Sp1 and Sp3 transcription factors synergistically regulateHGF receptor gene exp - 《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 13:23 |只看该作者 |倒序浏览 |打印

作者：XianghongZhang, YingjianLi, ChunsunDai, JunweiYang, PeterMundel,  YouhuaLiu作者单位：1 Department of Pathology, University ofPittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Department of Cell Biology, Peking Union MedicalCollege, Beijing 10000 China; and Department of Medicine, Albert Einstein College ofMedicine, Bronx, New York 10461



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      【摘要】( n0 J' W3 W- f; V
   Weinvestigated the expression pattern and underlying mechanism thatcontrols hepatocyte growth factor (HGF) receptor (c-met) expression innormal kidney and a variety of kidney cells. Immunohistochemical staining showed widespread expression of c-met in mouse kidney, apattern closely correlated with renal expression of Sp1 and Sp3transcription factors. In vitro, all types of kidney cells testedexpressed different levels of c-met, which was tightly proportional tothe cellular abundances of Sp1 and Sp3. Both Sp1 and Sp3 bound to themultiple GC boxes in the promoter region of the c-met gene.Coimmunoprecipitation suggested a physical interaction between Sp1 andSp3. Functionally, Sp1 markedly stimulated c-met promoter activity.Although Sp3 only weakly activated the c-met promoter, its combinationwith Sp1 synergistically stimulated c-met transcription.Conversely, deprivation of Sp proteins by transfection of decoy Sp1oligonucleotide or blockade of Sp1 binding with mithramycin A inhibitedc-met expression. The c-met receptor in all types of kidney cells wasfunctional and induced protein kinase B/Akt phosphorylation in adistinctly dynamic pattern after HGF stimulation. These resultsindicate that members of the Sp family of transcription factors play animportant role in regulating constitutive expression of the c-met gene in all types of renal cells. Our findings suggestthat HGF may have a broader spectrum of target cells and possess widerimplications in kidney structure and function than originally thought.
      【关键词】 hepatocyte growth factor Sp Sp gene regulation Akt kinase' \% {8 S/ @. c
   INTRODUCTION
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HEPATOCYTE GROWTH FACTOR (HGF) receptor is the product of the c-met protooncogene, which is a membrane-spanning protein that belongs to thereceptor tyrosine kinase superfamily ( 3, 35 ). The c-met gene was originally isolated from a human osteogenic sarcoma cell line that was treated in vitro with the chemical carcinogen N -methyl- N '-nitro- N -nitrosoguanidine( 37 ). Mature c-met protein is a 190-kDa,disulfide-linked heterodimer that consists of - and -subunits( 37 ). The -subunit is heavily glycosylated and iscompletely extracellular. The -subunit has an extracellular portionthat is involved in ligand binding and also has a transmembrane segmentand a cytoplasmic tyrosine kinase domain that contains multiplephosphorylation sites. Both subunits are encoded within a singleopen-reading frame and are produced from the proteolytic cleavage of a170-kDa precursor ( 30 ). On binding to HGF, the c-metreceptor undergoes autophosphorylation of the tyrosine residues in itscytoplasmic domain and initiates cascades of signal transduction eventsthat eventually lead to specific cellular responses ( 5, 31 ). It has been demonstrated that the HGF/c-met signalingsystem plays a vital role in cell survival, proliferation,migration, and differentiation in a wide spectrum of targettissues including kidneys ( 21, 28, 33 ).* }% ]8 i  k& W
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Because all biological activities of HGF are presumably mediated by asingle c-met receptor, its expression is likely one of the crucialcomponents that determine cell-type specificity and overall activity ofHGF actions. Earlier studies indicated that the c-met geneis predominantly expressed in epithelial cells from different organs,whereas its ligand is primarily derived from the mesenchyme( 46 ). This characteristic pattern of expression aswell as the pleiotrophic nature of its actions makes HGF an importantparacrine and/or endocrine mediator for mesenchymal/epithelial interactions, which are critical processes in organ development, tissueregeneration, and tumorigenesis under various physiological andpathological conditions. However, recent studies suggest that thec-met receptor is expressed at different levels in nonepithelial cellsas well. For instance, c-met expression is observed in endothelial cells, various types of blood cells, and glomerular mesangial cells( 4, 27, 52, 54 ). Because these cells also express HGF,these observations indicate that the autocrine pathway is anotherimportant mode of action for this paired receptor-ligand system, atleast in certain types of cells.
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The kidney is one of the organs in which the c-met receptor isabundantly expressed, although little is known about its function atnormal physiological settings ( 21, 31 ). Earlier studies ( 26, 40 ) revealed that c-met protein is primarilyexpressed in renal tubular epithelial cells along the entire nephron in normal rat kidney. Little or no c-met protein was observed in othertypes of cells (such as renal interstitial fibroblasts) aside fromrenal tubules. However, it remains a question whether these cells trulydo not express c-met or their expression level is instead below thedetection limits by conventional approaches. Furthermore, the molecularmechanism that governs the constitutive expression of the c-met gene in various types of kidney cells remains largely unknown.

In this study, we examined the expression pattern of the c-met receptorin normal adult kidneys and in a wide variety of kidney cells in vitro.We found that c-met is ubiquitously expressed in normal kidney in apattern that overlaps with that of the Sp family of transcriptionfactors. Both Sp1 and Sp3 proteins bound to the promoter region of the c-met gene and functionally activated its transcription. Alltypes of kidney cells tested in vitro expressed the functional c-metreceptor and induced protein kinase B (PKB)/Akt phosphorylation afterHGF stimulation.% ]6 j3 r3 P0 i! Q! I3 a5 p/ x" Q

MATERIALS AND METHODS

Animals. Male CD-1 mice (body wt 20-24 g) were purchased from HarlanSprague Dawley (Indianapolis, IN). The mice were housed in the animalfacilities of the University of Pittsburgh Medical Center and had freeaccess to food and water. Animals were treated humanely using approvedprocedures in accordance with the guidelines of the InstitutionalAnimal Use and Care Committee of the National Institutes of Health atthe University of Pittsburgh School of Medicine. The mice were killedby exsanguination while under general anesthesia. The kidneys wereremoved and immediately decapsulated. One part of the kidney was frozenin Tissue-Tek optimal cutting-temperature compound in preparation forcryosection. Another part was fixed in 10% neutral-buffered formalinand embedded in paraffin in preparation for histology andimmunohistochemical staining.1 i- X" v# B. C% Z0 U

