positive controlfor Egr-1 binding. The labeled probes were then gel purified and usedin EMSA as described previously ( 46 ). Four micrograms ofPoly(dI-dC)-Poly(dI-dC) (Pharmacia, Piscataway, NJ) were used as thenonspecific competitor in 10 µl reaction mixture. The bindingreactions were carried out at 37°C for 30 min before loading of 5%nondenaturing polyacrylamide (19:1, acrylamide:bisacrylamide) gels. Forcompetition experiments, 100-fold molar excess of unlabeleddouble-strand oligo was included in the reaction mixture except whereindicated otherwise. For supershift experiments, specific antibodiesagainst Sp1, Sp3, Egr-1, and normal control IgG (Santa Cruz) wereincubated with nuclear protein extracts for 30 min at 37°C beforeaddition of reaction buffer. Gels were run in 0.5× TBE buffer (0.045 MTris · borate, 0.001 M EDTA) at a constantvoltage of 190 V, dried, and autoradiographed with intensifying screens.6 ]8 p# V4 X$ L( m7 C' K

Immunoprecipitation. Renal epithelial mIMCD-3 cells grown on 100-mm plate were lysed on icein 1 ml RIPA buffer containing 1× PBS, 1% Nonidet P-40, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and1% protease inhibitors cocktail (Sigma). Whole cell lysates wereclarified by centrifugation at 12,000 g for 10 min at 4°C,and the supernatants were transferred into a fresh tube. To preclearcell lysates, 0.25 µg of normal rabbit IgG and 20 µl of protein A/GPlus-Agarose (Santa Cruz) were added into 1 ml of whole cell lysates.After incubation for 1 h at 4°C, supernatants were collected bycentrifugation at 1,000 g for 5 min at 4°C. Lysates wereimmunoprecipitated overnight at 4°C with 1 µg of anti-Sp1 andanti-Egr-1, respectively, followed by precipitation with 20 µl ofprotein A/G Plus-Agarose for 3 h at 4°C. After four washes withRIPA buffer, the immunoprecipitates were boiled for 5 min in SDS samplebuffer. The resulting precipitated complexes were separated onSDS-polyacrylamide gels and blotted with various antibodies asdescribed above.$ G' `3 K  ~3 y( \: L4 F
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RESULTS. x* t. \, J8 t% t3 d: ^

Oxidative stress inhibits c-met expression while concomitantlyinducing egr-1. We investigated the effects of oxidative stress on c-met gene expression in renal epithelial cells in vitro. As shown in Fig. 1, incubation of renal collecting ductepithelial cells (mIMCD-3) with 0.5 mM H 2 O 2 markedly suppressed the steady-state levels of c-met mRNA in atime-dependent manner. At 6 h after H 2 O 2 treatment, ~70% inhibition of c-met mRNA was observed in mIMCD-3cells, as determined by quantitatively measuring the intensity of thehybridization signal. This inhibition of c-met expression byH 2 O 2 was also dose dependent, and significantinhibition of c-met mRNA was observed at a concentration as low as 0.05 mM (data not shown). Of note, at the concentration of 0.5 mM as used inthis study, H 2 O 2 did not significantly causecell death, as the lactase dehydrogenase activity in the supernatant ofcontrol and H 2 O 2 -treated mIMCD-3 cell cultureis essentially identical (data not shown). H 2 O 2 also had no appreciable effects on housekeeping gene GAPDH expression.2 h6 `, m4 C) _3 R$ l& t" |, O

Fig. 1. Oxidative stress inhibits c-met expression andconcomitantly induces egr-1. Mouse renal inner medullary collectingduct epithelial cells (mIMCD-3) were treated with 0.5 mMH 2 O 2 in serum-free medium for various periodsof time as indicated. A : mRNA levels for c-met and egr-1were determined by Northern blot analyses, respectively. The blots werereprobed with GAPDH to ensure equal loading of each lane. Left : sizes of gene transcripts. B : graphicalpresentation of the c-met inhibition and egr-1 induction in mIMCD-3cells after oxidative stress. Relative abundances are reported withhighest level as 100. Data are presented after normalizing withGAPDH.
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Of interest, we found that the inhibition of c-met expression byoxidative stress was accompanied by a marked induction of an earlygrowth responsive gene (egr-1). Northern blot analysis indicated that0.5 mM H 2 O 2 induced a rapid increase in thesteady-state level of egr-1 mRNA, which peaked at 3 h afteroxidative stress (Fig. 1 ). A close correlation between c-metsuppression and egr-1 induction was observed in renal epithelial cellsat different time points after H 2 O 2 treatment(Fig. 1 ). Of note, the peak of egr-1 induction preceded the maximalinhibition of c-met gene expression in response to oxidativestress (Fig. 1 B ). In accordance with c-met suppression,induction of egr-1 expression mediated by oxidative stress was alsodose dependent in renal epithelial cells (data not shown).
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Similar to mRNA levels, c-met protein was also significantly reducedafter incubation with 0.5 mM H 2 O 2 in renalepithelial cells (Fig. 2 ). Western blotanalysis revealed that H 2 O 2 suppressed c-metprotein in a dose-dependent fashion. As shown in Fig. 2, inhibition ofc-met protein expression coincided with induction of Egr-1 proteinexpression in renal epithelial cells in response toH 2 O 2 treatment. This reciprocal associationbetween inhibition of c-met and induction of egr-1 by oxidative stresslikely implies that these two events may be somehow interconnected inrenal epithelial cells.

