CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury

Kameswaran Surendran1,2 and Theodore C. Simon1,3

1 Department of Pediatrics, 3 Department of Molecular Biology and Pharmacology, and 2 Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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C-type natriuretic peptide (CNP) regulates salt excretion, vascular tone, and fibroblast proliferation and activation. CNP inhibits fibroblast activation in vitro and fibrosis in vivo, but endogenous CNP gene (Nppc) expression during tissue fibrosis has not been reported. 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 cells during nephrogenesis. Wnt signaling components beta -catenin and T cell factor/lymphoid enhancer binding factor (TCF/LEF) specifically bound to cognate elements in the Nppc proximal promoter. Wnt-4, beta -catenin, and LEF-1 activated an Nppc transgene in cultured cells, and transgene activation by Wnt-4 and LEF-1 was dependent on the presence of intact cognate elements. These findings suggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependent manner after renal injury and thus may contribute to limiting renal fibrosis.

myofibroblast; nephrogenesis; tubulointerstitial fibrosis; C-type natriuretic peptide


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C-TYPE NATRIURETIC PEPTIDE (CNP) is one of a family of three related peptides that regulate fluid and electrolyte homeostasis (39). Expression of the CNP gene (Nppc) is detected at highest levels in the central nervous system, female reproductive tissues, and bone (7, 20). While CNP has vasodilatory action, circulating levels of CNP are low, and local autocrine and paracrine functions have been proposed (5). Mice with a targeted Nppc null mutation die during the neonatal period, with severe dwarfism due to a failure of endochondral ossification (7). One prominent role suggested for CNP is inhibition of myofibroblast function.

Myofibroblasts are cells that differentiate from fibroblasts after tissue injury and proliferate and gain smooth muscle cell features such as the expression of alpha -smooth muscle actin. Myofibroblasts are essential for wound matrix contraction and healing (46) and secrete high levels of collagen, fibronectin, and other extracellular matrix constituents. Myofibroblasts cease proliferation and disappear during normal wound healing (16) but can persist during chronic inflammation and injury. CNP may be part of the mechanisms by which myofibroblast activation is terminated. CNP inhibits hepatic myofibroblastic stellate cell (45) and fibroblast (6) proliferation, and fibroblasts express the CNP receptor, natriuretic peptide receptor B (NPR-B) (6). CNP administration prevents neointimal fibrosis and arterial smooth muscle cell proliferation following balloon angioplasty (15, 47). CNP inhibits basic fibroblast growth factor-, platelet-derived growth factor-, and epidermal growth factor-induced arterial smooth muscle cell proliferation (36). Administration of CNP after renal glomerular injury reduces glomerular fibrosis and limits the proliferation of alpha -smooth muscle actin-positive mesangial cells (4). These experiments demonstrate that exogenous CNP administration inhibits the proliferation and activation of smooth muscle cells and myofibroblasts and limits tissue fibrosis. However, expression and regulation of CNP gene (Nppc) expression after tissue injury have not been reported, and transcriptional regulation of Nppc has not been extensively studied (33). Here, we examined Nppc expression in a well-characterized mouse model of renal tubulointerstitial fibrosis: unilateral ureteral obstruction (UUO) (30).

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 is transdifferentiation 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 matrix deposition. By 4 wk after ligation, the entire kidney exhibits extensive fibrosis. We report that Nppc expression was induced after UUO, and in identical cell populations to those in which Wnt4 is induced.