Cell culture and treatment. Mouse inner medullary collecting duct epithelial cell line 3 (mIMCD-3),rat renal interstitial fibroblasts (NRK-49F), and Drosophila Schneider line 2 (SL-2) cells were obtained from the American TypeCulture Collection (Rockville, MD). The human kidney proximal tubularcell line (HKC) was provided by Dr. L. Racusen of Johns HopkinsUniversity. Rat glomerular mesangial cells were a gift of Dr. C. Wu ofthe University of Pittsburgh. The conditionally immortalized mousepodocyte cell line was established from the transgenic mouse thatcarries a thermosensitive variant of the simian virus 40 (SV40)promotor as described previously ( 34 ). mIMCD-3, HKC, andNRK-49F cells were maintained in a 1:1 DMEM/Ham's F-12 medium (LifeTechnologies, Grand Island, NY) mixture supplemented with 10% fetalbovine serum (FBS). Mesangial cells were cultured in RPMI 1640 mediumsupplemented with 20% FBS. To propagate podocytes, cells were culturedon type I collagen at 33°C in the RPMI 1640 medium supplemented with10% FBS and 10 U/ml mouse recombinant interferon (IFN)- (R & DSystems, Minneapolis, MN) to enhance the expression of athermosensitive T antigen. To induce differentiation, podocytes weregrown at 37°C in the absence of IFN- for 14 days undernonpermissive conditions ( 34 ). SL-2 cells were grown in Schneider's medium (Life Technologies) supplemented with 10% FBS. Forchemical blockade of Sp binding, mIMCD-3 cells were treated withmithramycin A (Sigma, St. Louis, MO) at different concentrations forvarious periods of time.
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Immunohistochemical staining. Kidney sections from paraffin-embedded tissues were prepared at 4-µmthickness using a routine procedure. Immunohistochemical localizationwas performed using the Vector MOM immunodetection kit (VectorLaboratories, Burlingame, CA) according to procedures describedpreviously ( 7 ). The primary antibody against mouse c-met(sc-8057) was obtained from Santa Cruz Biochemical (Santa Cruz, CA). Asa negative control, the primary antibody was replaced with nonimmunenormal IgG, and no staining occurred.2 k" K; y9 G6 i' A
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Frozen section and immunofluorescence staining. Cryosections were prepared at 5-µm thickness in a cryostat and werefixed in a cold 1:1 methanol-acetone mixture for 10 min at 20°C.Immunostaining was performed as described previously ( 51 ).Briefly, cryosections were incubated with 20% normal donkey serum inPBS for 30 min at room temperature to reduce background staining.Sections were washed with PBS and incubated with primary antibodies inPBS containing 1% BSA overnight at 4°C. The primary antibodiesagainst mouse c-met, Sp1 (sc-59), and Sp3 (sc-644) were obtained fromSanta Cruz Biochemical. Sections were then incubated for 1 h withaffinity-purified secondary antibodies (Jackson ImmunoResearchLaboratories, West Grove, PA) at a 1:100 dilution in PBS that contained1% BSA before being washed extensively with PBS. Slides were mountedwith antifade mounting media and examined on a Nikon Eclipse E600epifluorescence microscope (Melville, NY) equipped with a digital camera.
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Western blot analysis. Various types of kidney cells were lysed with SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue). Samples were heated at100°C for 5-10 min and were then loaded and separated on precast10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins wereelectrotransferred to a nitrocellulose membrane (Amersham, ArlingtonHeights, IL) in transfer buffer that contained 48 mMTris · HCl, 39 mM glycine, 0.037% SDS, and 20%methanol at 4°C for 1 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). The membranes were then incubated for 16 h at 4°Cwith various primary antibodies in blocking buffer that contained 5%milk at the dilutions specified by the manufacturers. Thephospho-specific Akt antibody (that detects Akt only when it isphosphorylated at specific sites) and the total Akt antibody (thatdetects Akt independently of phosphorylation state) were obtained fromCell Signaling (Beverly, MA). The antibodies against Sp1, Sp3, c-met, and actin were purchased from Santa Cruz Biochemical. The membranes were washed extensively in TBS buffer and were then incubated withhorseradish peroxidase-conjugated secondary antibody (Sigma) at adilution of 1:10,000 for 1 h at room temperature in 5% nonfat milk dissolved in TBS. Membranes were then washed with TBS buffer, andthe signals were visualized using an ECL system (Amersham).
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Preparation of nuclear protein extract. For preparation of nuclear protein extracts, mIMCD-3 cells in anexponential growth stage were washed twice with cold PBS and scrapedoff the plate with a rubber policeman. Cells were collected and thenuclei were isolated according to methods described elsewhere( 24 ). Briefly, the pelleted cells were resuspended in 4 volumes of buffer A that contained protease inhibitors: 20 mM HEPES, pH 7.9, 0.5 M sucrose, 1.5 mM NaCl, 60 mM KCl, 0.15 mMspermidine, 0.5 mM spermine, 0.5 mM EDTA, and 1 mM dithiothreitol plus2 µg each of leupeptin, soybean trypsin inhibitor, antipain, andchymostatin per milliliter. An equal volume of buffer A that contained 0.6% Nonidet P-40 was added with gentle mixing to lyse thecells. Immediately after lysis, the solution was diluted with 8 volumesof buffer A, and the nuclei were collected by centrifugation at 5,000 g for 30 min at 4°C. Nuclear protein wasextracted with 0.4 M KCl · TGM (10 mMTris · HCl, pH 7.6, 10% glycerol, 3 mM MgCl 2, and 3 mM EGTA) buffer that contained proteaseinhibitors as described. The lysate was centrifuged for 45 min at50,000 g at 4°C, and the supernatant was then collectedand dialyzed against 60 mM KCl · TGM buffer usinga mini-dialysis system (Life Technologies). The insoluble material wasremoved by centrifugation, and aliquots of protein extract were quicklyfrozen and stored at 80°C after the protein concentration had beendetermined using a bicinoninic acid (BCA) protein assay kit (Sigma).

Electrophoresis mobility shift assays. A DNA fragment (F1) corresponding to 217 to 49 of the 5'-flankingregion of the human c-met gene was isolated via PCRamplification of the c-met promoter as described previously( 23 ). The F1 fragment was labeled with 32 P byincluding -[ 32 P]dCTP (3,000 Ci/mmol; Amersham) in thePCR reactions. The labeled probes were then gel purified and used inthe electrophoresis mobility shift assays (EMSAs) as describedpreviously ( 24 ). The nonspecific competitor was 4 µg ofPoly(dI-dC) · Poly(dI-dC) (Pharmacia, Piscataway,NJ) added to 10 µl of reaction mixture. The binding reactions werecarried out at room temperature for 15 min before loading of 5%nondenaturing polyacrylamide (19:1 acrylamide-bisacrylamideratio) gels. For competition experiments, a 100-fold molar excessof unlabeled DNA fragments or double-stranded oligonucleotides (oligos)was included in the reaction mixture. Oligos were chemicallysynthesized by a commercial source (Life Technologies). Complementarystrands were annealed in a mixture of 10 mMTris · HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA byheating the mixture to 95°C and cooling it to room temperature over a period of 3 h. The sequences of the oligos used in thisstudy are shown in Table 1. Forsupershift experiments, specific antibodies against Sp1, Sp3, Egr-1,and normal control IgG (Santa Cruz) were incubated with nuclear proteinextracts for 15 min at room temperature before reaction buffer wasadded. Gels were run in 0.5× TBE (0.045 MTris · borate with 0.001 M EDTA) buffer at aconstant voltage of 190 V and were dried and autoradiographed withintensifying screens.
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Table 1. Oligonucleotide sequences used in this study% Y5 n7 e, r, u: c! |) r. Q4 g
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Immunoprecipitation. mIMCD-3 cells grown on 100-mm plates were lysed on ice in 1 ml of RIPAbuffer that contained 1× PBS, 1% Nonidet P-40, 0.1% SDS, 10 µg/mlphenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1%protease inhibitor cocktail (Sigma). Whole cell lysates were clarifiedby centrifugation at 12,000 g for 10 min at 4°C, and thesupernatants were transferred into fresh tubes. To preclear celllysates, 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 each of anti-Sp1,anti-Sp3, and normal IgG, which was followed by precipitation with 20 µl of protein A/G PLUS-agarose for 3 h at 4°C. After fourwashes with RIPA buffer, the immunoprecipitates were boiled for 5 minin SDS sample buffer. The resulting precipitated complexes wereseparated on SDS-polyacrylamide gels and blotted with variousantibodies as described.% ?/ x" [2 x9 L  \; S5 s
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Plasmid construction, transfection, and reporter gene assay. The 0.2met-chloramphenicol acetyltransferase (CAT) and 0.1met-CATchimeric plasmids, which contain 0.2 and 0.1 kb of the 5'-flanking region of the c-met gene, respectively, and the codingsequence for CAT, have been described elsewhere ( 23 ). The Drosophila SL-2 cells, which lack endogenous Sptranscription factors, were used for investigating the effects of Spproteins on c-met promoter activity ( 6 ). At24 h before transfection, the cells were seeded onto six-wellplates at 2 × 10 5 cells/well. Cells were thentransiently cotransfected with a constant amount of 0.2met-CAT or0.1met-CAT chimeric plasmids and an increasing amount of eitherpPac-Sp1 or pPac-Sp3 expression vectors under the control of insectactin promoter. The DNA-calcium phosphate method was used according tothe instructions of the CellPhect transfection kit (Pharmcia,Piscataway, NJ). Cells were incubated with DNA-calcium phosphatecoprecipitation buffer for 16 h and washed twice with serum-freemedium. Complete medium that contained 10% FBS was added, and thecells were incubated for an additional 24 h before harvest for CATassays. After being washed in PBS, the cells were pelleted, resuspendedin 150 µl of 0.25 M Tris · HCl at pH 7.5, anddisrupted by three freeze-thaw cycles. The protein suspension wasclarified by centrifugation at 15,000 g for 5 min at 4°C,and the supernatant was collected and assayed for CAT activity by aprocedure described previously ( 23 ). Because Sp proteinsare known to activate the transcription of many internal controlreporter vectors driven under the SV40 early promoter and the thymidinekinase promoter ( 6, 16 ), the relative CAT activity in thisstudy was reported after normalization for protein concentration.Protein concentration was determined using a BCA protein assay kit(Sigma). All experiments were repeated at least three times to ensurereproducibility. For deprivation of endogenous Sp proteins in renalepithelial cells with decoy oligos, mIMCD-3 cells were cotransfectedwith CAT reporter plasmids and either 20- or 50-fold molar excess ofwild-type or mutant Sp1 oligos. At 36 h after transfection, cellswere harvested, and CAT activities were determined.