Fig. 2. Close association between inhibition of c-met andinduction of Egr-1 by oxidative stress in renal epithelial cells. ThemIMCD-3 cells were treated with different concentrations ofH 2 O 2 as indicated in serum-free medium for6 h. A : Western blot analysis demonstrates a reciprocalassociation between c-met suppression and Egr-1 induction in renalepithelial cells. Cell lysates were immunoblotted with antibodiesagainst c-met and Egr-1, respectively. B : graphicalpresentation of the c-met inhibition and egr-1 induction in mIMCD-3cells after oxidative stress.
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Ectopic expression of egr-1 suppresses endogenous c-met expressionin renal epithelial cells. To test a direct relevance of Egr-1 induction and c-met suppression, weinvestigated the effects of ectopic expression of exogenous Egr-1 onc-met expression in renal tubular epithelial cells by transfecting theEgr-1 expression vector. As shown in Fig. 3, forced expression of exogenous Egr-1suppressed endogenous c-met expression in mIMCD-3 cells in adose-dependent fashion. Quantitative determination of the relativeabundances exhibited a closely inverse relationship between c-met andEgr-1 proteins in mIMCD-3 cells following transfection of differentamounts of Egr-1 expression vector (Fig. 3 C ). Similarly,expression of exogenous Egr-1 also inhibited endogenous c-metexpression in HKC cells (Fig. 3, B and D ). Thusthese results establish that Egr-1 may directly mediate the suppressionof c-met expression by oxidative stress in renal tubular epithelialcells.

Fig. 3. Exogenous Egr-1 inhibits c-met expression in renalepithelial cells. Renal epithelial mIMCD-3 ( A ) and humankidney proximal tubular cells (HKC; B ) were transientlytransfected with increasing amounts of Egr-1 expression vector,respectively. Twenty-four hours after transfection, the cell lysateswere prepared and immunoblotted with antibodies against c-met, Egr-1,and actin, respectively. C and D : graphicalpresentation of the c-met and Egr-1 levels in mIMCD-3 ( C )and HKC ( D ) cells after transfection. Relative abundancesare reported with highest level as 1.0. Data are presented afternormalizing with actin.3 t. D# f0 l6 C( N0 l# r# @

Promoter region responsible for c-met inhibition by oxidativestress contains both Egr-1- and Sp1-binding sites. To further investigate how Egr-1 mediates c-met inhibition by oxidativestress in renal epithelial cells, we defined the cis -acting element(s) responsible for c-met inhibition in the promoter region ofthe c-met gene. Various lengths of the c-met promoter region coupled with the firefly luciferase reporter gene were transfected intorenal epithelial mIMCD-3 cells. The luciferase activities weredetermined in the transfected cells after incubation in the absence orpresence of 0.5 mM H 2 O 2 for 24 h. As shownin Fig. 4, H 2 O 2 significantly suppressed the reporter activities of pGL3-0.7met and pGL3-0.2met constructs that contain 0.7- and 0.2-kb5'-flanking regions of the c-met gene, respectively.However, no significant inhibition of reporter activity was observedafter transfection with pGL3-0.1met construct in response toH 2 O 2 treatment (1.0 ± 0.03 in control vs.0.58 ± 0.06 in H 2 O 2 group, P = 0.692, n = 3). Hence, the cis -acting element responsible for c-met inhibition waslargely localized at a 156-bp region corresponding to nucleotide position 223 to 68 of c-met gene. Sequence analysisrevealed that there are an imperfect Egr-1- and three Sp1-binding sites within this region (Fig. 4 C ) ( 18 ), which led usto speculate that alterations in cellular abundance or activity ofEgr-1 and/or Sp proteins might be involved in mediating c-metinhibition in response to oxidative stress.
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Fig. 4. Localization of the cis -acting element responsible formediating c-met inhibition by oxidative stress. A : variouschimeric reporter constructs containing different lengths of the c-metpromoter region coupled with firefly luciferase (Luc) reporter genewere transfected into renal epithelial mIMCD-3 cells. For internalcontrol of transfection efficiency, Renilla reniformis luciferase under thymidine kinase promoter was also cotransfected intomIMCD-3 cells. The transfected cells were then treated without or with0.5 mM H 2 O 2 for 24 h. B :relative luciferase activity (with pGL3-0.1met as 1.0) waspresented after normalization of transfection efficiency. Data arepresented as means ± SE from 3 independent experiments.** P C : sequence of the cis -acting region responsible for mediating c-met inhibitionby oxidative stress. Three Sp1 sites as well as an imperfect Egr-1 siteare indicated.% _: f' N* p$ K! R  ^5 ^; ~. Z

