CNP gene expression is activated by Wnt signaling andcorrelates with Wnt4 expres



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发表于 2009-4-21 13:35 |显示全部帖子

作者：KameswaranSurendran,  Theodore C.Simon,作者单位：1 Department of Pediatrics, Department of Molecular Biology and Pharmacology, and Division of Biology and Biomedical Sciences,Washington University School of Medicine, St. Louis, Missouri 63110
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      【摘要】9 p+ m1 v2 v. G* {& O
   C-typenatriuretic peptide (CNP) regulates salt excretion, vascular tone, andfibroblast proliferation and activation. CNP inhibits fibroblastactivation in vitro and fibrosis in vivo, but endogenous CNP gene( Nppc ) expression during tissue fibrosis has not beenreported. We determined that Nppc is induced in renal tubular epithelia and then in interstitial myofibroblasts after unilateral ureteral obstruction (UUO). Induction of Nppc occurred in identical cell populations to those in which Wnt4 is induced after renal injury. In addition, Nppc was activated in Wnt4- expressing cellsduring nephrogenesis. Wnt signaling components -catenin and T cellfactor/lymphoid enhancer binding factor (TCF/LEF) specifically bound tocognate elements in the Nppc proximal promoter. Wnt-4, -catenin, and LEF-1 activated an Nppc transgene incultured cells, and transgene activation by Wnt-4 and LEF-1 wasdependent on the presence of intact cognate elements. These findingssuggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependentmanner after renal injury and thus may contribute to limiting renal fibrosis. ( Q& k. u, q# s! b# M0 d6 J, `
      【关键词】 myofibroblast nephrogenesis tubulointerstitial fibrosis Ctypenatriuretic peptide8 Y  k1 E  A5 y" R2 x# i
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C-TYPE NATRIURETIC PEPTIDE (CNP) is one of a family of three related peptides thatregulate fluid and electrolyte homeostasis ( 39 ).Expression of the CNP gene ( Nppc ) is detected at highest levels in the central nervous system, female reproductive tissues, andbone ( 7, 20 ). While CNP has vasodilatory action,circulating levels of CNP are low, and local autocrine and paracrinefunctions have been proposed ( 5 ). Mice with a targeted Nppc null mutation die during the neonatal period, withsevere dwarfism due to a failure of endochondral ossification( 7 ). One prominent role suggested for CNP is inhibition ofmyofibroblast function.; \7 h$ x; z! j) @/ T

Myofibroblasts are cells that differentiate from fibroblasts aftertissue injury and proliferate and gain smooth muscle cell features suchas the expression of -smooth muscle actin. Myofibroblasts areessential for wound matrix contraction and healing( 46 ) and secrete high levels of collagen, fibronectin, andother extracellular matrix constituents. Myofibroblasts ceaseproliferation and disappear during normal wound healing( 16 ) but can persist during chronic inflammation andinjury. CNP may be part of the mechanisms by which myofibroblastactivation is terminated. CNP inhibits hepatic myofibroblastic stellatecell ( 45 ) and fibroblast ( 6 ) proliferation, and fibroblasts express the CNP receptor, natriuretic peptide receptorB (NPR-B) ( 6 ). CNP administration prevents neointimal fibrosis and arterial smooth muscle cell proliferation following balloon angioplasty ( 15, 47 ). CNP inhibits basicfibroblast growth factor-, platelet-derived growth factor-, andepidermal growth factor-induced arterial smooth muscle cellproliferation ( 36 ). Administration of CNP after renalglomerular injury reduces glomerular fibrosis and limits theproliferation of -smooth muscle actin-positive mesangial cells( 4 ). These experiments demonstrate that exogenous CNPadministration inhibits the proliferation and activation of smoothmuscle cells and myofibroblasts and limits tissue fibrosis. However,expression and regulation of CNP gene ( Nppc ) expressionafter tissue injury have not been reported, and transcriptionalregulation of Nppc has not been extensively studied( 33 ). Here, we examined Nppc expression in awell-characterized mouse model of renal tubulointerstitial fibrosis:unilateral ureteral obstruction (UUO) ( 30 ).$ a- S7 o) L" U. R5 H- Z, I5 O