Statistical analysis. Quantitation of the Western blots was performed by measuring theintensity of the hybridization signals with the use of NIH Imageanalysis software. Data were expressed as means ± SE. Statistical analyses of the data were carried out by t -test with the useof SigmaStat software (Jandel Scientific, San Rafael, CA). P was considered significant.
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RESULTS
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Overlapping expression pattern of c-met and Sp family proteins innormal kidney. The expression of c-met protein in normal mouse kidney was examined byimmunohistochemical staining using a specific antibody against c-met.As shown in Fig. 1, the c-met protein waswidely expressed in normal mouse kidney. All tubular epithelial cells along the entire nephron were positive for c-met protein, with highlevels observed in distal tubules and collecting duct epithelia. Weakstaining was also noticeable in the glomeruli, which was most likelypresent in glomerular visceral epithelial cells (podocytes) andmesangial cells. In contrast, c-met receptor staining in the renal interstitium of normal mouse kidney was extremely weak or nondetectable. When c-met antibody was replaced by normal IgG, nostaining occurred (Fig. 1 B ), suggesting the specificity of c-met staining. These results indicate that c-met protein isconstitutively expressed in normal adult kidney in a relativelyubiquitous fashion with different levels in distinct types of kidneycells.

Fig. 1. Immunohistochemical localization of c-met protein innormal mouse kidney. A : ubiquitous presence of c-met proteinin normal kidney is shown. Specific staining for c-met protein wasvisible in renal tubules along the entire nephron. Weak staining wasalso observed in renal glomeruli (arrow). B : as a negativecontrol, no staining was observed in the kidney when c-met primaryantibody was replaced with normal IgG. Scale bar, 20 µm.; q' c+ @& z! w

The widespread expression pattern of c-met in normal kidney led us tospeculate that there might be ubiquitous trans -acting factor(s) responsible for its transcriptional activation. Earlier studies ( 23 ) revealed the presence of multiple GC boxes(Sp1 binding sites) in the promoter region of the c-met gene, which potentially implicates the Sp family of transcriptionfactors in regulating c-met expression in the kidney.Therefore, we examined the expression patterns of Sp1 and Sp3transcription factors and the relationships with c-met in normal kidneyvia double immunofluorescence staining for simultaneous detection ofboth c-met and Sp proteins. As shown in Fig. 2, there was close correlation betweenthe expression patterns of c-met protein and the Sp family oftranscription factors in normal adult kidney. In kidney sections, c-metprotein expression completely overlapped with the presence of nuclearSp1 protein (Fig. 2 ). Similarly, Sp1 and Sp3 proteins were alsoconcomitantly expressed in the nuclei of various kidney cells,suggesting a possibly functional coupling and cooperation with oneanother. Except on rare occasions in which some cells expressed moreSp1 than Sp3 or vice versa, Sp1 and Sp3 were largely expressed in comparable amounts in many cells of normal adult kidney (Fig. 2 ).2 Z( ^9 q# R0 D7 D! C

Fig. 2. Colocalization of c-met protein with Sp family oftranscription factors in normal mouse kidney. Representativemicrographs demonstrate a close association of c-met protein with Sp1and Sp3 transcription factors. Immunofluorescence staining for both Sp1(red) and c-met (green) in cryosections of normal mouse kidney( top ). Merging of two micrographs ( top right )illustrates the overlapping pattern of c-met and Sp1 localization.Immunofluorescence staining for Sp1 (red) and Sp3 (green) in normalkidney ( bottom ). Merging of two micrographs ( bottomright ) demonstrates a largely overlapping pattern of Sp1 and Sp3.Cell nuclei with approximately equal amounts of both Sp1 and Sp3(yellow) are indicated (arrowheads). Scale bar, 20 µm.
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Correlation of c-met level with Sp family protein abundance invarious types of kidney cells in vitro. We next examined c-met protein expression in a wide variety of kidneycells in vitro by using Western blot analysis. As shown in Fig. 3, all types of kidney cells tested,including glomerular mesangial cells, podocytes, proximal tubularepithelial cells, collecting duct epithelial cells, and renalinterstitial fibroblasts, expressed different levels of c-met protein.A single band of 145-kDa -subunit of c-met protein was observed inall types of kidney cells in polyacrylamide gels under reducingconditions. This observation is consistent with the in vivo data, whichdemonstrates widespread expression of c-met protein in normal adultkidney as described (see Fig. 1 ). Western blot analysis also exhibited that all of the kidney cells tested expressed distinctive levels of Sp1and Sp3 transcription factors. A doublet of Sp1 protein thatrepresented a different phosphorylated status ( 15 ) at~95 kDa was detected in all renal cells. Sp3 displayed two doublets at 124 and 84 kDa, respectively. Presumably, these different isoforms are derived from distinct internal translation initiations ( 18, 49 ). Intriguingly, a plot of the abundances of c-met, Sp1, and Sp3 proteins indicates that there is a remarkably tight correlation between the c-met and Sp protein levels in various types of kidney cells in vitro (Fig. 3 C ). These results establish that c-metreceptor levels in diverse types of kidney cells are in proportionalto, and are likely dictated by, the endogenous abundance of the Sp family of transcription factors.6 S! Z; w9 Y! A( V! B
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Fig. 3. Close correlation of c-met expression with the proteinlevels of Sp1 and Sp3 transcription factors in various types of kidneycells. Cell lysates from major types of kidney cells, including humanproximal tubular cells (HKC), mouse inner medullary collecting ductepithelial cells (mIMCD-3), rat renal interstitial fibroblasts(NRK-49F), rat mesangial cells (RMC), and mouse glomerular visceralepithelial cells (podocyte), were immunoblotted with specificantibodies against c-met, Sp1, Sp3, and actin. A :representative Western blot analysis of five different types of kidneycells. B : Western blot analysis of c-met and Sp proteinexpression of nondifferentiated (ND) and differentiated (D) podocytes.Positive signals with each antibody are indicated ( right ) asare molecular sizes (in kDa; left ). C : linearregression of the abundance of c-met protein vs. the combined levels ofSp1 and Sp3 in various types of kidney cells. Quantitative data fromdensitometric analysis of Western blots were plotted after correctionwith actin to ensure equal loading. Regression-line equation andcorrelation coefficient are shown.