Egr-1 inhibits c-met promoter activity but does not directly bindto the cis-acting element. To establish the functional significance of Egr-1 in controlling c-met gene transcription, we examined the effects ofoverexpression of Egr-1 on c-met promoter activity by cotransfectingthe Egr-1 expression vector and c-met promoter reporter construct intorenal epithelial cells. As shown in Fig. 5, transfection of increasing amounts ofEgr-1 expression vector and pGL3-0.2met reporter construct thatcontains an imperfect Egr-1-binding site suppressed c-met promoteractivity in a dose-dependent manner, suggesting that Egr-1 is criticalfor c-met transcriptional suppression.
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Fig. 5. Exogenous Egr-1 functionally suppresses c-met promoteractivity in renal epithelial cells. Chimeric luciferase reporterconstructs containing different lengths (0.2 and 0.1 kb) of c-metpromoter region were cotransfected with increasing amounts of Egr-1expression vector into mIMCD-3 cells. The relative luciferase activitywas presented after normalization with Renilla reniformis luciferase for transfection efficiency. Coexpression of pEgr-1 andpGL3-0.1met constructs did not significantly reduce reporteractivity (not shown). Data were presented as means ± SE from 3 independent experiments. ** P+ u3 H9 t# E5 t; L9 L* V: t
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We further investigated a potential interaction between Egr-1 proteinand the cis -element in the c-met promoter. As the imperfect Egr-1-binding site partially overlaps with Sp1 site, one would speculate that increased Egr-1 in renal epithelial mIMCD-3 cells afterH 2 O 2 treatment might compete with Sp1 forbinding to that region. To further explore this possibility, we usedoligo corresponding to this overlapping Sp1/Egr-1 site as a probe inEMSA. As shown in Fig. 6 A,three major shifted bands were observed when the oligo was incubatedwith nuclear protein extract from H 2 O 2 -treatedmIMCD-3 cells. Competition with unlabeled wild-type Sp1/Egr-1 oligo,but not mutant Sp1, largely abolished the binding. To determine the identity of these shifted bands, a supershift approach with specific antibodies was used. Shown in Fig. 6 B, antibodies againstSp1 and Sp3, but not Egr-1, caused a further shift in polyacrylamide gels, indicating that Egr-1 does not bind to this imperfect Egr-1 sitewith a single mutation. Of note, double-strand oligo containing aperfect Egr-1-binding site did recognize Egr-1 protein in EMSA (Fig. 6 C ). That Egr-1-binding activity was markedly induced in mIMCD-3 cells treated with H 2 O 2 (Fig. 6 C ). Therefore, Egr-1 appears to inhibit c-met gene transcription by a mechanism independent of direct binding to itsimperfect cis -element in c-met promoter.
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Fig. 6. Egr-1 does not bind to c-met gene promoter region. A and B :EMSA demonstrates that Egr-1 does not bind to the imperfectEgr-1-binding site of c-met promoter. Double-strand Sp1/Egr-1oligonucleotide (oligo) corresponding to 86~ 61 of the c-metpromoter region that contains a perfect Sp1 site overlapped with animperfect Egr-1 site (with a single mutation) was labeled with 32 P and incubated with the nuclear protein extract derivedfrom mIMCD-3 cells treated with 0.5 mM H 2 O 2. A : for competition experiments, a 100-fold excess ofunlabeled probes was included in the binding mixtures. Oligo containingmutant Sp1-binding site was described previously ( 46 ) andalso used as a competitor. B : for supershift experiments,nuclear protein extracts were incubated with antibodies against normalIgG, Sp1, Sp3, or Egr-1, respectively. C : Egr-1 in renalepithelial cells recognizes and binds to its cognate cis -acting element. Oligo containing 2 perfect Egr-1-bindingsites was incubated with nuclear protein extracts derived from eithercontrol or H 2 O 2 -treated mIMCD-3 cells.Competition and supershift experiments were also carried out asindicated. Bottom : sequences of the probes. Arrows indicatebinding complexes. Arrowheads denote supershiftedcomplexes.
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Induction of Egr-1 by oxidative stress attenuates the binding ofSp1 to its cognate site but does not affect the cellular abundance ofSp proteins. Given that Egr-1 does not bind to the c-met promoter, we next comparedthe binding ability of Sp1 protein to the Sp1/Egr-1 site under basaland H 2 O 2 -treated conditions. As shown in Fig. 7, A and C,induction of Egr-1 by oxidative stress significantly suppressed thebinding of Sp proteins to the c-met promoter. Weak Sp1 and Sp3 bindingwas observed when nuclear extract rich in Egr-1 fromH 2 O 2 -treated mIMCD-3 cells was used, suggestingthat a high level of Egr-1 may interfere with Sp protein binding. Of interest, preabsorption of the nuclear extract fromH 2 O 2 -treated mIMCD-3 cells with anti-Egr-1antibody tended to restore the binding ability of Sp proteins (Fig. 7, B and D ). This implies that a high level of Egr-1induced by oxidative stress is likely responsible for the attenuationof Sp proteins binding to c-met promoter in renal epithelial cells.
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Fig. 7. Oxidative stress attenuates Sp1 binding to the c-metpromoter region, but it does not affect Sp1 expression in renalepithelial cells. A : EMSA demonstrates that oxidative stressattenuated Sp1 binding to its cognate site. Nuclear protein extracts(NPE) derived from either control orH 2 O 2 -treated mIMCD-3 cells were incubated withthe probe containing Sp1-binding site in the presence of differentamounts of unlabeled probe (folds) as a competitor. B :preabsorption of Egr-1 with anti-Egr-1 antibody restores Sp1 binding.Nuclear protein extracts were incubated with anti-Egr-1 antibody beforeEMSA. C and D : graphical presentations of therelative abundance of Sp1 binding to its cognate sequence asdemonstrated in A and B, respectively. Relativeabundances are reported with highest level as 100. E