Ligation of a single ureter results in extensive tubular cell damage,followed by macrophage influx and a fibrotic response ( 9, 10, 30 ). By 1 wk, smooth muscle actin-positive interstitial fibroblasts are apparent. One source of interstitial fibroblasts istransdifferentiation from tubular epithelial cells ( 23 ), which acquire expression of smooth muscle actin and other myofibroblast markers while still part of the tubular structure ( 31 ).Fibrosis continues to progress, with increasing epithelial loss,accumulation of interstitial myofibroblasts, and extracellular matrixdeposition. By 4 wk after ligation, the entire kidney exhibitsextensive fibrosis. We report that Nppc expression wasinduced after UUO, and in identical cell populations to those in which Wnt4 is induced.
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The Wnt genes code for secreted glycoproteins that actnear their site of synthesis and are ligands for the frizzledfamily of receptors ( 34, 51 ). Wnts regulate target genetranscription through multiple intracellular signaling pathways( 29 ), with the best-studied pathway involving an increasein the half-life of cytosolic -catenin ( 2, 53 ). -Catenin regulates gene transcription by translocating to thenucleus and interacting with transcription factors of the T cellfactor/lymphoid enhancer binding factor (TCF/LEF) family ( 2, 3 ). These factors are a subfamily of high-mobility group factorsthat bind to specific DNA sequence elements ( 17, 50 ). -Catenin contains no DNA binding domain but a transcriptionalactivational domain. Wnt-4 is one member of the Wnt family that plays acritical role in genitourinary development. Wnt4 isactivated in induced metanephric mesenchyme and is required formetanephric mesenchymal condensation ( 43 ). Wnt-4 isexpressed in the developing Müllerian structures and is requiredfor female development ( 48 ). Wnt-4 is also expressed inadult reproductive tissues including uterus ( 28 ), ovary( 19 ), and breast ( 14 ). We and others havefound that Wnt-4 is induced after renal injury leading to interstitialfibrosis ( 32, 44 ). Wnt4 is first activated inthe collecting duct epithelium after UUO and then in the accumulatinginterstitial myofibroblasts ( 44 ). We report our findingthat Nppc is induced in identical cell populations and atthe same time as Wnt4 in the injured kidney and test the hypothesis that Wnt signaling regulates Nppc expression.3 A% v, H* I+ M* P1 N4 C2 W

MATERIALS AND METHODS
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Animals, ureteral obstruction, and tissue preparation. Approval was obtained from the Washington University InstitutionalAnimal Care and Use Committee for all experiments involving animals.UUO was performed on anesthetized FVB/N and C57BL6/J mice of 6-7wk of age and was accomplished by surgical cautery of the left renalureter ~15 mm from the renal pelvis. The obstructed kidneys wereharvested at 7 or 14 days after obstruction. Uninjured kidneys wereharvested from 7-wk-old mice. Obstructed kidneys were frozen in OCTembedding medium for in situ hybridization. At least two obstructedkidneys were examined at each time point by in situ hybridization.Mouse embryos at day 17.5 of gestation ( E17.5 )were harvested after timed mating of 129SV/J mice.7 t! ~9 D  p6 M6 W
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RNA in situ hybridization. Wnt4 and Nppc mRNA transcripts were detected inmurine samples by in situ hybridization of tissue sections. Thetemplate used to produce murine Wnt4 riboprobes was a giftfrom Andy McMahon ( 43 ). The in situ Wnt4 antisense riboprobe spans nucleotides 803 to 366 (GenBank NM009523).The murine Nppc exon 2 was amplified by PCR from FVB/N mousegenomic DNA. Primers used to generate the Nppc template were5'-AGGTCCCGAGAACCCCG (sense) and 5'-ATGGAGCCGATCCGGTCC (antisense) togenerate a probe that complements nucleotides 1566-1721 (GenBank D28873 ). The PCR products were ligated into the pZero 2.0 bacterialplasmid (Invitrogen), and the sequence was verified. Radiolabeledantisense riboprobes for in situ hybridization were transcribed usinglinearized cDNA templates, [ - 33 P]UTP (Amersham), andthe Promega in vitro transcription system. In situ hybridization wasperformed utilizing 10- to 12-µm sections of OCT-embedded kidneys asdescribed ( 44 ).2 q% V0 r1 W( \: P  e( o+ |

Transient transfection assays and cell culture. A transgene was constructed to include the Nppc sequencespanning nucleotides 1209 to  56 relative to the start site oftranscription (GenBank U62939, nucleotides 6-1271). The fifty-sixtranscribed nucleotides are outside the CNP open reading frame. The Nppc promoter sequence was amplified by PCR from FVB/N mousegenomic DNA, using primers 5'-TAATGGTACCCATGTCCATCCCAGCAGTCTTCC (sense)and 5'-TGTGAAGCTTGGATTGCCAAGCGAGCACAG (antisense). The amplimer wasdigested with Kpn I and Hin dIII, and ligated intopGL3 Basic vector (Promega) cut with the same enzymes. The resultingplasmid contains the Nppc sequences 1209 to  56, includingthe transcriptional start site, linked to firefly luciferase cDNA and atranscriptional stop site. This transgene is termed 1209CNP.Modifications in the Nppc promoter region of 1209CNPdetailed in Fig. 4 A were introduced utilizing the StratageneQuickChange Site-Directed Mutagenesis Kit according to themanufacturer's instructions. A murine Wnt-4 mammalian cell expressionplasmid was purchased from Upstate Biotechnology and contains the Wnt-4open reading frame fused with the murine Wnt1 transcript5'-untranslated sequences to increase Wnt-4 production ( 53 ). The expression plasmid for full-length -cateninplasmid in pCDNA3 was a gift from Stephen Byers ( 13 ). Theexpression plasmid for full-length LEF-1 was a kind gift from HansClevers. All transfections included plasmid pRL-TK (Promega) as acontrol for transfection efficiency, and this plasmid expresses Renilla luciferase from a viral promoter.; a. _; Z* @& c+ b1 L