The Wnt genes code for secreted glycoproteins that act near their site of synthesis and are ligands for the frizzled family of receptors (34, 51). Wnts regulate target gene transcription through multiple intracellular signaling pathways (29), with the best-studied pathway involving an increase in the half-life of cytosolic beta -catenin (2, 53). beta -Catenin regulates gene transcription by translocating to the nucleus and interacting with transcription factors of the T cell factor/lymphoid enhancer binding factor (TCF/LEF) family (2, 3). These factors are a subfamily of high-mobility group factors that bind to specific DNA sequence elements (17, 50). beta -Catenin contains no DNA binding domain but a transcriptional activational domain. Wnt-4 is one member of the Wnt family that plays a critical role in genitourinary development. Wnt4 is activated in induced metanephric mesenchyme and is required for metanephric mesenchymal condensation (43). Wnt-4 is expressed in the developing Müllerian structures and is required for female development (48). Wnt-4 is also expressed in adult reproductive tissues including uterus (28), ovary (19), and breast (14). We and others have found that Wnt-4 is induced after renal injury leading to interstitial fibrosis (32, 44). Wnt4 is first activated in the collecting duct epithelium after UUO and then in the accumulating interstitial myofibroblasts (44). We report our finding that Nppc is induced in identical cell populations and at the same time as Wnt4 in the injured kidney and test the hypothesis that Wnt signaling regulates Nppc expression.


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Animals, ureteral obstruction, and tissue preparation. Approval was obtained from the Washington University Institutional Animal Care and Use Committee for all experiments involving animals. UUO was performed on anesthetized FVB/N and C57BL6/J mice of 6-7 wk of age and was accomplished by surgical cautery of the left renal ureter ~15 mm from the renal pelvis. The obstructed kidneys were harvested at 7 or 14 days after obstruction. Uninjured kidneys were harvested from 7-wk-old mice. Obstructed kidneys were frozen in OCT embedding medium for in situ hybridization. At least two obstructed kidneys 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.

RNA in situ hybridization. Wnt4 and Nppc mRNA transcripts were detected in murine samples by in situ hybridization of tissue sections. The template used to produce murine Wnt4 riboprobes was a gift from 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 mouse genomic DNA. Primers used to generate the Nppc template were 5'-AGGTCCCGAGAACCCCG (sense) and 5'-ATGGAGCCGATCCGGTCC (antisense) to generate a probe that complements nucleotides 1566-1721 (GenBank D28873). The PCR products were ligated into the pZero 2.0 bacterial plasmid (Invitrogen), and the sequence was verified. Radiolabeled antisense riboprobes for in situ hybridization were transcribed using linearized cDNA templates, [alpha -33P]UTP (Amersham), and the Promega in vitro transcription system. In situ hybridization was performed utilizing 10- to 12-µm sections of OCT-embedded kidneys as described (44).

Transient transfection assays and cell culture. A transgene was constructed to include the Nppc sequence spanning nucleotides -1209 to +56 relative to the start site of transcription (GenBank U62939, nucleotides 6-1271). The fifty-six transcribed nucleotides are outside the CNP open reading frame. The Nppc promoter sequence was amplified by PCR from FVB/N mouse genomic DNA, using primers 5'-TAATGGTACCCATGTCCATCCCAGCAGTCTTCC (sense) and 5'-TGTGAAGCTTGGATTGCCAAGCGAGCACAG (antisense). The amplimer was digested with KpnI and HindIII, and ligated into pGL3 Basic vector (Promega) cut with the same enzymes. The resulting plasmid contains the Nppc sequences -1209 to +56, including the transcriptional start site, linked to firefly luciferase cDNA and a transcriptional stop site. This transgene is termed -1209CNP. Modifications in the Nppc promoter region of -1209CNP detailed in Fig. 4A were introduced utilizing the Stratagene QuickChange Site-Directed Mutagenesis Kit according to the manufacturer's instructions. A murine Wnt-4 mammalian cell expression plasmid was purchased from Upstate Biotechnology and contains the Wnt-4 open reading frame fused with the murine Wnt1 transcript 5'-untranslated sequences to increase Wnt-4 production (53). The expression plasmid for full-length beta -catenin plasmid in pCDNA3 was a gift from Stephen Byers (13). The expression plasmid for full-length LEF-1 was a kind gift from Hans Clevers. All transfections included plasmid pRL-TK (Promega) as a control for transfection efficiency, and this plasmid expresses Renilla luciferase from a viral promoter.