Comparison of the c-met receptor levels from podocytes of thedifferentiated and nondifferentiated states uncovered that c-met receptor expression was repressed in differentiated podocytes (Fig. 3 B ). This inhibition of c-met expression during podocyte differentiation was accompanied by decreased Sp1 and Sp3 protein levels(Fig. 3 B ). To further explore the correlation between c-met expression and Sp protein abundances, we examined the kinetics of c-metand Sp protein expression during podocyte differentiation induced byswitching podocyte culture to 37°C in the absence of INF- ( 34 ). As shown in Fig. 4,c-met inhibition occurred in the first 4 days of podocytedifferentiation, and levels were sustained beyond this time point.Again, there was a strong association between c-met and Sp proteinlevels during this period of podocyte differentiation( r 2 = 0.94).% l4 F/ f. H9 F
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Fig. 4. Concomitant alterations of c-met expression with Sp1 andSp3 transcription factors during podocyte differentiation. Mousepodocyte differentiation was initiated by switching the culture from 33 to 37°C in the absence of interferon- under nonpermissiveconditions. At various time points (in days) as indicated, podocyteswere harvested and the cell lysate was immunoblotted with specificantibodies against c-met, Sp1, Sp3, and actin. Identification( right ) and molecular sizes (in kDa; left ) of theproteins are indicated.2 @1 l# o- w! X3 Z  p: A: A

Binding of Sp1 and Sp3 to GC boxes of c-met promoter region. To examine the possibility of Sp proteins participating in theregulation of the c-met gene, we first determined whether Sp proteins interact with the c-met promoter by performing EMSA using aDNA fragment that contained three cis -acting Sp1 sites (GCboxes) from the c-met gene. When the DNA fragment wasincubated with nuclear protein extract derived from mIMCD-3 cells,multiple DNA-protein complexes were formed that had retarded migration,which resulted in three shifted bands in polyacrylamide gels undernondenaturing conditions (C1-C3; see Fig. 5 A ). These binding complexeswere largely abolished by using an unlabeled F1 fragment itself as acompetitor in the incubation. Under the same conditions, the complexeswere also completely abrogated in the presence of a 100-fold molarexcess of the double-stranded oligo that corresponds to the Sp1 site ofthe c-met gene. The binding complexes were intact whenincubated with a 100-fold molar excess of the mutated Sp1 oligo inwhich two GGs were substituted with two TTs in the Sp1 binding region.Other oligos with unrelated sequences such as activator protein 1 (AP-1), nuclear factor- B (NF- B), and cAMP response element didnot interrupt the formation of the binding complexes (Fig. 5 A ), suggesting the specificity of these DNA-protein interactions. Supershift assay with specific antibodies revealed thatthese binding complexes were contributed by Sp1 and Sp3 transcription factors (Fig. 5 B ). Incubation with Sp1 antibody caused afurther shift of the C2 complex and the formation of supershifted bands (SS1 and SS2), whereas the Sp3 antibody resulted in a supershift of theC1 and C3 complexes and formation of SS3 and SS2 (Fig. 5 B ).As expected, incubation with normal IgG or other unrelated antibodiessuch as anti-Egr-1 transcription factor did not cause either asupershift or inhibition of the complex formation.
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Fig. 5. Electrophoresis mobility shift assay (EMSA) demonstrates that bothSp1 and Sp3 proteins bind to the GC boxes in the c-met promoter region. A : 32 P-labeled DNA fragment (F1) correspondingto the nucleotide sequence 217 to 50 of the c-met promoter regionwas incubated with nuclear protein extract (NPE) from mIMCD-3 cells inEMSA reaction mixture. Three major DNA-protein complexes (designatedC1, C2, and C3) were formed between renal epithelial cell nuclearproteins and c-met promoter. Competition for binding was performed byincluding a 100-fold molar excess of unlabeled F1 itself oroligonucleotides corresponding to Sp1, mutant Sp1 (Sp1m), activatorprotein 1 (AP-1), nuclear factor- B (NF- B), and cAMP responseelement (CRE) as indicated. Sequences of these oligonucleotides arepresented in Table 1. B : identification of these DNA-proteincomplexes (C1-C3) was characterized by supershift analysis withspecific antibodies and normal rabbit IgG. Supershifted protein-DNAcomplexes with Sp1 antibody are designated as SS1 and SS2, and thosewith Sp3 antibody are denoted as SS3 and SS2 (arrows).
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Careful examination of the supershift data revealed that band SS2 wasformed in the reactions involving both anti-Sp1 and anti-Sp3 antibodies(Fig. 5 B, lanes 3 and 4 ). Theseresults implied that SS2 could be attributable to an interactingcomplex that potentially consisted of Sp1, Sp3, and IgG, which suggeststhat two members of Sp family proteins (Sp1 and Sp3) may physically interact with each other. To test this hypothesis, we performed additional coimmunoprecipitation experiments to demonstrate a directinteraction between Sp1 and Sp3 in renal epithelial cells. As shown inFig. 6 A, when cell lysatederived from mIMCD-3 cells was immunoprecipitated with Sp1 antibody,Sp3 protein was detected in the precipitated complexes. Similarly, Sp1protein was also found in the complexes immunoprecipitated by Sp3antibody (Fig. 6 B ). Thus there is a physical interactionbetween Sp1 and Sp3 proteins that could potentially lead to afunctional cooperation in regulating c-met transcription inrenal epithelial cells.0 X& `* g, q- a$ M9 R7 W# C( ~9 _

Fig. 6. Physical interaction between Sp1 and Sp3 proteins inrenal epithelial cells was demonstrated by coimmunoprecipitation.mIMCD-3 epithelial cells were immunoprecipitated (IP) with Sp1 and Sp3antibodies or normal rabbit IgG. Resulting complexes were separated onpolyacrylamide gels and immunoblotted (IB). A :immunoprecipitation with Sp1 antibody and blotting with Sp3 and Sp1antibodies. B : immunoprecipitation with Sp3 antibody andblotting with Sp1 and Sp3 antibodies. Coimmunoprecipitated Sp1 and Sp3were evident in both experiments.8 ~3 @$ S6 b! |- k
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Functional cooperation among members of Sp family in activatingc-met transcription. To determine the function of Sp1 and Sp3 proteins in regulating c-metexpression, we transfected the 0.2met-CAT reporter construct thatcontains multiple Sp1 binding sites with expression vector for Sp1 andSp3 in Drosophila SL-2 cells. Because Drosophila cells lack endogenous Sp activity, the cells provide a sensitive and reliable in vivo assay system for investigating the effects of Spproteins on gene transcription. As shown in Fig. 7 A, Sp1 dramatically activatedc-met promoter activity in a dose-dependent manner. An ~18-foldinduction in reporter gene activity was observed after cotransfectionof SL-2 cells with 0.2met-CAT plasmid and 3 µg of Sp1 expressionvector pPac-Sp1. Sp3 alone also induced, to a much less extent, c-metpromoter activity. Cotransfection of 3 µg of Sp3 expression vectorpPac-Sp3 with 0.2met-CAT plasmid into SL-2 cells resulted in anapproximately eightfold induction of reporter activity (Fig. 7 A ).( Q+ t" k6 f4 J/ [
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Fig. 7. Sp1 and Sp3 synergistically activate c-met promoteractivity. Reporter 0.2met-chloramphenicol acetyltransferase (CAT)plasmid that contains the c-met promoter sequence corresponding to 223 to  60 linked to the coding region of the CAT gene wastransiently cotransfected with Sp1 and Sp3 expression vectors into Drosophila Schneider cells. A : cell homogenateswere prepared 48 h after cotransfection with a fixed amount (3 µg) of 0.2met-CAT reporter plasmid and increasing amounts ofindividual pPac-Sp1 and pPac-Sp3 expression vectors as indicated. B : cells were cotransfected with 3 µg of 0.2met-CATreporter construct and various amounts of individual pPac-Sp1 andpPac-Sp3 plasmids or in combination as indicated. Relative CATactivities (fold induction, with transfection of 0.2met-CAT plasmidalone taken as 1.0) are presented as means ± SE from 3 independent experiments. * P P6 x# q; X2 ^# Q
- [( E- z% X9 I
To investigate the potentially functional interaction between Sp1 andSp3 proteins in activating c-met transcription, wecotransfected SL-2 cells with the 0.2met-CAT construct plus both Sp1and Sp3 expression vectors. As presented in Fig. 7 B, thecombination of Sp1 and Sp3 resulted in a dramatically synergisticinduction of c-met gene transcription. The magnitude of theCAT reporter induction that was elicited by the combined Sp1 and Sp3was greater than the additive effect obtained by Sp1 and Sp3individually. Hence, both Sp1 and Sp3 activate c-met transcription in a functionally cooperative manner.* F, D" t& C" `; N/ m' N: S) u# u