RatB1a cells were cultured in DMEM plus 10% fetal bovine serumand nonessential amino acids. NRK52 E cells (ATCC) were cultured inDMEM adjusted to contain 1.5g/l sodium bicarbonate, 4 mM L -glutamine, 1 mM sodium pyruvate, and 5% fetal bovineserum. Transient transfections in RatB1a cells were performed insix-well plates with 8-10 × 10 4 cellsseeded/well 18-24 h before transfection. Transient transfection inNRK52E cells was done in six-well plates with 5 × 10 4 cells seeded/well 18-24 h before transfection. All transient transfections were performed using the Superfect reagent (Qiagen) following procedures recommended by the manufacturer. Briefly, plasmidDNA was incubated with the Superfect reagent in DMEM for 5 min, afterwhich the appropriate culture medium was added. This mixture was thenadded directly to cells from which the medium had been removed. Cellswere covered with the transfection mixture for 2 h, after whichthe solution was replaced with the appropriate culture medium. Celllysates were prepared for expression analysis between 36 and 48 hafter removal of the transfection mixture. Firefly luciferase and Renilla luciferase reporter activities were determined usingDual Luciferase Assay System reagents and protocol (Promega).Luciferase activity was quantified in a luminometer immediately aftercell lyses. The total amount of DNA transfected per well was keptconstant in all transfections performed in a single experiment,utilizing appropriate "empty" expression plasmids. All assays wereperformed in triplicate for each condition in each experiment, and allcell transfection experiments were repeated at least twice.

Gel mobility shift assays. DNA for gel mobility shift assays (GMSAs) was produced by annealing twocomplementary single-strand synthetic oligonucleotides to produce 15-bpdouble-strand DNA (oligomers). An oligomer containing an optimalTCF/LEF binding site: 5'-CC CTTTGAT CTTACC ( 26 ) was radiolabeled with [ - 32 P]ATP using polynucleotidekinase (Roche). GMSAs were performed as described ( 41 )with the following exceptions: 25 ng of herring sperm DNA (Sigma) wereincluded in all reactions instead of poly(dI. dC), and 375 ng of bovine serum albumin and a 128-fold molar excess of competitoroligonucleotide were included in each binding mixture. Three microgramsof nuclear extract from SW480 cells were used per reaction. The SW480nuclear protein extracts were prepared by ultracentrifugation( 11 ). GMSA supershift assays included 0.75 µg ofanti- -catenin antibody ( C19220, Transduction Laboratories) or 0.75 µg of anti-vimentin antibody (V6630, Sigma) in each binding mixture.A 128-fold molar excess of various 15-bp oligomers was utilizedto compete with the radiolabeled optimal TCF/LEF binding site oligomer.These competitor oligomers were derived from the core and flankingsequences corresponding to each of the six potential Tcf/Lef sites inthe 1209CNP transgene (see Fig. 5 A ). Control competitionswere performed using a mutated optimal TCF/LEF binding site oligomer,5'-CC CTTTG GC CTTACC ( 26 ), as well asmutated oligomers corresponding to the modified Nppc Tcf/Lefsites (see Fig. 5 A ).
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Nppc is induced during renal tubulointerstitial disease in cellsthat express Wnt4. Previous studies have defined an antifibrotic role for exogenous CNP( 15, 47 ), but Nppc expression during tissuefibrosis has not been defined. Therefore, we examined the expressionpattern of Nppc during renal tubulointerstitial diseaseprogression after UUO. Nppc mRNA was not detected by in situhybridization in the uninjured kidneys of normal adult mice (Fig. 1 A ). One week after UUO,strong Nppc expression was induced in the renal cortex (Fig. 1 B ). High-power views revealed that expression was confinedto collecting duct epithelial cells (Fig. 1, C - E ). This expression pattern is identicalto that of Wnt4 after UUO ( 44 ), where cortical expression in collecting duct epithelial cells was defined by thepresence of aquaporin-3 ( 44 ). In situ hybridization was performed to compare cellular localization of Wnt4 and Nppc transcripts. One week after UUO, Wnt4 wasinduced in the same collecting duct epithelial cells that support Nppc expression (Fig. 1 ). Nppc may be active in agreater number of the collecting duct epithelial cells than Wnt4, perhaps reflecting paracrine action of this secreted protein. In the uninjured kidney, Wnt4 was detected only inpapillary collecting duct epithelial cells (Fig. 1 F ), incontrast to Nppc, which was not detected. Wnt4 isinitially activated in the collecting duct epithelium and later in thesurrounding interstitial cells ( 44 ). Two weeks after UUO, Wnt4 expression is evident in interstitial myofibroblasts( 44 ), and Nppc activation was observed inidentical cell populations (Fig. 2 ).
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Fig. 1. Nppc is induced in renal tubules 1 wk afterunilateral ureteral obstruction (UUO) and in the same cells in which Wnt4 is induced. RNA in situ hybridization was performed todetermine whether Nppc expression is induced in the murinekidney after UUO. Sections were hybridized with a probe specific for Nppc, then counterstained with hematoxylin and eosin. Nppc mRNA is not detected in the adult uninjured kidney( A ) but is induced in a punctate pattern in the cortex 1 wkafter UUO ( B ). C : higher power view. Comparisonof high-power darkfield view ( D ) and brightfield view( E ) reveals induced Nppc expression is incollecting duct epithelial cells. Adjacent sections were hybridizedwith a specific probe for Wnt4 ( F - I ). A and F, B and G, C and H, D and I, and E and J :images of the same region in adjacent sections. Wnt4 isexpressed in the collecting ducts found in the renal papillae (arrow in F ) of an uninjured mouse kidney, and Wnt4 expression is induced in cortical regions in the 1-wk obstructed kidney( G ). Nppc mRNA expression ( B and C ) and Wnt4 mRNA expression ( G and H ) are evident in strikingly similar patterns on adjacentsections (arrows and arrowheads in B and G ).Arrows in C and H, renal tubules that expressboth Wnt4 and Nppc.3 y( ^- Q: V# G+ i5 J; Q