xidant stress does not affect endogenous Sp1 and Sp3 expression inrenal epithelial cells. The mIMCD-3 cells were treated with 0.5 mMH 2 O 2 in serum-free medium for various periodsof duration as indicated. The cell lysates were immunoblotted withantibodies against Sp1 and Sp3, respectively.
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We next explored the possibility of whether a decreased Sp levelaccounts for c-met inhibition after oxidative stress, because earlierstudies showed that both Sp1 and Sp3 play a crucial role inestablishing c-met constitutive expression in the kidney( 46 ). As shown in Fig. 7 E, no significantchange was found in cellular levels of Sp1 protein in renal epithelialcells at different time points after 0.5 mMH 2 O 2 treatment, as revealed by Western blot analysis. H 2 O 2 also did not significantlyaffect the expression of Sp3 protein. Thus it is obvious that c-metsuppression by oxidative stress is independent of the alteration incellular levels of Sp proteins.% h; A: ^) T3 D$ l; e

Physical interaction of Egr-1 with Sp1. The observation that Egr-1 does not directly bind to the c-met promoter(Fig. 6 ) raises an intriguing possibility that Egr-1 may affect thebinding of Sp proteins to the c-met promoter through protein-proteininteraction. To test this hypothesis, we examined the interactionbetween Egr-1 and Sp1 by a coimmunoprecipitation approach usingspecific antibodies against Egr-1 and Sp1. As shown in Fig. 8, when cell lysates derived from eithercontrol or H 2 O 2 -treated renal epithelialmIMCD-3 cells were immunoprecipitated with anti-Sp1 antibody, Egr-1 wasdetected in the precipitated complexes (Fig. 8 A ). Theabundance of Egr-1 present in the precipitated complexes was largelyproportional to the cellular Egr-1 level afterH 2 O 2 treatment in a time-dependent manner.Similarly, Sp1 protein was also detected in the reciprocal experimentswhen cell lysates were immunoprecipitated with Egr-1 antibody (Fig. 8 B ), suggesting that Egr-1 binds to Sp1 through aprotein-protein interaction. However, in control mIMCD-3 cells that arepoor in Egr-1, Sp1 was not found in the Egr-1 immunoprecipates (Fig. 8 B ). Therefore, it is conceivable that after oxidativestress, increased Egr-1 tends to bind Sp1 protein via protein-proteininteraction, leading to sequestration of Sp1 for c-met genetranscriptional activation.
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Fig. 8. Egr-1 physically interacts with Sp1 transcription factor.Renal epithelial mIMCD-3 cells were treated with 0.5 mMH 2 O 2 for various periods of time as indicated.The cell lysates were immunoprecipitated (IP) with specific antibodiesagainst Sp1 ( A ) and Egr-1 ( B ), respectively. Theimmunoprecipitates were separated on a SDS-PAGE and immunoblotted (IB)with anti-Sp1 and anti-Egr-1 antibodies, respectively.5 H9 D# T6 d' G