Deprivation of Sp family proteins by decoy oligos in renalepithelial cells inhibits c-met promoter activity. To further confirm the importance of the Sp family proteins inregulating c-met transcription, we cotransfected the0.2met-CAT reporter construct to mIMCD-3 cells with decoy oligocorresponding to the Sp1 binding site, which competes with c-metpromoter Sp1 sites for binding with cellular trans -acting Spproteins. This strategy presumably leads to a decrease in theavailability of the cellular Sp proteins to the c-met promoter. Asshown in Fig. 8, introduction of the Sp1decoy oligo markedly inhibited c-met gene transcription in adose-dependent fashion. Cotransfection of a 50-fold molar excess of Sp1decoy oligo together with the 0.2met-CAT plasmid suppressed c-metpromoter activity by ~70%. In fact, the CAT reporter gene activitywas reduced by Sp1 decoy to a level similar to that elicited by the0.1met-CAT plasmid in which three Sp1 binding sites were deleted.However, transfection of the mutant Sp1 oligo that failed to bind Spproteins due to mutations in the Sp1 binding region (see Fig. 5 ) didnot significantly affect 0.2met-CAT reporter activity in renalepithelial cells. Thus the abundance of endogenous cellular Sp familyproteins likely dictates the level of c-met expression in kidney cells.4 w; D/ c9 ]. z* l

Fig. 8. Deprivation of Sp proteins by cotransfection of decoyoligonulceotides inhibits c-met promoter activity. A :schematic representation of chimeric reporter plasmids 0.2met-CAT and0.1met-CAT. Three Sp1 sites were located at the c-met promoter regionbetween nucleotides 68 and 223 (ovals). B : mIMCD-3 cellswere cotransfected with 0.2met-CAT plasmid and a 20- or 50-fold molarexcess of Sp1 or mutant Sp1 decoy oligos (Sp1mut oligo), respectively,as indicated. Cells were also transfected with 0.1met-CAT construct asa control in which the three Sp1 sites of the c-met promoter regionwere deleted. Relative CAT activities (with 0.2met-CAT plasmid alonetaken as 100) are presented as means ± SE from 3 independentexperiments. ** P
& @6 d  F& m4 a! c1 B
Blockade of Sp protein binding inhibits c-met expression in renalepithelial cells. We next investigated the effects of blockade of Sp binding via chemicalantagonist in renal epithelial cells on c-met receptor expression.mIMCD-3 cells were treated with mithramycin A, a potent inhibitor of Spbinding ( 39, 42 ), for various periods of time at differentconcentrations. Because c-met expression is primarily regulated at thetranscriptional level in mIMCD-3 cells ( 25 ), the effect ofthe Sp inhibitor on c-met expression was determined by measuring theprotein levels using Western blot analysis after various treatments. Asshown in Fig. 9, mithramycin A markedly inhibited c-met expression in mIMCD-3 cells in a dose-dependent manner.At concentrations as low as 10 8 80% after a 24-h incubation. Thekinetics of c-met inhibition by mithramycin A are presented in Fig. 9 B. Blockade of Sp binding significantly inhibited c-metexpression as early as 12 h after incubation with the chemicalantagonist. Of note, the inhibitory effect of mithramycin A wasspecific, because expression of other genes such as actin was notblocked by this chemical inhibitor (Fig. 9 ). These results suggest thatthe binding of Sp proteins to cognate cis -acting elements isessential for constitutive expression of the c-met gene inrenal epithelial cells.) x3 h* s) A1 R: v4 v
- D# c( s! M& H+ O- I  o) G1 @
Fig. 9. Chemical blockade of Sp1 binding with mithramycin Ainhibits c-met expression in renal epithelial cells in dose- andtime-dependent manners. A : mIMCD-3 cells were incubated withvarious concentrations of mithramycin A for 24 h. B :Cells were incubated with 10 7 M of mithramycin A fordifferent periods of time. Cell lysates were separated onpolyacrylamide gels and immunoblotted with c-met antibody. The sameblots were reprobed with actin to ensure equal loading of thesamples.

Functionality of c-met receptor in different types of kidney cellsin vitro. The ubiquitous expression pattern of c-met in the kidney prompted us toinvestigate whether the c-met receptor is functional in all types ofkidney cells. To this end, we studied the phosphorylation andactivation of PKB/Akt kinase, which is a major signaling protein in thepathway leading to cell survival ( 12, 36 ), in various types of kidney cells after HGF stimulation. Consistent with a previousreport ( 22 ), HGF induced marked Akt phosphorylation asearly as 5 min after stimulation in proximal tubular epithelial cells,and this induction was sustained to at least 1.5 h after HGFincubation (Fig. 10 ). In addition toproximal tubular epithelial cells, all other types of kidney cellstested, including glomerular mesangial cells, podocytes, collectingduct epithelial cells, and renal interstitial fibroblasts, responded toHGF stimulation and induced Akt phosphorylation and activation (Fig. 10 ). Therefore, the c-met receptor is indeed functional in all types ofkidney cells tested and responds to HGF stimulation to initiate signal transduction events that lead to cell survival.& v: r9 j1 T" W% P7 t, d9 l' v
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Fig. 10. Activation of protein kinase B/Akt in various types ofkidney cells in response to HGF stimulation. Major types of kidneycells, including RMC cells, NRK-49F cells, mIMCD-3 cells, HKC cells,mouse differentiated glomerular podocytes (D), and nondifferentiatedpodocytes (ND) were incubated with 20 ng/ml human recombinant HGF forvarious periods of time as indicated. A : cell lysates wereimmunoblotted with antibodies against phospho-specific Akt or totalAkt, respectively. Molecular size markers (in kDa) are shown. B : dynamic patterns of Akt phosphorylation and activation invarious types of kidney cells after HGF stimulation. Quantitative datafrom densitometric analysis of Western blots were plotted aftercorrection with total Akt.