Fig. 2. Expression of Nppc and Wnt4 isinduced in renal inner medullary myofibroblasts after 2 wk of UUO. Insitu hybridization was performed in adjacent sections of kidneys 2 wkafter UUO to detect Nppc and Wnt4 cellularexpression patterns. A - C : Nppc expression. D - F : Wnt4 expression.Arrows in A and D, inner medullary region withoverlapping Nppc and Wnt4 expression,respectively. B and C : higher power views of theinner medullary region shown in A. E and F : higher power views of C. Expression of both Wnt4 and Nppc is visible in interstitial cells.Arrows in B and C and E and F,interstitial cells that express Nppc and Wnt4,respectively.
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Wnt4 and Nppc are expressed in the same cell populations duringrenal development. The precise temporal and spatial coincidence of Wnt4 and Nppc induction after renal damage suggest the possibilitythat both genes may be induced during renal development at the time Wnt4 is activated. The expression pattern of Nppc mRNA was determined in murine embryonic kidneys at 17.5 days ofgestation ( E17.5 ) and compared with that of Wnt4.Nephrogenesis is initiated when groups of metanephric mesenchymal cellsare induced by the ureteric bud to form condensates, which convert totubular epithelial structures and mature into nephrons. Nephrogenesisin the developing kidney is an ongoing process, and the E17.5 kidney contains nephrons at all stages ofnephrogenesis. Nppc mRNA (Fig. 3, D - F ) wasdetected in the developing kidney in the nephrogenic zone. Wnt4 is expressed in these same cells (Fig. 3, A - C ), as well as in the central stromal cells where Nppc is not expressed (Fig. 3, A vs. D ). Wnt4 expression is initially induced in thecondensing metanephric mesenchyme and remains active during metanephriccondensation and conversion of condensates to renal epithelial vesiclesand then comma- and S-shaped bodies. The earliest detectable Nppc expression during nephrogenesis occurs in metanephricmesenchymal condensates (Fig. 3 F ) in which Wnt4 is also expressed (Fig. 3 C ).$ J2 s2 ~5 ~" h" A& T; r7 O3 _
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Fig. 3. Nppc is expressed during renal development andin the same cells as Wnt4. In situ hybridization for Wnt4 ( A - C ) and Nppc ( D - F ) mRNA expression in sections of mouseembryonic kidneys at 17.5 days of gestation. A and D : darkfield images of adjacent sections. B and E : brightfield images of adjacent sections. Wnt4 is expressed in the central stromal cells (arrow in A ) andin the nephrogenic zone (*). Nppc is also expressed in thenephrogenic zone (*) but not central stroma ( D ). B and E : expression of Wnt4 and Nppc in metanephric mesenchymal condensates in adjacentsections of developing mouse kidneys. C and F :higher power views of B and E, respectively,where the arrows indicate the same metanephric condensate exhibiting Wnt4 and Nppc expression, arrowheads indicate anearby branching ureteric duct, and * indicate the same position ineach section.
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Wnt-4 transactivates the Nppc promoter in cultured cells. Wnt4 and Nppc are expressed in the same cellsafter renal damage, suggesting that Wnt-4 may regulate Nppc expression. Wnt-4 stabilizes cytosolic -catenin in cultured RatB1acells ( 44 ) and might activate Nppc through thispathway in these cells. The RatB1a cells resemble the renalmyofibroblasts in which Wnt4 is activated in that they arenontransformed fibroblasts that express high levels of -smoothmuscle actin protein and collagen 1 (I) mRNA (data notshown). A transgene was constructed from murine Nppc nucleotides 1209 to  56 relative to the start site of transcription linked to the firefly luciferase coding region as a reporter( 1209CNP). Transient transfections were performed in the presence andabsence of a Wnt-4 expression construct to determine the effectof Wnt-4 on Nppc activity (Fig. 4 ). Expression of Wnt-4 resulted in an increase in Nppc transgene activity.
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Fig. 4. Wnt-4 activates the Nppc transgene in culturedmyofibroblasts. An Nppc reporter transgene ( 1209CNP) wasutilized to determine the effect of Wnt-4 on Nppc activation. 1209CNP was constructed with a luciferase reporter drivenby nucleotides 1209 to  53 of the Nppc gene, relative tothe start site of transcription. Cells were treated, with each wellreceiving 1 µg 1209CNP transgene plasmid, 0.25 µg pRL-TK(transfection efficiency control), and 2 µg Wnt-4 expression plasmidor 2 µg control empty expression plasmid. Activity of the transgenein each well was normalized to the activity of pRL-TK, the constitutiveexpression plasmid. Values are normalized to the unstimulated 1209CNPactivity, each bar represents the average of values obtained from 3 wells, and the error bar indicates the upper limit of 1 SD.* Signicantly different ( P t -test.. N: M3 Z0 l) R9 [0 C
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TCF/LEF family factors bind to sites in the Nppc promoter. The possibility that Wnt-4 directly regulates Nppc expression was explored. The canonical Wnt signaling pathway regulates gene transcription through binding of -catenin to TCF/LEFtranscription factors, which recognize specific binding sites in theregulatory sequences of target genes. The start site of Nppc transcription has been defined for the murine gene ( 20 ),and we located six potential TCF/LEF binding sites in the 1,200 nucleotides 5' to the transcriptional start. These sites differed fromthe consensus TCF/LEF binding sequence, 5'-CTTTGWW-3' ( 17, 49, 50 ), by one or two residues (Fig. 5 A ), although the coresequence of site 1 is identical to that of a proven TCF/LEFbinding site in the c- jun regulatory region( 27 ). Thus Wnt-4 might directly activate Nppc transcription through the localization of -catenin bound to aTcf/Lef family member to the Nppc promoter." G5 l3 a3 b* Q4 q" m