DISCUSSION
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HGF receptor signaling plays a critical role in controllingdiverse types of cellular responses such as cell survival,proliferation, migration, and differentiation ( 17, 25, 26, 42 ). Given the importance of the c-met receptor in cellphysiology, it is easy to realize that its expression is tightlycontrolled under normal physiological conditions. Aberrant regulationof the c-met expression has been implicated in developing various typesof tumors in transgenic animal models and in patients ( 2, 4, 7, 12, 40 ). Although many cytokines, growth factors, and hormoneshave been demonstrated to upregulate c-met expression in a wide varietyof cells including renal epithelial cells ( 21, 27 ), littleis known as to the extracellular cues that suppress its expression( 35 ). In this report, we demonstrated that oxidative stress suppresses c-met expression in renal epithelial cells in a time-and dose-dependent fashion. This inhibition of c-met expression byoxidant stress is likely mediated by the interplay between Sp1 andEgr-1 transcription factors, in which induced Egr-1 sequesters Sp1'sfunction as a transcriptional activator of the c-met gene through physical interaction.  B. M+ ^1 M1 V4 ?/ H. U7 [
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One of the intriguing features in renal epithelial cells afteroxidative stress is the concomitant and reciprocal expression of c-metand Egr-1 transcription factors (Figs. 1 and 2 ). This inverseassociation between c-met and Egr-1 was later confirmed by ectopicexpression of Egr-1 via transfection (Fig. 3 ). Egr-1 is the prototypicmember of the Egr family of transcription factors whose expression israpidly induced in response to various stimuli ( 31, 47 ).Egr-1 contains a DNA binding domain that is homologous to that found inSp1 and recognizes and binds to the consensus GC-rich sequence5'-GCGGGGGCG-3' of its target genes in a zinc-dependent manner( 11, 15 ). Although Egr-1 binding usually results in activation of gene transcription, it also suppresses gene transcription depending on specific context of the promoters ( 1, 33 ). As to the c-met gene, we found that Egr-1 was unable to bind tothe c-met promoter (Fig. 6 ) but markedly suppressed its transcription (Fig. 5 ) and protein expression (Fig. 3 ), despite robust induction ofEgr-1 transcript (Fig. 1 ), protein (Fig. 2 ), and its DNA-binding activity (Fig. 6 C ) in renal epithelial cells afterH 2 O 2 treatment. The inability of Egr-1 to bindto the cis -element is likely attributable to the existenceof an imperfect match (8 nucleotides of 9) in the putative Egr-1 siteof the c-met promoter. This underscores that a single mutation in thecore sequence of the Egr-1-binding site can cripple the interactionbetween the trans -acting factor and cis -acting element.* O1 C' _4 P- o! P' S
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The involvement of Sp1 in mediating oxidative stress-triggered c-metsuppression is initially hinted by the identification of a cis -acting H 2 O 2 -responsive region,which harbors three Sp1-binding sites and an imperfect Egr-1 site (Fig. 4 ). Moreover, DNA-protein interaction studies reveal an attenuatedbinding of Sp proteins (Sp1 and Sp3) to their cognate sites when usingnuclear extract from H 2 O 2 -treated renalepithelial cells (Fig. 7 ). It should be noted that there is anadditional perfect Sp1-binding site located at the nucleotide positionof  29 to  34 relative to the c-met transcriptional initiation site( 18 ), which could explain the marginal reduction inluciferase activity in the pGL3-0.1met construct afterH 2 O 2 treatment (Fig. 4 ). These observations areconsistent with earlier studies demonstrating that the Sp family oftranscription factors is essential for establishing the constitutiveexpression of c-met gene in diverse types of cells( 18, 36, 37, 46 ). For instance, c-met expression in normaladult kidney is largely overlapped with Sp1 and Sp3, and the abundanceof c-met is tightly proportional to endogenous cellular levels of Spproteins ( 46 ). Of importance, both Sp1 and Sp3 proteins trans -activate the c-met promoter in a highly synergisticway. However, treatment of renal epithelial cells withH 2 O 2 induced no alteration in cellular levels of both Sp1 and Sp3 (Fig. 7 E ). Such observation establishesthat oxidant stress suppresses c-met expression by a mechanisminvolving a possible interplay between Sp proteins and induced Egr-1rather than any alteration in cellular level of Sp proteins.1 N0 u0 h1 L# k& p% {- M2 y  p
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Interplay between Egr-1 and Sp1 transcription factors has beendescribed to mediate the transcriptional regulation of many genes( 1, 10, 16, 30, 41 ). Extensive studies indicate that themolecular mechanism governing the interaction between Egr-1 and Sp1 isoverwhelmingly controlled through directly competing for binding to theoverlapping cis -acting element. In many promoters containingGC-rich elements, Sp1- and Egr-1-binding sites often are partiallyoverlapped by sharing several nucleotides in their respective corebinding sequence. Thus overexpression or induction of one factor,leading to an increased cellular concentration, will result in"displacement" of another factor for binding to the overlappingsites. Such displacement of Sp1 by Egr-1, or vice versa, could lead toeither transcriptional activation or repression, depending on aparticular promoter and cellular content. For example, Egr-1 competeswith Sp1 protein for an overlapping region in the promoter ofplatelet-derived growth factor A and functions as a positive activator( 16 ). Thus the Egr-1/Sp1 displacement mechanism may be animportant regulatory pathway in the control of inducible geneexpression ( 16 ). Contrary to this report, Egr-1 has been shown to act as a negative regulator and represses Sp1-mediated activation of many genes including protein-tyrosine phosphatase 1B( 10 ), Epstein-Barr virus C ( 30 ), and 1 -adrenergic receptor ( 1 ). These studiessuggest that Egr-1 and Sp1 often elicit opposite regulation ofparticular genes by competing for binding to an overlapping site andthereby displacing with each other. Hence, competition for DNA bindingbetween inducible Egr-1 and constitutive Sp1 may provide a definedmeans of transcriptional regulation. Such a mechanism, however, couldnot account for the regulation of c-met expression in renal epithelialcells after oxidative stress, because Egr-1 does not directly bind tothe c-met promoter (Fig. 6 ).
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The action of Egr-1 on gene transcription is also regulated through itsphosphorylation by an inducible casein kinase II ( 13, 38, 45 ). This represents an alternative mechanism implicating theinterplay between Egr-1 and Sp1 in modulating gene transcription, besides the displacement of DNA binding. Previous studies show thatestrogen blocks macrophage colony-stimulating factor (M-CSF) geneexpression by decreasing casein kinase II-dependent phosphorylation ofEgr-1 ( 38 ). Phosphorylated Egr-1, possibly due toconformational changes of specific domains that modulate the affinityof Egr-1 for Sp1, binds less ardently to Sp1, which leads to higherlevels of free Sp1 for stimulating activation of the M-CSF gene( 38 ). Therefore, posttranslational modification of Egr-1also plays a critical role in regulating gene transcription bymodulating the affinity of Egr-1 for Sp1. However, because it has notbeen tested whether oxidative stress affects the phosphorylation of Egr-1 in this study, it remains elusive whether this mode of regulation is related to c-met suppression by oxidative stress in renal epithelial cells.9 g, ^+ o% e/ s/ S9 W
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Our findings in the present study unravel a distinctive mode ofinteraction between Egr-1 and Sp1 transcription factors, in which Egr-1sequesters Sp1 for its transcriptional activation of the c-met gene. The sequestration of Sp1 by Egr-1 is most likely mediated through protein-protein interaction. This conclusion isconsistent with, and supported by, several lines of observations. First, despite increased amounts of Egr-1 expression after oxidative stress in renal epithelial cells, Egr-1 did not directly bind to thec-met promoter. This excludes the possibility of a direct "displacement" of Sp1 by increasing Egr-1 afterH 2 O 2 treatment via competition for binding.Second, physical interaction between Egr-1 and Sp1 is clearly evident,as illustrated by coimmunoprecipitation (Fig. 8 ). It is unlikely thatformation of the Egr-1/Sp1 complex requires the binding of bothproteins to the cis -acting DNA element in the c-metpromoter, because such a complex was absent in the EMSA studies (Fig. 6 ). Third, ectopic expression of Egr-1 functionally suppresses c-metpromoter and protein expression in renal epithelial cells, a scenarioresembling that in renal epithelial cells where Egr-1 is abundantfollowing oxidative stress. Because Egr-1 suppression of c-metexpression does not require its binding to DNA element, our findings onEgr-1 sequestration of Sp1 may have broad implications in theregulation of any genes that are transcriptionally controlled byubiquitous Sp1 and potentially provide a generalized mechanism elucidating how inducible Egr-1 protein controls the expression of awide variety of genes, regardless of whether they have an Egr-1-bindingsite in their promoter regions.