We found that the Akt phosphorylation and activation induced by HGF indifferent types of kidney cells displayed distinctly dynamic patterns(Fig. 10 ). The activation of Akt kinase by HGF took place in glomerularmesangial cells (RMC) and renal interstitial fibroblasts (NRK-49F) in asimilarly dynamic fashion (Fig. 10 ). The Akt kinase was activatedmarkedly and rapidly (as early as 5 min), but this induction wastransient, and the phosphorylated Akt abundance quickly returned towardbaseline level after 1 h (Fig. 10 ). The dynamics of Akt activationin mIMCD-3 cells and differentiated podocytes were comparable todelayed responses starting at 30 and 60 min, respectively, after HGFstimulation. Conversely, HGF induced rapid and sustained Aktphosphorylation in HKC cells and nondifferentiated podocytes.1 J; }$ t  O3 E" N- ]' R- X2 {% z& K7 |

DISCUSSION. L: t3 G( u" ]% ~; y

HGF and its specific c-met receptor are classically considered asa paired signaling system for mediating signal exchange betweenmesenchyme and epithelia via paracrine actions ( 46, 47 ).This assumption is largely based on observations that the c-metreceptor is predominantly expressed in epithelial cells, whereas itsligand is mainly produced by mesenchyme-derived cells ( 46 ). In this study, we demonstrate that almost all kidneycells in normal adult animals express different levels of c-metreceptor protein. In vitro, all types of kidney cells tested, including glomerular mesangial cells, podocytes, proximal tubular epithelial cells, collecting duct epithelial cells, and interstitial fibroblasts, expressed the functional c-met receptor and responded to HGFstimulation to induce PKB/Akt phosphorylation. Although the presentstudy has not included renal glomerular and vascular endothelial cells, studies elsewhere indicate that endothelial cells also express thefunctional c-met receptor and respond to HGF stimulation( 52 ). Altogether, these results suggest that c-metreceptor expression in the kidney is widespread and ubiquitous. Becauseit is the receptor that determines the target specificity of HGFactions, our results suggest that HGF may have a broader spectrum oftarget cells in the kidney and thereby possess wider implications in kidney structure and function than previously envisioned.

Although the c-met receptor has been demonstrated to be expressed inrenal tubular epithelial cells, its presence and function in othertypes of cells such as interstitial fibroblasts are uncertain. Incontradiction to the present study, earlier observations often suggested an absence of c-met receptors in renal interstitial cells innormal rats ( 26 ). This discrepancy is probablyattributable to the low sensitivity of the immunohistochemical stainingapproach that was employed previously. Consistent with this notion,extremely weak or no staining for the c-met receptor was also observedin the interstitium of mouse kidney in this study (see Fig. 1 ). The finding of ubiquitous expression of c-met in the kidney is supported byseveral lines of evidence. First, c-met receptor protein is detectablein all types of homogenous kidney cell populations via immunoblotting,a more sensitive detection approach (see Fig. 3 ). Second, c-metreceptor expression in the kidney is clearly controlled by Sp1 and Sp3transcription factors (see Figs. 5-8 ), whose expression in turn isubiquitous. Third, all types of kidney cells tested in vitro retain thec-met receptor and undergo a cascade of signal transduction eventsleading to Akt kinase phosphorylation in response to HGF stimulation(see Fig. 10 ). It is of interest to note that despite the very lowabundance of c-met receptors in NRK-49F cells and glomerular mesangialcells, the magnitude of the cellular responses, such as Akt activationafter HGF stimulation, in these cells is compatible with that in othertypes of cells in which the c-met receptor is highly expressed (seeFig. 10 ). This obvious irrelevance of c-met abundance to cellularresponse insinuates that low levels of c-met receptors in these cellsare not a limiting factor for optimal biological actions of HGF.; S3 \3 N! E$ j# J/ o% l) A" u
" m1 ]# h% v! l
Because kidney cells with mesenchymal phenotypes such as interstitialfibroblasts and mesangial cells presumably express HGF ( 52 ), the presence of c-met receptors in these types ofcells suggests an autocrine loop formation with simultaneous expression of both the receptor and its ligand in the same cell. Theseobservations expand the modes of HGF action in normal kidney beyond thewell-described paracrine and endocrine mechanisms. The physiologicalsignificance of HGF autocrine action is largely unknown. It isplausible to speculate that the autocrine signaling of HGF may be oneof the pathways essential for the development and maintenance of normal kidney structure and function. In this regard, previous studies indicate that HGF and c-met are coexpressed in early metanephrogenic mesenchyme, which leads to the promotion of mesenchymal-to-epithelial cell transdifferentiation during nephrogenesis ( 1, 50 ). Similarly, both c-met and HGF are significantlyinduced in rat glomerular mesangial cells in response to interleukin-6stimulation ( 27 ).; q# }* R8 ]. o

In light of the widespread expression of the c-met gene inthe kidney, it is not surprising to find that c-met is expressed in apattern that is overlapped with the ubiquitous Sp family oftranscription factors. The importance of Sp proteins in the constitutive expression of c-met in the kidney is established byseveral lines of observations in this study. These include a tightcorrelation between c-met receptor abundance and the levels of Spproteins in various types of kidney cells (see Fig. 3 ). This intrinsicinterconnection between c-met and Sp proteins is also evident duringpodocyte differentiation (see Fig. 4 ). Hence, cellular endogenous Spprotein level probably is a key molecular determinant for thedifferential expression of c-met in diverse types of kidney cells. Insupport of this, Sp proteins are found to bind to the promoter regionof the c-met gene and to functionally activate c-met transcription. Conversely, deprivation of Sp proteins by a decoy strategy and blockade of Sp binding by chemical antagonist inhibits c-met expression in renal epithelial cells. Although thepresent study uses the human c-met promoter, previous studies by Seoland colleagues ( 44, 45 ) have demonstrated that the two Sp1binding sites in the mouse c-met gene are also critical forestablishing basal c-met expression as well as for modulating induced c-met transcription, suggesting that there is littlemechanistic difference in the regulation of c-met transcription by Sp proteins in different species. Accordingly, arecent report demonstrates that inhibition of c-met expression in humanprimary hepatocytes by IFN- is mediated by decreased binding of Sp1to the c-met promoter ( 41 ). In this context, it shouldalso be noted that Sp1 -knockout embryos were severelydefective in development and all died around day 11 ofgestation ( 29 ), a time point precisely before the death of c-met -knockout embryos at days 13 and 14 ( 2 ).2 f" J. |9 T, Q, E  F4 g8 i' W
; n% m2 f! W# X, G' A& `
The Sp family of transcription factors consists of at least fourmembers with distinct expression patterns and diverse functions in thedifferent types of cells ( 49 ). All four proteins share similar structural features, including a highly conserved DNA bindingdomain that consists of three zinc fingers close to the COOH-terminalregion ( 49 ). Sp1 is the prototype of this family and isexpressed ubiquitously. Sp1 functions as a transcriptional activatorfor a large number of genes implicated in cell-cycle regulation,hormonal activation, and embryogenic development. Sp2 exhibits asignificant structural difference from other members of the Sp familyand does not bind to the GC box but to a GT-rich element in thepromoter region of the T-cell receptor gene ( 19 ). Littleis known regarding Sp2 tissue distribution and its function. AlthoughSp3 is also ubiquitously expressed, the expression of Sp4 is tissuespecific and largely restricted to the brain ( 48 ). Becauseboth Sp3 and Sp1 are often present in the same cell and areindistinguishable in DNA binding specificity, Sp3 is generally considered to be an antagonist for Sp1 by functionally suppressing Sp1-mediated gene activation ( 10, 11, 17 ). However,several reports also suggest a positive regulation of gene expression by Sp3 in different circumstances. For instance, Sp3 has been shown toactivate the promoter of the human 2 (I) collagen gene and the mouse growth-hormone receptor gene ( 8, 13, 53 ). Inthe present study, our data suggest that Sp3 alone activates c-met gene transcription, although to much less extent thanSp1. In addition, Sp3 not only fails to inhibit Sp1-activated c-met gene transcription, but also actually enhancesSp1-induced c-met expression in a synergistic fashion (see Fig. 7 ).These observations underline that members of the Sp family oftranscription factors interact with one another to produce eitherpositive or negative regulation of a particular gene, depending on thecontext of specific promoter and cellular environments.* }, Y+ E8 y! T( e1 Q