Fig. 5. The Nppc promoter contains 6 potential T cellfactor/lymphoid enhancer binding factor (Tcf/Lef) binding sites. A : comparison with a TCF/LEF family core consensus bindingsequence revealed 6 potential Tcf/Lef binding sites in the Nppc promoter nucleotides 1209 to  56 relative to thestart site of transcription. Sites were sequentially numbered toindicate proximal-to-distal location relative to the start site oftranscription. The table lists the TCF/LEF consensus binding sitesequence and the sequences of oligomers containing each Nppc site and an optimal TCF/LEF binding site oligomer used in gel mobilityshift assays (GMSAs). Bold letters indicate variation from the TCF/LEFconsensus sequence in the Nppc sites. "Mutations" arenucleotides substituted in oligomers to destroy TCF/LEF binding. B : GMSAs were performed with the radiolabeled optimalTCF/LEF binding site oligomer indicated in A and nuclearextracts from SW480 cells. Two prominent complexes form, A and B in lane 1. Complex B is the transactivationalcomplex containing TCF/LEF and -catenin, as verified by alteredmobility in the presence of anti- -catenin antibody ( lane3, complex B vs. C ) but not in the presenceof a control antibody (anti-vimentin, lane 2 ). C :affinity of the complex containing TCF/LEF and -catenin for the Nppc binding sites was determined by GMSA competitions. Lanes 1-14 : GMSAs performed in the presence of 128-foldmolar excess of unlabeled oligomers representing potential Tcf/Lefbinding sites present in the Npp c regulatory region or anoptimal TCF/LEF binding site. Lane 1 demonstrates that theinclusion of an excess of unlabeled optimal TCF/LEF binding siteoligomer eliminates formation of complex B. Inclusion of anexcess of unlabeled mutated TCF/LEF binding site oligomer does noteliminate complex B formation. Similar competitions wereperformed with native and mutant oligomers for each of the potential Nppc Tcf/Lef binding sites, as indicated in lanes3-14. Oligomers derived from the native but not mutant Nppc Tcf/Lef sites 1 and 2 couldcompete for complex formation as well as the optimal TCF/LEF bindingsite oligomer.$ n) `1 |# o4 J5 r* _" G  U+ t" P8 h