RatB1a cells were cultured in DMEM plus 10% fetal bovine serum and nonessential amino acids. NRK52 E cells (ATCC) were cultured in DMEM adjusted to contain 1.5g/l sodium bicarbonate, 4 mM L-glutamine, 1 mM sodium pyruvate, and 5% fetal bovine serum. Transient transfections in RatB1a cells were performed in six-well plates with 8-10 × 104 cells seeded/well 18-24 h before transfection. Transient transfection in NRK52E cells was done in six-well plates with 5 × 104 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, plasmid DNA was incubated with the Superfect reagent in DMEM for 5 min, after which the appropriate culture medium was added. This mixture was then added directly to cells from which the medium had been removed. Cells were covered with the transfection mixture for 2 h, after which the solution was replaced with the appropriate culture medium. Cell lysates were prepared for expression analysis between 36 and 48 h after removal of the transfection mixture. Firefly luciferase and Renilla luciferase reporter activities were determined using Dual Luciferase Assay System reagents and protocol (Promega). Luciferase activity was quantified in a luminometer immediately after cell lyses. The total amount of DNA transfected per well was kept constant in all transfections performed in a single experiment, utilizing appropriate "empty" expression plasmids. All assays were performed in triplicate for each condition in each experiment, and all cell transfection experiments were repeated at least twice.

Gel mobility shift assays. DNA for gel mobility shift assays (GMSAs) was produced by annealing two complementary single-strand synthetic oligonucleotides to produce 15-bp double-strand DNA (oligomers). An oligomer containing an optimal TCF/LEF binding site: 5'-CCCTTTGATCTTACC (26) was radiolabeled with [gamma -32P]ATP using polynucleotide kinase (Roche). GMSAs were performed as described (41) with the following exceptions: 25 ng of herring sperm DNA (Sigma) were included in all reactions instead of poly(dI.dC), and 375 ng of bovine serum albumin and a 128-fold molar excess of competitor oligonucleotide were included in each binding mixture. Three micrograms of nuclear extract from SW480 cells were used per reaction. The SW480 nuclear protein extracts were prepared by ultracentrifugation (11). GMSA supershift assays included 0.75 µg of anti-beta -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 utilized to compete with the radiolabeled optimal TCF/LEF binding site oligomer. These competitor oligomers were derived from the core and flanking sequences corresponding to each of the six potential Tcf/Lef sites in the -1209CNP transgene (see Fig. 5A). Control competitions were performed using a mutated optimal TCF/LEF binding site oligomer, 5'-CCCTTTGGCCTTACC (26), as well as mutated oligomers corresponding to the modified Nppc Tcf/Lef sites (see Fig. 5A).


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Nppc is induced during renal tubulointerstitial disease in cells that express Wnt4. Previous studies have defined an antifibrotic role for exogenous CNP (15, 47), but Nppc expression during tissue fibrosis has not been defined. Therefore, we examined the expression pattern of Nppc during renal tubulointerstitial disease progression after UUO. Nppc mRNA was not detected by in situ hybridization in the uninjured kidneys of normal adult mice (Fig. 1A). One week after UUO, strong Nppc expression was induced in the renal cortex (Fig. 1B). High-power views revealed that expression was confined to collecting duct epithelial cells (Fig. 1, C-E). This expression pattern is identical to that of Wnt4 after UUO (44), where cortical expression in collecting duct epithelial cells was defined by the presence of aquaporin-3 (44). In situ hybridization was performed to compare cellular localization of Wnt4 and Nppc transcripts. One week after UUO, Wnt4 was induced in the same collecting duct epithelial cells that support Nppc expression (Fig. 1). Nppc may be active in a greater 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 in papillary collecting duct epithelial cells (Fig. 1F), in contrast to Nppc, which was not detected. Wnt4 is initially activated in the collecting duct epithelium and later in the surrounding interstitial cells (44). Two weeks after UUO, Wnt4 expression is evident in interstitial myofibroblasts (44), and Nppc activation was observed in identical cell populations (Fig. 2).