An interesting and novel finding in this study is the synergisticaction of Sp1 and Sp3 in activating c-met genetranscription. Apart from the cotransfection data presented in Fig. 7 B, Western blot analysis also reveals that the differencesin c-met levels among diverse types of kidney cells are greater than ineither Sp1 or Sp3 alone (see Fig. 3 ). This suggests a favorableinteraction between cellular Sp1 and Sp3 proteins in establishing theconstitutive expression of c-met in the kidney. In accordance withthis, Sp1 and Sp3 are largely expressed in an identical pattern withcomparable abundances in different types of normal kidney cells (seeFig. 2 ). Although the molecular mechanism that underlies thesynergistic activation of the c-met gene by Sp1 and Sp3remains unknown, physical interaction between two members of the Spfamily may be of importance for such functional cooperation. Sp1 isknown to be capable of forming homotypic interactions that lead tomultimeric complexes ( 38 ), which mediate transcriptionalsynergism among multiple GC boxes. Moreover, many heterotypicinteractions of Sp1 with diverse types of transcription factors, suchas YY1, E2F, and Smad2/3, just to name a few, have been documented( 9, 14, 20, 32, 43 ). Indeed, a direct interaction betweenSp1 and Sp3 in renal epithelial cells has been demonstrated bycoimmunoprecipitation with either Sp1 or Sp3 antibodies (see Fig. 6 ).In addition, supershift experiments using antibodies against either Sp1or Sp3 (see Fig. 5 ) exhibit the presence of an additional complex (SS2)with identical size that presumably consists of Sp1, Sp3, andIgG. Therefore, heterotypic interaction between Sp1 and Sp3occurs in renal epithelial cells and is probably critical for thesynergistic activation of c-met gene transcription.0 L9 q# W4 Z- ?2 ^9 j3 D1 Z3 p4 W

The finding of ubiquitous expression of c-met receptors in varioustypes of renal cells suggests a broader spectrum of target cells forHGF in normal kidney than previously thought. Perhaps moresurprisingly, the functional response to HGF by a particular type ofcells appears to be unrelated to the cellular abundance of c-metreceptors (see Fig. 10 ). These observations lead one to rethink the HGFbiology in the kidney. It now appears certain that HGF signalingpossesses important biological activities not only in tubularepithelial cells, where the c-met receptor is robustly expressed, butalso in the cells with low abundance of c-met receptors, such asglomerular mesangial cells and interstitial fibroblasts. Undoubtedly,one of the great challenges in the future is to determine the exactfunction of HGF signaling in a particular type of renal cells in thecontext of whole kidney in vivo.; N! O: v: D1 x. u7 z$ v6 I
; O5 W. d- c: H! {! f
In summary, the c-met receptor is expressed in normal adult kidney andin diverse types of kidney cells in a ubiquitous fashion. Theconstitutive expression of the c-met gene is closelycorrelated with and primarily mediated by the synergistic actions oftranscription factors Sp1 and Sp3. Physiologically, the c-met receptorin all types of kidney cells tested is functional and initiates acascade of signal transduction events leading to Akt phosphorylation in response to HGF. In view of the fact that the receptor determines thetarget specificity of the ligand, these results suggest that HGF mayhave broader implications in kidney structure and function undervarious physiological and pathological conditions.

ACKNOWLEDGEMENTS

The authors thank Drs. R. Tjian and G. Suske for providing the Sp1and Sp3 expression plasmids.
      【参考文献】: Z1 e- A: U, H$ h2 _
1. Andermarcher, E,Surani MA,andGherardi E. Co-expression of the HGF/SF and c-met genes during early mouse embryogenesis precedes reciprocal expression in adjacent tissues during organogenesis. Dev Genet 18:254-266,1996  .

0 E# }5 H/ ?5 r% m
2. Bladt, F,Riethmacher D,Isenmann S,Aguzzi A,andBirchmeier C. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376:768-771,1995 .5 O* _, q7 }' r, s4 [( s

0 p! C) X7 a* l& V
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 .' R5 O8 Z5 R% B8 Z+ w. p2 b0 J0 r
) p/ i" S, {" l
, c! d8 ^! s  H& \- b# ~' \

4. Chen, Q,DeFrances MC,andZarnegar R. Induction of met proto-oncogene (hepatocyte growth factor receptor) expression during human monocyte-macrophage differentiation. Cell Growth Differ 7:821-832,1996 ." ]- V' ~% J1 M3 {

' I( u& ?) f+ ]" W+ ]9 a. a  |

5. Comoglio, P. Pathway specificity for Met signaling. Nat Cell Biol 3:E161-E162,2001  .& a; I! @" Q( |9 C

% ^' X- k- C. F9 m1 F, {9 e. v* v

6. Courey, AJ,andTjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887-898,1988  .8 M) k/ T7 ^, h+ C4 Y, _9 H
/ U8 ~( _) Y; a, G! P

7. 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 .
2 m4 G' D! g* i5 M- I/ q

8. Denson, LA,Menon RK,Shaufl A,Bajwa HS,Williams CR,andKarpen SJ. TNF- downregulates murine hepatic growth hormone receptor expression by inhibiting Sp1 and Sp3 binding. J Clin Invest 107:1451-1458,2001  .$ ?, e7 K* Q6 U9 P7 P! b3 d. ^. A
0 f# B# A( C+ c, M1 R

9. Galvin, KM,andShi Y. Multiple mechanisms of transcriptional repression by YY1. Mol Cell Biol 17:3723-3732,1997 .
1 C) g6 w$ k& x9 ]' x5 Q: k

10. Ghayor, C,Chadjichristos C,Herrouin JF,Ala-Kokko L,Suske G,Pujol JP,andGalera P. Sp3 represses the Sp1-mediated transactivation of the human COL2A1 gene in primary and de-differentiated chondrocytes. J Biol Chem 276:36881-36895,2001 .5 r6 o  M- A3 I8 W

) O  D3 S4 K0 K& e2 O

11. Hagen, G,Muller S,Beato M,andSuske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13:3843-3851,1994  .% z! b+ \; R) o" J1 k& u9 b5 x& q

& c. B" e! V" J, ?) w5 @/ V

12. Hiromura, K,Monkawa T,Petermann AT,Durvasula RV,andShankland SJ. Insulin is a potent survival factor in mesangial cells: role of the PI3-kinase/Akt pathway. Kidney Int 61:1312-1321,2002  .3 T5 y( P' d3 J; Z  J

0 I3 X( @/ p# Q" c
6 H$ U6 A4 o) b" f3 Z
13. Ihn, H,andTrojanowska M. Sp3 is a transcriptional activator of the human 2 (I) collagen gene. Nucleic Acids Res 25:3712-3717,1997 .( ~0 Y: G' M6 e/ W; z" e
# e: ]. Y/ z2 z0 \

14. Inagaki, Y,Nemoto T,Nakao A,Dijke P,Kobayashi K,Takehara K,andGreenwel P. Interaction between GC box binding factors and Smad proteins modulates cell lineage-specific 2 (I) collagen gene transcription. J Biol Chem 276:16573-16579,2001 .) b* X9 e7 ?1 f
% b8 |' Y  a4 H  y( x. D* H# F8 E# X" v

15. Jackson, SP,MacDonald JJ,Lees-Miller S,andTjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 63:155-165,1990  .- }9 ]( r0 j  n, Z+ w8 H

, N0 U4 x( I5 i
  R; W' I# f) f0 }
16. Jones, KA,Yamamoto KR,andTjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42:559-572,1985  .% z* y& h9 d0 _/ s5 n

9 o) C# a  X7 J+ l2 Z

17. Kennett, SB,Moorefield KS,andHorowitz JM. Sp3 represses gene expression via the titration of promoter-specific transcription factors. J Biol Chem 277:9780-9789,2002 .