GMSAs were performed to determine the relative affinity of the sixpotential Nppc Tcf/Lef sites for a TCF- -catenin complex. TCF- -catenin complexes were derived from SW480 cells, a human colonic carcinoma cell line with high levels of endogenous TCF-4 and -catenin ( 27 ). Nuclear extracts from SW480 cells form a ternary complex with TCF/LEF binding sequences, consisting of theoligomer, TCF, and -catenin ( 27 ). This complex formedwith a radiolabeled oligonucleotide containing the optimal TCF/LEF sequence (Fig. 5 A ), as indicated by a specific supershiftwith anti- -catenin antibody (Fig. 5 B, complexB ). An additional, unidentified complex also formed ( complexA ). GMSA competitions were performed with a radiolabeled optimalTCF/LEF binding sequence oligomer and SW480 nuclear extracts todetermine whether complex formation occurred with the Nppc sites. Specific complexes are identified by comparing GMSA thatincluded a 128-fold molar excess of unlabeled optimal binding siteoligomer (Fig. 5 C, lane 1 ) and GMSA that includeda 128-fold molar excess of an oligomer identical except for mutation oftwo residues to abolish TCF/LEF factor binding ( lane 2 ).Complete loss of complex B in the presence of excess oligomer with an authentic TCF/LEF binding site but not in the presenceof excess oligomer without a functional TCF/LEF binding site indicatesthat this complex contains TCF/LEF factors.

GMSA competition assays were performed to determine the relativeaffinity of the complex containing TCF/LEF and -catenin for the Nppc Tcf/Lef binding site oligomers (Fig. 5 C ).Nonradiolabeled oligomers containing the Nppc Tcf/Lef site 1 sequence (Fig. 5 C, lane 3 ) or site 2 sequence ( lane 5 ) competed as well as theoptimal TCF/LEF binding site oligomer ( lane 1 ) for thecomplex containing TCF/LEF and -catenin ( lane 2, complex B ). The nonradiolabeled oligomers containing mutated Nppc Tcf/Lef site 1 sequence ( lane 4 )or mutated Nppc Tcf/Lef site 2 sequence( lane 6 ) demonstrated a significantly lower affinity for thecomplex containing TCF/LEF and -catenin compared with the oligomerscontaining the native sequences of these Nppc Tcf/Lef sites.Thus Nppc Tcf/Lef sites 1 and 2 interact in vitro with a nuclear protein complex containing TCF/LEF and -catenin with similar affinity to that of an optimal site. Nppc sites 3-6 demonstrated significantlyless affinity than the optimal site for complex formation.6 k, |1 w9 k, s2 d2 L. N
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LEF-1 transactivates the Nppc promoter through interaction withTcf/Lef binding sites. The functional significance of the six potential Nppc Tcf/Lef binding sites was determined utilizing transient transfection assays in cultured cells. Transfections were performed in cultured RatB1a cells, which have properties of myofibroblasts, and also a ratkidney tubular epithelial cell line, NRK52E ( 8 ). The renalepithelial cell line and myofibroblast cell line were chosen for thetransfection assays because Wnt4 and Nppc are initially activated in the renal epithelial cells (Fig. 1 ) and thenin interstitial myofibroblasts (Fig. 2 ). The ability of Wnt signalingpathway components to transactivate the 1209CNP transgene was tested in the cultured cell lines. Both -catenin and LEF-1 transactivated the 1209CNP transgene in both cell lines (Fig. 6, A and B ).
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Fig. 6. LEF-1 transactivates the Nppc transgene incultured cells. Transient transfections were utilized to demonstratethat LEF-1 transactivates the 1209CNP transgene in RatB1a cells( A ) and NRK52E cells ( B ). The 1209CNP transgeneis constructed with a luciferase reporter driven by nucleotides 1209to  53 of the Nppc gene. Cells were treated with atransfection mixture containing 1 µg of 1209CNP transgene plasmidplus expression plasmids for Wnt signaling components. All transfectionmixtures contained 0.25 µg of pRL-TK to control for transfectionefficiency and empty expression plasmid to keep the amount of DNAconstant. The transgene was active in both cell lines withoutstimulation ( A and B ). Inclusion of 2 µg -catenin expression plasmid in the transfection mixture increased 1209CNP activity significantly in RatB1a cells ( A,* P B,* P 1209CNP activitysignificantly in RatB1a cells ( A, * P B, * P 1209CNP activity and are expressed asaverage, with error bars indicating SD and significance calculated byStudent's t -test. C and D : mutationsof the Tcf/Lef binding sites reduce 1209CNP activation by LEF-1. Nppc transgene activation by 2 µg of LEF-1 expressionplasmid was determined for 1209CNP and 1209CNP with mutations toabolish Tcf/Lef binding sites. Transfections in RatB1a cells( C ) and in NRK52E cells ( D ) revealed asignificant loss of activation when Tcf/Lef sites 1 and 2 were mutated or when all sites were mutated(* P A and B. E : mutations of the Tcf/Lef binding sites reduce 1209CNPactivation by Wnt-4. Nppc transgene activation by 2 µg ofWnt-4 expression plasmid was determined for 1209CNP and 1209CNPwith mutations to abolish Tcf/Lef binding sites 1 and 2. Transfections in RatB1a cells revealed a significantactivation of the native transgene by Wnt-4 (* P A and B, except that 6 individual wells were utilized toobtain each value.7 k4 y, y: m7 @$ s: [( A7 b
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Transactivation assays were performed with 1209CNP transgenes inwhich the TCF/LEF sites were destroyed by site-directed mutagenesis todetermine whether LEF-1 transactivation of 1209CNP is mediatedthrough the six TCF/LEF elements. LEF-1 transactivation was comparedamong the native transgene, a transgene with sites 1 and 2 mutagenized, and a transgene with all six sitesmutagenized. Sites 1 and 2 demonstratedsignificantly greater Tcf/Lef binding in the GMSA than the other foursites. Mutagenesis was accomplished with a 2-bp change to the coreTCF/LEF consensus sequence of each site, as shown in Fig. 5 A. LEF-1 activation of 1209CNP was significantly reducedin both cell lines by modification of Tcf/Lef sites 1 and 2 (Fig. 6, C and D ) compared with thenative 1209CNP transgene. Mutagenesis of all six sites did not resultin a further decrease in 1209CNP transactivation by LEF-1 (Fig. 6, C and D ). These results indicate sites1 and 2 bind Tcf-Lef complexes in vitro and mediate Nppc transgene activation by LEF-1 in cells.7 @1 P+ f3 u5 d3 C3 g