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Fig. 1.   Nppc is induced in renal tubules 1 wk after unilateral ureteral obstruction (UUO) and in the same cells in which Wnt4 is induced. RNA in situ hybridization was performed to determine whether Nppc expression is induced in the murine kidney 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 wk after UUO (B). C: higher power view. Comparison of high-power darkfield view (D) and brightfield view (E) reveals induced Nppc expression is in collecting duct epithelial cells. Adjacent sections were hybridized with 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 is expressed 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 adjacent sections (arrows and arrowheads in B and G). Arrows in C and H, renal tubules that express both Wnt4 and Nppc.



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Fig. 2.   Expression of Nppc and Wnt4 is induced in renal inner medullary myofibroblasts after 2 wk of UUO. In situ hybridization was performed in adjacent sections of kidneys 2 wk after UUO to detect Nppc and Wnt4 cellular expression patterns. A-C: Nppc expression. D-F: Wnt4 expression. Arrows in A and D, inner medullary region with overlapping Nppc and Wnt4 expression, respectively. B and C: higher power views of the inner 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.

Wnt4 and Nppc are expressed in the same cell populations during renal development. The precise temporal and spatial coincidence of Wnt4 and Nppc induction after renal damage suggest the possibility that 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 of gestation (E17.5) and compared with that of Wnt4. Nephrogenesis is initiated when groups of metanephric mesenchymal cells are induced by the ureteric bud to form condensates, which convert to tubular epithelial structures and mature into nephrons. Nephrogenesis in the developing kidney is an ongoing process, and the E17.5 kidney contains nephrons at all stages of nephrogenesis. Nppc mRNA (Fig. 3, D-F) was detected 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 the condensing metanephric mesenchyme and remains active during metanephric condensation and conversion of condensates to renal epithelial vesicles and then comma- and S-shaped bodies. The earliest detectable Nppc expression during nephrogenesis occurs in metanephric mesenchymal condensates (Fig. 3F) in which Wnt4 is also expressed (Fig. 3C).


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Fig. 3.   Nppc is expressed during renal development and in the same cells as Wnt4. In situ hybridization for Wnt4 (A-C) and Nppc (D-F) mRNA expression in sections of mouse embryonic 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) and in the nephrogenic zone (*). Nppc is also expressed in the nephrogenic zone (*) but not central stroma (D). B and E: expression of Wnt4 and Nppc in metanephric mesenchymal condensates in adjacent sections 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 a nearby branching ureteric duct, and * indicate the same position in each section.

Wnt-4 transactivates the Nppc promoter in cultured cells. Wnt4 and Nppc are expressed in the same cells after renal damage, suggesting that Wnt-4 may regulate Nppc expression. Wnt-4 stabilizes cytosolic beta -catenin in cultured RatB1a cells (44) and might activate Nppc through this pathway in these cells. The RatB1a cells resemble the renal myofibroblasts in which Wnt4 is activated in that they are nontransformed fibroblasts that express high levels of alpha -smooth muscle actin protein and collagen alpha 1(I) mRNA (data not shown). 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 and absence of a Wnt-4 expression construct to determine the effect of 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 cultured myofibroblasts. An Nppc reporter transgene (-1209CNP) was utilized to determine the effect of Wnt-4 on Nppc activation. -1209CNP was constructed with a luciferase reporter driven by nucleotides -1209 to +53 of the Nppc gene, relative to the start site of transcription. Cells were treated, with each well receiving 1 µg -1209CNP transgene plasmid, 0.25 µg pRL-TK (transfection efficiency control), and 2 µg Wnt-4 expression plasmid or 2 µg control empty expression plasmid. Activity of the transgene in each well was normalized to the activity of pRL-TK, the constitutive expression plasmid. Values are normalized to the unstimulated -1209CNP activity, 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 < 0.02) by Student's t-test.