In summary, we showed herein that oxidative stress suppresses c-metexpression in renal epithelial cells in a time- and dose-dependent fashion. We demonstrate that Egr-1, the product of an inducible immediate-early responsive gene, is a key mediator of c-met inhibition, primarily by its sequestration of Sp1 as a transcriptional activator for the c-met gene via physical interaction. The results ofthis study provide the mechanistic insights into the transcriptional repression of the c-met gene and will form the basis forfurther analyses of the regulation of this important signaling molecule under normal and pathological settings.
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ACKNOWLEDGEMENTS2 ?4 ~3 C9 O/ y( U" \7 o  }
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We thank Drs. R. Tjian and F. Rauscher III for generously providingplasmid vectors.3 F, W! r$ P, f5 ^; w8 s& H
      【参考文献】1 ^* e! G" `- o" W
1. Bahouth, SW,Beauchamp MJ,andVu KN. Reciprocal regulation of (1)-adrenergic receptor gene transcription by Sp1 and early growth response gene 1: induction of EGR-1 inhibits the expression of the (1)-adrenergic receptor gene. Mol Pharmacol 61:379-390,2002 .+ x+ d1 I( e* J# f
3 g+ }/ b' E7 J* q; N
# [0 D' Z) }+ P! t% G* q
, [3 p+ s" g) k5 S
2. Bell, A,Chen Q,DeFrances MC,Michalopoulos GK,andZarnegar R. The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice. Oncogene 18:887-895,1999  .# B+ U6 w, d4 y- }7 S, [. }( d

3 H7 w* g! U$ Z# U
3. Bottaro, DP,Rubin JS,Faletto DL,Chan AM,Kmiecik TE,Vande Woude GF,andAaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251:802-804,1991 .
" q8 H  \- _* ~; l5 \% L

4. Cheng, HL,Trink B,Tzai TS,Liu HS,Chan SH,Ho CL,Sidransky D,andChow NH. Overexpression of c-met as a prognostic indicator for transitional cell carcinoma of the urinary bladder: a comparison with p53 nuclear accumulation. J Clin Oncol 20:1544-1550,2002 .9 ]( F/ H) t# s- L

, ~+ Z4 ~5 |5 J4 `! P0 ?
5. Comoglio, P. Pathway specificity for Met signalling. Nat Cell Biol 3:E161-E162,2001  .* g8 G% c7 T4 f0 p" o" D

& C1 n% k- S, V) _' K9 C2 g' O

6. Dai, C,Yang J,andLiu Y. Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice. J Am Soc Nephrol 13:411-422,2002 .
! M- r$ @! z: v  m* V

" P1 o2 A* F* [8 Y
7. Danilkovitch-Miagkova, A,andZbar B. Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest 109:863-867,2002  .
6 \$ c8 w/ C) r7 [/ F& }, g

8. D'Errico, A,Fiorentino M,Ponzetto A,Daikuhara Y,Tsubouchi H,Brechot C,Scoazec JY,andGrigioni WF. Liver hepatocyte growth factor does not always correlate with hepatocellular proliferation in human liver lesions: its specific receptor c-met does. Hepatology 24:60-64,1996.

- G9 ]3 S3 }: S7 v1 ]
9 k) I$ H5 x2 z
9. Dignam, JD,Lebovitz RM,andRoeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475-1489,1983 .

1 p8 l$ K8 ]5 }# q$ c/ G
10. Fukada, T,andTonks NK. The reciprocal role of Egr-1 and Sp family proteins in regulation of the PTP1B promoter in response to the p210 Bcr-Abl oncoprotein-tyrosine kinase. J Biol Chem 276:25512-25519,2001 .

3 \( B1 g1 q7 G, Q0 P, j
11. Gashler, A,andSukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol 50:191-224,1995  .