To determine whether Wnt-4 activation of the Nppc transgenewas dependent on intact Tcf/Lef binding sites, Wnt-4 activation of thenative transgene and the transgene with Tcf/Lef sites 1 and 2 mutagenized was tested in RatB1a cells (Fig. 6 E ). Wnt-4 significantly activated the native transgene butnot the mutagenized transgene. The mutagenized transgene exhibited adecreased transcriptional activity in the absence of Wnt-4 comparedwith the native transgene, consistent with constitutive activity of the -catenin and Tcf-Lef pathways in these cells. We have detectedcytosolic -catenin in these cells ( 44 ), and theartificial -catenin/TCF/LEF target gene TOPFLASH ( 25 )exhibits twice the activity in RatBa1 cells as an identical gene withmutagenized TCF/LEF binding sites (data not shown). We were unable todemonstrate Wnt activation of any gene in NRK52E cells, indicating thatthese cells may have a defect in Wnt signaling components such as the receptor.

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We have identified Nppc as a gene regulated by Wnt-4,LEF-1, and -catenin. The cell transfection assays and GMSAsdemonstrate that the Wnt- -catenin-LEF-1 signaling pathway directlystimulates Nppc through interaction with Tcf/Lef sites1 and 2. Mutagenesis of Tcf/Lef sites 1 and 2 decreased but did not eliminate 1209CNP transactivationby LEF-1, but no additional loss of transactivation occurred when allsix potential Tcf/Lef sites were mutagenized. These results are similarto those reported for TCF/LEF/ -catenin regulation of Axin2, where the promoter contains eight potential Tcf/Lefsites and mutation of all sites together reduces but does not eliminate -catenin activation of an Axin2 transgene( 24 ). TCF/LEF family members bind to DNA and achievetranscriptional regulation of target genes through interaction withcoactivators such as -catenin or corepressors such as grouchohomologues ( 2 ). We observed that LEF-1 alone achievessignificant activation of the Nppc transgene in both celllines, in agreement with previous studies showing that overexpressionof LEF-1 can mimic Wnt signaling ( 3, 38 ). The Nppc Tcf/Lef element binds TCF4 and is activated by LEF-1,but the identity of Tcf/Lef family members in the kidney and theiractivity during injury and repair remain unknown. This pathway plays acritical role in nephrogenesis, where Wnt-4 signaling is required, andpossibly in nephrogenic differentiation because PKD1 is atarget of Wnt -catenin/TCF/LEF signaling.; W. ~/ t, x+ t  A* N
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We have linked the induction of Nppc expression to Wnt4 expression in renal development and after renal injury,in that Nppc is expressed in cells that also express Wnt4. Wnt4 is activated in the collecting ductepithelium and later in the surrounding interstitial cells( 44 ) after renal damage, and Nppc was induced in identical cell populations and at the same time as Wnt4.These results are consistent with regulation of Nppc transcription by Wnt4, because Wnts bind tightly to the cellsurface and are believed to act in an autocrine or paracrine fashion( 34 ). Other Wnts are also present in the kidney and couldpotentially regulate Nppc expression through the samesignaling pathway ( 32 ) in addition to or in conjunctionwith Wnt-4.' q  @4 A0 Y9 B  O/ j' t/ D! V, D1 _
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Nppc activation is not detected in the papillary epithelialcells that express Wnt4 in the normal kidney, implying thatWnt-4 is not sufficient for Nppc activation in the papillarycollecting ducts. Other regulators present in the wounded kidney may berequired in addition to Wnt4 to activate Nppc. Itis also possible that inhibitors of Wnt signaling are active in thenormal kidney to suppress Nppc activation. A role for CNP inwound healing is consistent with activation that is dependent onmediators present only during wounding. Renal damage initiated bynumerous processes results in Wnt4 activation throughout thecollecting ducts ( 44 ), suggesting a role for Wnt-4 incollecting duct function. Wnt-4 regulation of Nppc providesprecedent for involvement of Wnt-4 in regulating other molecules thatcontrol electrolyte balance. It would be of interest to screen forWnt-inducible genes in papillary collecting duct epithelial cells todetect other potential targets, because some of these may be active inthe normal papillary collecting ducts. Wnt-4 may regulate genes in thenormal kidney distinct from those it regulates in response to renalinjury. Similarly, Wnt4 expression in the normal papillarycollecting ducts may be regulated by a mechanism different from the onethat activates Wnt4 expression in response to collectingduct injury. Alternatively, the most distal collecting ducts in thenormal kidney may be subject to a greater degree of stress than therest of the collecting ducts, and Wnt4 expression may be aresponse to recurring mild injuries in these cells.5 v  |$ a! X; E3 v