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 beta -catenin to TCF/LEF transcription factors, which recognize specific binding sites in the regulatory 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 from the consensus TCF/LEF binding sequence, 5'-CTTTGWW-3' (17, 49, 50), by one or two residues (Fig. 5A), although the core sequence of site 1 is identical to that of a proven TCF/LEF binding site in the c-jun regulatory region (27). Thus Wnt-4 might directly activate Nppc transcription through the localization of beta -catenin bound to a Tcf/Lef family member to the Nppc promoter.


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Fig. 5.   The Nppc promoter contains 6 potential T cell factor/lymphoid enhancer binding factor (Tcf/Lef) binding sites. A: comparison with a TCF/LEF family core consensus binding sequence revealed 6 potential Tcf/Lef binding sites in the Nppc promoter nucleotides -1209 to +56 relative to the start site of transcription. Sites were sequentially numbered to indicate proximal-to-distal location relative to the start site of transcription. The table lists the TCF/LEF consensus binding site sequence and the sequences of oligomers containing each Nppc site and an optimal TCF/LEF binding site oligomer used in gel mobility shift assays (GMSAs). Bold letters indicate variation from the TCF/LEF consensus sequence in the Nppc sites. "Mutations" are nucleotides substituted in oligomers to destroy TCF/LEF binding. B: GMSAs were performed with the radiolabeled optimal TCF/LEF binding site oligomer indicated in A and nuclear extracts from SW480 cells. Two prominent complexes form, A and B in lane 1. Complex B is the transactivational complex containing TCF/LEF and beta -catenin, as verified by altered mobility in the presence of anti-beta -catenin antibody (lane 3, complex B vs. C) but not in the presence of a control antibody (anti-vimentin, lane 2). C: affinity of the complex containing TCF/LEF and beta -catenin for the Nppc binding sites was determined by GMSA competitions. Lanes 1-14: GMSAs performed in the presence of 128-fold molar excess of unlabeled oligomers representing potential Tcf/Lef binding sites present in the Nppc regulatory region or an optimal TCF/LEF binding site. Lane 1 demonstrates that the inclusion of an excess of unlabeled optimal TCF/LEF binding site oligomer eliminates formation of complex B. Inclusion of an excess of unlabeled mutated TCF/LEF binding site oligomer does not eliminate complex B formation. Similar competitions were performed with native and mutant oligomers for each of the potential Nppc Tcf/Lef binding sites, as indicated in lanes 3-14. Oligomers derived from the native but not mutant Nppc Tcf/Lef sites 1 and 2 could compete for complex formation as well as the optimal TCF/LEF binding site oligomer.

GMSAs were performed to determine the relative affinity of the six potential Nppc Tcf/Lef sites for a TCF-beta -catenin complex. TCF-beta -catenin complexes were derived from SW480 cells, a human colonic carcinoma cell line with high levels of endogenous TCF-4 and beta -catenin (27). Nuclear extracts from SW480 cells form a ternary complex with TCF/LEF binding sequences, consisting of the oligomer, TCF, and beta -catenin (27). This complex formed with a radiolabeled oligonucleotide containing the optimal TCF/LEF sequence (Fig. 5A), as indicated by a specific supershift with anti-beta -catenin antibody (Fig. 5B, complex B). An additional, unidentified complex also formed (complex A). GMSA competitions were performed with a radiolabeled optimal TCF/LEF binding sequence oligomer and SW480 nuclear extracts to determine whether complex formation occurred with the Nppc sites. Specific complexes are identified by comparing GMSA that included a 128-fold molar excess of unlabeled optimal binding site oligomer (Fig. 5C, lane 1) and GMSA that included a 128-fold molar excess of an oligomer identical except for mutation of two 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 presence of excess oligomer without a functional TCF/LEF binding site indicates that this complex contains TCF/LEF factors.