7 L* L3 s" E0 i, \1 \

12. Horiguchi, N,Takayama H,Toyoda M,Otsuka T,Fukusato T,Merlino G,Takagi H,andMori M. Hepatocyte growth factor promotes hepatocarcinogenesis through c-met autocrine activation and enhanced angiogenesis in transgenic mice treated with diethylnitrosamine. Oncogene 21:1791-1799,2002  .
* ?; P' f  ^, X% x: t- H

( B4 e' A1 X6 z. ?3 r
13. Jain, N,Mahendran R,Philp R,Guy GR,Tan YH,andCao X. Casein kinase II associates with Egr-1 and acts as a negative modulator of its DNA binding and transcription activities in NIH 3T3 cells. J Biol Chem 271:13530-13536,1996 .
& Y& T) n2 E' D7 A- D
/ l6 N% O1 z. b7 g4 f

14. Joannidis, M,Spokes K,Nakamura T,Faletto D,andCantley LG. Regional expression of hepatocyte growth factor/c-met in experimental renal hypertrophy and hyperplasia. Am J Physiol Renal Fluid Electrolyte Physiol 267:F231-F236,1994 .
( w2 u. p) m: Q: K
7 d$ L: |' P. j0 k/ W2 m

15. Khachigian, LM,andCollins T. Early growth response factor 1: a pleiotropic mediator of inducible gene expression. J Mol Med 76:613-616,1998  .; P" @0 H/ I, s8 R: q. Y
  ?& {# R/ N* f% g

16. Khachigian, LM,Williams AJ,andCollins T. Interplay of Sp1 and Egr-1 in the proximal platelet-derived growth factor A-chain promoter in cultured vascular endothelial cells. J Biol Chem 270:27679-27686,1995 .' U9 q4 F7 k) ], i
6 X: e# Z: t! u& Z: K' _
( l) i- e: ]. f% I

17. Liu, Y. Hepatocyte growth factor and the kidney. Curr Opin Nephrol Hypertens 11:23-30,2002  .

8 c% K6 e, q' i9 h

18. Liu, Y. The human hepatocyte growth factor receptor gene: complete structural organization and promoter characterization. Gene 215:159-169,1998  .
4 g* R4 T' K$ e. l! w

19. Liu, Y,Beedle AB,Lin L,Bell AW,andZarnegar R. Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene. Mol Cell Biol 14:7046-7058,1994 .$ y# ?' D  R$ a  w6 Y
$ a0 E4 ~2 S# G2 o( |+ d2 Y

4 e0 D. Y+ `  u/ E
20. Liu, Y,Rajur K,Tolbert E,andDworkin LD. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. Kidney Int 58:2028-2043,2000  .' c. Q& O; k" R! _  s- m

3 ]; Y4 D! X# g# J) f" d7 ~
21. Liu, Y,Tolbert EM,Lin L,Thursby MA,Sun AM,Nakamura T,andDworkin LD. Upregulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure. Kidney Int 55:442-453,1999  .  O8 M$ }$ n- y: o" s1 P! n
6 w1 K* u" U1 _4 y1 Q* k
1 s8 x. k( G% k1 [
0 J. U0 z8 _. u, Q0 `4 h( Y
22. Liu, Y,Tolbert EM,Sun AM,andDworkin LD. In vivo and in vitro evidence for increased expression of HGF receptor in kidney of diabetic rat. Am J Physiol Renal Fluid Electrolyte Physiol 271:F1202-F1210,1996 .

% [1 i7 S  S& f& ]

23. Liu, Y,Tolbert EM,Sun AM,andDworkin LD. Primary structure of rat HGF receptor and induced expression in glomerular mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 271:F679-F688,1996 ./ n3 Y! ^( G7 y. o' q$ K& [
. e+ U) [; m) K! P9 O* y

' m; w6 F8 l% c; W' h
24. Maina, F,Pante G,Helmbacher F,Andres R,Porthin A,Davies AM,Ponzetto C,andKlein R. Coupling met to specific pathways results in distinct developmental outcomes. Mol Cell 7:1293-1306,2001  .3 ?: @" _) j; W  t# i! w7 E
: F, p* o8 M2 I) |2 W: i
: z) W9 Y1 m8 }
7 f0 B4 E6 r! M6 q, Z
25. Matsumoto, K,andNakamura T. Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases. Kidney Int 59:2023-2038,2001  .
& K; h2 L5 v% K: ?6 J3 Q
- U# y6 W+ c9 r( D' ?
  t+ v1 e: I; Q) g* \
26. Michalopoulos, GK,andDeFrances MC. Liver regeneration. Science 276:60-66,1997 .

27. Moghul, A,Lin L,Beedle A,Kanbour-Shakir A,DeFrances MC,Liu Y,andZarnegar R. Modulation of c-MET proto-oncogene (HGF receptor) mRNA abundance by cytokines and hormones: evidence for rapid decay of the 8 kb c-MET transcript. Oncogene 9:2045-2052,1994  .
7 N( z4 n; \. n
) h8 B  c0 a) B+ c) o8 e
: J# r1 S" w2 f' d  k; w4 P1 k. ]( @
28. Morello, S,Olivero M,Aimetti M,Bernardi M,Berrone S,Di Renzo MF,andGiordano S. MET receptor is overexpressed but not mutated in oral squamous cell carcinomas. J Cell Physiol 189:285-290,2001  .3 P3 p% S: U+ }  {  c/ A: d
4 F+ s! m8 d/ l+ V! Y6 d( V6 Y

29. Naldini, L,Vigna E,Narsimhan RP,Gaudino G,Zarnegar R,Michalopoulos GK,andComoglio PM. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 6:501-504,1991  .
& R& r: ~) z- _* E; J' t! c

9 J' Y! d7 c, k7 h' }
30. Nilsson, T,Zetterberg H,Wang YC,andRymo L. Promoter-proximal regulatory elements involved in oriP-EBNA1- independent and -dependent activation of the Epstein-Barr virus C promoter in B-lymphoid cell lines. J Virol 75:5796-5811,2001 ., \0 v3 ^  z. o6 @" {

0 b$ B& S9 A% {& j

31. Ohba, M,Shibanuma M,Kuroki T,andNose K. Production of hydrogen peroxide by transforming growth factor- 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol 126:1079-1088,1994 .
- h; @4 Q$ S2 m5 b" v4 v- Y
/ k$ P4 Y3 p+ ?3 j* _2 b  P

32. Park, M,Dean M,Kaul K,Braun MJ,Gonda MA,andVande Woude G. Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc Natl Acad Sci USA 84:6379-6383,1987 .