18. Kennett, SB,Udvadia AJ,andHorowitz JM. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res 25:3110-3117,1997 .: D4 ~7 f/ T  b% h8 n* ^7 G
) `3 ?# _8 t0 T4 B/ F
1 V, i! Y4 \9 A& ?; N3 g

19. Kingsley, C,andWinoto A. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol 12:4251-4261,1992 .

- ]: Y0 {; u2 S6 ~5 Q

20. Lai, CF,Feng X,Nishimura R,Teitelbaum SL,Avioli LV,Ross FP,andCheng SL. Transforming growth factor- upregulates the 5 integrin subunit expression via Sp1 and Smad signaling. J Biol Chem 275:36400-36406,2000 ., R$ S- f5 {  A
- \0 L4 T( S* Y" B% J+ |
; p0 g8 i! B2 A9 Y$ L
# J7 W! ^( r6 l% Y; `/ E0 q" ^
21. Liu, Y. Hepatocyte growth factor and the kidney. Curr Opin Nephrol Hypertens 11:23-30,2002  .
8 ^2 w; B9 C) R+ c) u6 C. q

22. Liu, Y. Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms. Am J Physiol Renal Physiol 277:F624-F633,1999 .

' U+ p: v2 ]( r: t

23. Liu, Y. The human hepatocyte growth factor receptor gene: complete structural organization and promoter characterization. Gene 215:159-169,1998  .
- f9 s. r( d9 Z
9 f7 H8 d3 y. x# O  J9 ^0 ]$ d

24. 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 .
3 G3 W5 x6 w$ l
- u9 r( \1 h: d: \1 ^! r, d! c+ ^
' o  a4 o* Q; I. [# V
25. Liu, Y,Tolbert EM,Lin L,Thursby MA,Sun AM,Nakamura T,andDworkin LD. Up-regulation 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  .- \9 t; U! q7 O3 P
$ \+ J+ Y: Q1 y. o

26. 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 .

: w! B0 K6 N. U, [7 ~' R
27. 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 .

8 b' U/ B: u( H- v% E- `8 L9 C* o
28. 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  .
. r" l  w9 S5 S
1 m7 v# Z: j; P0 }
' I9 Y. Q  O  n( f, X( P
29. Marin, M,Karis A,Visser P,Grosveld F,andPhilipsen S. Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell 89:619-628,1997  .
' I$ @- ~( G& p  p2 M

' o& g0 \" I4 h
30. Mark, MR,Lokker NA,Zioncheck TF,Luis EA,andGodowski PJ. Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding. J Biol Chem 267:26166-26171,1992 .

9 l# d  u3 K/ ~, e
% F7 v- x) \, F$ a, q3 Y# @: F
31. Matsumoto, K,andNakamura T. Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases. Kidney Int 59:2023-2038,2001  .

1 q; K/ K  E( `! _2 y5 Z7 \9 ~

32. Melnikova, IN,Lin HR,Blanchette AR,andGardner PD. Synergistic transcriptional activation by Sox10 and Sp1 family members. Neuropharmacology 39:2615-2623,2000  .
/ W2 B# O5 ]" ^% A6 d1 ~& }9 R

33. Michalopoulos, GK,andDeFrances MC. Liver regeneration. Science 276:60-66,1997 .

" \1 R) ~% ^; ]* m% c; r2 g& R

34. Mundel, P,Reiser J,Zuniga Mejia Borja A,Pavenstadt H,Davidson GR,Kriz W,andZeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236:248-258,1997  .

35. 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  .- z; K; D$ C; i) {! N- Q/ j
' n8 e; E; B6 y% e; x

36. Nicholson, KM,andAnderson NG. The protein kinase B/Akt signaling pathway in human malignancy. Cell Signal 14:381-395,2002  .
9 t3 k- {% F* X! ^4 [8 a
$ Y! C: a# F5 M9 H) T  z2 T

37. 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 .

9 I4 _0 V" H) J/ m
! |' E- `- p2 S  x* u
38. Pascal, E,andTjian R. Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev 5:1646-1656,1991 .( r6 n) ]. E0 K9 l# u, X8 q* `6 [, [: S

6 l7 [5 k" }, G5 t' L: D
' `2 |$ g2 S& {; e6 q
39. Poncelet, AC,andSchnaper HW. Sp1 and Smad proteins cooperate to mediate transforming growth factor- 1-induced 2 (I) collagen expression in human glomerular mesangial cells. J Biol Chem 276:6983-6992,2001 .# n% v! o* I9 H" f! _8 Y2 [
7 a7 f- v: E. L% ?% u

40. 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 .
/ D% j: ~! S  r

41. 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 down-regulates c-met in primary human hepatocytes. Gastroenterology 122:1020-1034,2002 .
$ A; g9 c! ]# @2 F
& n6 }3 Y6 T& I+ g# `0 N3 ]/ ^4 D# q' r

42. Ray, R,Snyder RC,Thomas S,Koller CA,andMiller DM. Mithramycin blocks protein binding and function of the SV40 early promoter. J Clin Invest 83:2003-2007,1989  .

6 _4 \8 G1 k- |7 p( p
43. Rotheneder, H,Geymayer S,andHaidweger E. Transcription factors of the Sp1 family: interaction with E2F and regulation of the murine thymidine kinase promoter. J Mol Biol 293:1005-1015,1999  .
' t% C. c6 i5 t9 m3 N( o

44. 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  .3 W$ l3 F6 s0 H: j/ V% j

: H: z2 T) R0 ^# }- X" X
4 C; S- k: v. J: d9 L9 @) f, d
45. 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 .

3 R3 K9 C( Z! [- e
46. Sonnenberg, E,Meyer D,Weidner KM,andBirchmeier C. Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development. J Cell Biol 123:223-235,1993 .# c8 Z7 [' g7 y7 Z3 k$ E8 N: z9 g

! N- x- b0 z' M  G$ u+ \
47. Stewart, F. Roles of mesenchymal-epithelial interactions and hepatocyte growth factor-scatter factor (HGF-SF) in placental development. Rev Reprod 1:144-148,1996 .& m# d% h2 a) Y: G$ U
# D/ m  w2 o8 R0 T, A

48. Supp, DM,Witte DP,Branford WW,Smith EP,andPotter SS. Sp4, a member of the Sp1-family of zinc finger transcription factors, is required for normal murine growth, viability, and male fertility. Dev Biol 176:284-299,1996  .+ h; @* g+ x3 p: b+ v/ z' ^
8 Y5 h! k  Q% p, x+ ~
8 t8 ^' i# s6 R1 l- }9 {
+ B  w) c" O4 _  I' d# r! ]' V
49. Suske, G. The Sp-family of transcription factors. Gene 238:291-300,1999  .6 H4 D; J& [; W4 }0 B

2 h- Y4 O8 Y. X* z" l
50. Woolf, AS,Kolatsi-Joannou M,Hardman P,Andermarcher E,Moorby C,Fine LG,Jat PS,Noble MD,andGherardi E. Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros. J Cell Biol 128:171-184,1995 .+ S2 ^! y, b" N
7 g, u( F) |% H5 T  t" a* P
/ [. r; g) t1 S
8 S4 T5 j( R2 J5 l1 @5 Q+ y
51. 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 .8 d' a0 Q& Q% D/ F, L% r
1 \  t$ p+ R# r0 m7 d! F3 a

) n$ h! G# E8 D& @% l2 ?
52. Yo, Y,Morishita R,Yamamoto K,Tomita N,Kida I,Hayashi S,Moriguchi A,Kato S,Matsumoto K,Nakamura T,Higaki J,andOgihara T. Actions of hepatocyte growth factor as a local modulator in the kidney: potential role in pathogenesis of renal disease. Kidney Int 53:50-58,1998  .

% ~6 `4 l$ R- X5 W  Y# w
8 S* v0 Y  m# w0 Y0 q& x
53. Yu, JH,Schwartzbauer G,Kazlman A,andMenon RK. Role of the Sp family of transcription factors in the ontogeny of growth hormone receptor gene expression. J Biol Chem 274:34327-34336,1999 .
/ f. Q0 S3 w; F3 e- X

54. Zarnegar, R,andMichalopoulos GK. The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J Cell Biol 129:1177-1180,1995 .

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