GMSA competition assays were performed to determine the relative affinity of the complex containing TCF/LEF and beta -catenin for the Nppc Tcf/Lef binding site oligomers (Fig. 5C). Nonradiolabeled oligomers containing the Nppc Tcf/Lef site 1 sequence (Fig. 5C, lane 3) or site 2 sequence (lane 5) competed as well as the optimal TCF/LEF binding site oligomer (lane 1) for the complex containing TCF/LEF and beta -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 the complex containing TCF/LEF and beta -catenin compared with the oligomers containing 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 beta -catenin with similar affinity to that of an optimal site. Nppc sites 3-6 demonstrated significantly less affinity than the optimal site for complex formation.

LEF-1 transactivates the Nppc promoter through interaction with Tcf/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 rat kidney tubular epithelial cell line, NRK52E (8). The renal epithelial cell line and myofibroblast cell line were chosen for the transfection assays because Wnt4 and Nppc are initially activated in the renal epithelial cells (Fig. 1) and then in interstitial myofibroblasts (Fig. 2). The ability of Wnt signaling pathway components to transactivate the -1209CNP transgene was tested in the cultured cell lines. Both beta -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 in cultured cells. Transient transfections were utilized to demonstrate that LEF-1 transactivates the -1209CNP transgene in RatB1a cells (A) and NRK52E cells (B). The -1209CNP transgene is constructed with a luciferase reporter driven by nucleotides -1209 to +53 of the Nppc gene. Cells were treated with a transfection mixture containing 1 µg of -1209CNP transgene plasmid plus expression plasmids for Wnt signaling components. All transfection mixtures contained 0.25 µg of pRL-TK to control for transfection efficiency and empty expression plasmid to keep the amount of DNA constant. The transgene was active in both cell lines without stimulation (A and B). Inclusion of 2 µg beta -catenin expression plasmid in the transfection mixture increased -1209CNP activity significantly in RatB1a cells (A, *P < 0.003) and NRK52E cells (B, *P < 0.004). Inclusion of 1 µg of LEF-1 expression plasmid in the transfection mixture increased -1209CNP activity significantly in RatB1a cells (A, *P < 0.0003) and NRK52E cells (B, *P < 0.0002). Transfections were performed in triplicate for each condition, and the experiment was repeated twice with similar results. Values are normalized to the unstimulated -1209CNP activity and are expressed as average, with error bars indicating SD and significance calculated by Student's t-test. C and D: mutations of the Tcf/Lef binding sites reduce -1209CNP activation by LEF-1. Nppc transgene activation by 2 µg of LEF-1 expression plasmid was determined for -1209CNP and -1209CNP with mutations to abolish Tcf/Lef binding sites. Transfections in RatB1a cells (C) and in NRK52E cells (D) revealed a significant loss of activation when Tcf/Lef sites 1 and 2 were mutated or when all sites were mutated (*P < 0.01). The LEF-1-induced fold-activation of each transgene was calculated by setting the unstimulated activity of each transgene at 1 unit of activity. Values are expressed and error and significance calculated as for A and B. E: mutations of the Tcf/Lef binding sites reduce -1209CNP activation by Wnt-4. Nppc transgene activation by 2 µg of Wnt-4 expression plasmid was determined for -1209CNP and -1209CNP with mutations to abolish Tcf/Lef binding sites 1 and 2. Transfections in RatB1a cells revealed a significant activation of the native transgene by Wnt-4 (* P < 0.003) but not the mutagenized transgene. Transgene activity is normalized to the activity of the unstimulated native transgene. Values are expressed and error and significance calculated as for A and B, except that 6 individual wells were utilized to obtain each value.