Nppc induction after injury suggests the presence of apathway for termination of wound healing and myofibroblast activation after injury. Extensive investigation has established that exogenous CNP inhibits myofibroblast proliferation and activation ( 6, 45 ), as well as prevents neointimal fibrosis after arterial injuries and glomerular fibrosis after renal injuries ( 4, 15, 47 ). Renal fibrosis induced by UUO does not resolve due to the sustained and continuing injury caused by permanent ureteral ligation, but Nppc induction may represent an attempt to control theongoing fibrosis. Furthermore, CNP produced in the kidney may circulate and contribute to the vascular phenotype that accompanies chronic renaldisease. CNP levels are increased in the plasma of diabetic rats( 40 ) and humans with chronic renal failure( 22 ). It would be of interest to test the requirement for Nppc in termination of myofibroblast activity after injury.This could be accomplished by comparing the extent of renalinterstitial cell proliferation and amount of extracellular matrixaccumulation after renal injury in normal mice to that in Nppc null mice rescued by CNP transgene expression in thebone ( 7 ). The potential of CNP to modify fibroticprogression could be determined by performing similar experiments inmice treated with exogenous CNP ( 4 ).
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A wider role for CNP as a mediator of Wnt-4 action is suggested bycoincident expression of the two genes at other sites. Both Wnt4 and Nppc are expressed in the growth plateof bone. Proliferating chondrocytes normally exit the cell cycle during endochondral ossification to become prehypertrophic chondrocytes andthen hypertrophic chondrocytes. Misexpression of Wnt-4 in chick embryolimb cartilage elements accelerates cell cycle exit of proliferatingchondrocytes, possibly through activation of the -catenin-LEF-1signal transduction pathway ( 18 ). Nppc mRNA isexpressed in the proliferating chondrocytes and prehypertrophic chondrocytes ( 7 ). Nppc / miceare defective in longitudinal bone growth due to a reduction in therate of maturation of proliferating chondrocytes to hypertrophic chondrocytes ( 7 ). These observations are consistent withWnt-4 regulating Nppc expression during chondrocytematuration. It will be of interest to determine whether themisexpression of Wnt-4 in the chick cartilage elements results inincreased or premature Nppc expression in proliferatingchondrocytes. Wnt4 and Nppc are both expressed inthe endometrium, and levels of both are coincidently regulatedthroughout the murine estrus cycle ( 1, 28, 37 ). It hasbeen suggested that CNP may mediate the tissue remodeling that occursin both wound healing and the estrus cycle ( 20 ). CNP mightalso mediate similar remodeling during renal development, whereconversion of metanephric mesenchyme to epithelium is dependent onWnt-4.

CNP may inhibit cellular proliferation through elevated cGMP signaling.Binding of CNP to its receptor activates the NPR-B intracellularguanylyl cyclase domain, resulting in increased cytosolic cGMP.Increased cGMP activates intracellular signaling pathways that regulatecellular proliferation. Mice with a null mutation in the cGMP-dependentprotein kinase II gene exhibit defective chondrocyte maturation( 35 ), a finding consistent with the phenotype of the Nppc null mutation. In addition, CNP inhibits culturedhepatic myofibroblast cell proliferation through a mechanism dependenton protein kinase G activation by cGMP ( 45 ). CNP may alsoinhibit cellular proliferation through cGMP inhibition of the MAPkinase pathway to limit growth factor stimulation of cell proliferation( 6, 45 ). Another mechanism by which CNP may inhibitcellular proliferation is through activation of the growtharrest-specific homeobox gene Gax and cyclin-dependent kinase inhibitor p21 ( 12, 52 ). CNP induces Gax expression in smooth muscle cells ( 52 ), and overexpressionof Gax inhibits cell proliferation in a p21-dependent manner( 42 ). Mice with a null mutation in the p21 genehave increased renal interstitial cell proliferation and higher numbersof interstitial myofibroblasts 3 days after UUO compared with that ofwild-type mice ( 21 ). It is possible that Nppc activation after renal injury may limit renal interstitial cellproliferation and the extent of fibrosis through activation of p21., |6 B* I. Q& N4 e" ]* k

ACKNOWLEDGEMENTS

The authors thank Jan Kitajewski for the RatB1a cells, KatherineLee and Lora Staloch for technical assistance, and David Wilson andJohn Majors for review of the manuscript.
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