33. Porntadavity, S,Xu Y,Kiningham K,Rangnekar VM,Prachayasitikul V,andSt Clair DK. TPA-activated transcription of the human MnSOD gene: role of transcription factors Sp-1 and Egr-1. DNA Cell Biol 20:473-481,2001  .
% B& b( V" D9 F% n' i# a
6 H' [: v, y# s+ G+ j
. x: Q( a) ~1 ]6 }0 P6 B% w7 K! z
34. Rabkin, R,Fervenza F,Tsao T,Sibley R,Friedlaender M,Hsu F,Lassman C,Hausmann M,Huie P,andSchwall RH. Hepatocyte growth factor receptor in acute tubular necrosis. J Am Soc Nephrol 12:531-540,2001 .+ \4 |4 z7 u% D" k8 m- I" q
- g, i7 O  `  b5 a, u* i, ^

35. Radaeva, S,Jaruga B,Hong F,Kim WH,Fan S,Cai H,Strom S,Liu Y,El-Assal O,andGao B. Interferon- activates multiple STAT signals and downregulates c-Met in primary human hepatocytes. Gastroenterology 122:1020-1034,2002 .
+ p) i2 k; p+ r' N8 G

. k. F, a, c# H3 D# j# S
36. Seol, DW,Chen Q,andZarnegar R. Transcriptional activation of the hepatocyte growth factor receptor (c-met) gene by its ligand (hepatocyte growth factor) is mediated through AP-1. Oncogene 19:1132-1137,2000  .
; _2 ~6 D/ {* T, E/ Q7 e% ~6 @

37. Seol, DW,andZarnegar R. Structural and functional characterization of the mouse c-met proto-oncogene (hepatocyte growth factor receptor) promoter. Biochim Biophys Acta 1395:252-258,1998 .

! y! K/ `% l9 M$ `. D5 o* D" O
38. Srivastava, S,Weitzmann MN,Kimble RB,Rizzo M,Zahner M,Milbrandt J,Ross FP,andPacifici R. Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1. J Clin Invest 102:1850-1859,1998  .

8 G6 ^2 D  G% X, m& J8 U2 j! s
39. Sweeney, P,El-Naggar AK,Lin SH,andPisters LL. Biological significance of c-met overexpression in papillary renal cell carcinoma. J Urol 168:51-55,2002  .
- J5 n( g7 G. @1 K6 |
& Z- F( z5 A$ U9 N; Q' s6 {

40. Takayama, H,LaRochelle WJ,Sharp R,Otsuka T,Kriebel P,Anver M,Aaronson SA,andMerlino G. Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proc Natl Acad Sci USA 94:701-706,1997 .
3 h4 h2 Q- o3 Y$ H% x$ l. s0 B

* i0 H; }! q: a5 {- J
41. Thottassery, JV,Sun D,Zambetti GP,Troutman A,Sukhatme VP,Schuetz EG,andSchuetz JD. Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene. J Biol Chem 274:3199-3206,1999 .
: i6 V" k) N# g5 |7 S7 U* d, [: f; i0 o
% b9 M% }! c! |- `

42. Wang, X,DeFrances MC,Dai Y,Pediaditakis P,Johnson C,Bell A,Michalopoulos GK,andZarnegar R. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 9:411-421,2002  .! J/ \1 a# U3 \+ n

4 e% U+ u: O, e6 }4 r0 u7 G
43. Yang, J,andLiu Y. Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol 13:96-107,2002 .0 H& |% u3 a8 ~7 \# b, A: ?

2 l1 g5 B1 I/ Q; Q
: z- m# @3 e: Z* R* f
44. Yang, J,Shultz RW,Mars WM,Wegner RE,Li Y,Dai C,Nejak K,andLiu Y. Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy. J Clin Invest 110:1525-1538,2002  .

; F( E' _) n  v. c( J8 N; ]! e6 z

45. Zdunek, M,Silbiger S,Lei J,andNeugarten J. Protein kinase CK2 mediates TGF- 1 -stimulated type IV collagen gene transcription and its reversal by estradiol. Kidney Int 60:2097-2108,2001  .1 Y# t5 q7 @5 Z/ e. p

! [% j, j! h; Z2 u
0 K* t' N: L* [; V* g2 j( q
46. Zhang, X,Li Y,Dai C,Yang J,Mundel P,andLiu Y. Sp1 and Sp3 transcription factors synergistically regulate HGF receptor gene expression in kidney. Am J Physiol Renal Physiol 284:F82-F94,2003 .

0 O) ?& o+ T1 D, c

47. Zhang, Z,andCohen DM. Hypotonicity increases transcription, expression, and action of Egr-1 in murine renal medullary mIMCD3 cells. Am J Physiol Renal Physiol 273:F837-F842,1997 .

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