Transactivation assays were performed with -1209CNP transgenes in which the TCF/LEF sites were destroyed by site-directed mutagenesis to determine whether LEF-1 transactivation of -1209CNP is mediated through the six TCF/LEF elements. LEF-1 transactivation was compared among the native transgene, a transgene with sites 1 and 2 mutagenized, and a transgene with all six sites mutagenized. Sites 1 and 2 demonstrated significantly greater Tcf/Lef binding in the GMSA than the other four sites. Mutagenesis was accomplished with a 2-bp change to the core TCF/LEF consensus sequence of each site, as shown in Fig. 5A. LEF-1 activation of -1209CNP was significantly reduced in both cell lines by modification of Tcf/Lef sites 1 and 2 (Fig. 6, C and D) compared with the native -1209CNP transgene. Mutagenesis of all six sites did not result in a further decrease in -1209CNP transactivation by LEF-1 (Fig. 6, C and D). These results indicate sites 1 and 2 bind Tcf-Lef complexes in vitro and mediate Nppc transgene activation by LEF-1 in cells.

To determine whether Wnt-4 activation of the Nppc transgene was dependent on intact Tcf/Lef binding sites, Wnt-4 activation of the native transgene and the transgene with Tcf/Lef sites 1 and 2 mutagenized was tested in RatB1a cells (Fig. 6E). Wnt-4 significantly activated the native transgene but not the mutagenized transgene. The mutagenized transgene exhibited a decreased transcriptional activity in the absence of Wnt-4 compared with the native transgene, consistent with constitutive activity of the beta -catenin and Tcf-Lef pathways in these cells. We have detected cytosolic beta -catenin in these cells (44), and the artificial beta -catenin/TCF/LEF target gene TOPFLASH (25) exhibits twice the activity in RatBa1 cells as an identical gene with mutagenized TCF/LEF binding sites (data not shown). We were unable to demonstrate Wnt activation of any gene in NRK52E cells, indicating that these cells may have a defect in Wnt signaling components such as the receptor.


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ABSTRACT
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REFERENCES

We have identified Nppc as a gene regulated by Wnt-4, LEF-1, and beta -catenin. The cell transfection assays and GMSAs demonstrate that the Wnt-beta -catenin-LEF-1 signaling pathway directly stimulates Nppc through interaction with Tcf/Lef sites 1 and 2. Mutagenesis of Tcf/Lef sites 1 and 2 decreased but did not eliminate -1209CNP transactivation by LEF-1, but no additional loss of transactivation occurred when all six potential Tcf/Lef sites were mutagenized. These results are similar to those reported for TCF/LEF/beta -catenin regulation of Axin2, where the promoter contains eight potential Tcf/Lef sites and mutation of all sites together reduces but does not eliminate beta -catenin activation of an Axin2 transgene (24). TCF/LEF family members bind to DNA and achieve transcriptional regulation of target genes through interaction with coactivators such as beta -catenin or corepressors such as groucho homologues (2). We observed that LEF-1 alone achieves significant activation of the Nppc transgene in both cell lines, in agreement with previous studies showing that overexpression of 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 their activity during injury and repair remain unknown. This pathway plays a critical role in nephrogenesis, where Wnt-4 signaling is required, and possibly in nephrogenic differentiation because PKD1 is a target of Wnt beta -catenin/TCF/LEF signaling.

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 duct epithelium 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 cell surface and are believed to act in an autocrine or paracrine fashion (34). Other Wnts are also present in the kidney and could potentially regulate Nppc expression through the same signaling pathway (32) in addition to or in conjunction with Wnt-4.

Nppc activation is not detected in the papillary epithelial cells that express Wnt4 in the normal kidney, implying that Wnt-4 is not sufficient for Nppc activation in the papillary collecting ducts. Other regulators present in the wounded kidney may be required in addition to Wnt4 to activate Nppc. It is also possible that inhibitors of Wnt signaling are active in the normal kidney to suppress Nppc activation. A role for CNP in wound healing is consistent with activation that is dependent on mediators present only during wounding. Renal damage initiated by numerous processes results in Wnt4 activation throughout the collecting ducts (44), suggesting a role for Wnt-4 in collecting duct function. Wnt-4 regulation of Nppc provides precedent for involvement of Wnt-4 in regulating other molecules that control electrolyte balance. It would be of interest to screen for Wnt-inducible genes in papillary collecting duct epithelial cells to detect other potential targets, because some of these may be active in the normal papillary collecting ducts. Wnt-4 may regulate genes in the normal kidney distinct from those it regulates in response to renal injury. Similarly, Wnt4 expression in the normal papillary collecting ducts may be regulated by a mechanism different from the one that activates Wnt4 expression in response to collecting duct injury. Alternatively, the most distal collecting ducts in the normal kidney may be subject to a greater degree of stress than the rest of the collecting ducts, and Wnt4 expression may be a response to recurring mild injuries in these cells.

Nppc induction after injury suggests the presence of a pathway 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 the ongoing fibrosis. Furthermore, CNP produced in the kidney may circulate and contribute to the vascular phenotype that accompanies chronic renal disease. 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 renal interstitial cell proliferation and amount of extracellular matrix accumulation after renal injury in normal mice to that in Nppc null mice rescued by CNP transgene expression in the bone (7). The potential of CNP to modify fibrotic progression could be determined by performing similar experiments in mice treated with exogenous CNP (4).

A wider role for CNP as a mediator of Wnt-4 action is suggested by coincident expression of the two genes at other sites. Both Wnt4 and Nppc are expressed in the growth plate of bone. Proliferating chondrocytes normally exit the cell cycle during endochondral ossification to become prehypertrophic chondrocytes and then hypertrophic chondrocytes. Misexpression of Wnt-4 in chick embryo limb cartilage elements accelerates cell cycle exit of proliferating chondrocytes, possibly through activation of the beta -catenin-LEF-1 signal transduction pathway (18). Nppc mRNA is expressed in the proliferating chondrocytes and prehypertrophic chondrocytes (7). Nppc-/- mice are defective in longitudinal bone growth due to a reduction in the rate of maturation of proliferating chondrocytes to hypertrophic chondrocytes (7). These observations are consistent with Wnt-4 regulating Nppc expression during chondrocyte maturation. It will be of interest to determine whether the misexpression of Wnt-4 in the chick cartilage elements results in increased or premature Nppc expression in proliferating chondrocytes. Wnt4 and Nppc are both expressed in the endometrium, and levels of both are coincidently regulated throughout the murine estrus cycle (1, 28, 37). It has been suggested that CNP may mediate the tissue remodeling that occurs in both wound healing and the estrus cycle (20). CNP might also mediate similar remodeling during renal development, where conversion of metanephric mesenchyme to epithelium is dependent on Wnt-4.

CNP may inhibit cellular proliferation through elevated cGMP signaling. Binding of CNP to its receptor activates the NPR-B intracellular guanylyl cyclase domain, resulting in increased cytosolic cGMP. Increased cGMP activates intracellular signaling pathways that regulate cellular proliferation. Mice with a null mutation in the cGMP-dependent protein kinase II gene exhibit defective chondrocyte maturation (35), a finding consistent with the phenotype of the Nppc null mutation. In addition, CNP inhibits cultured hepatic myofibroblast cell proliferation through a mechanism dependent on protein kinase G activation by cGMP (45). CNP may also inhibit cellular proliferation through cGMP inhibition of the MAP kinase pathway to limit growth factor stimulation of cell proliferation (6, 45). Another mechanism by which CNP may inhibit cellular proliferation is through activation of the growth arrest-specific homeobox gene Gax and cyclin-dependent kinase inhibitor p21 (12, 52). CNP induces Gax expression in smooth muscle cells (52), and overexpression of Gax inhibits cell proliferation in a p21-dependent manner (42). Mice with a null mutation in the p21 gene have increased renal interstitial cell proliferation and higher numbers of interstitial myofibroblasts 3 days after UUO compared with that of wild-type mice (21). It is possible that Nppc activation after renal injury may limit renal interstitial cell proliferation and the extent of fibrosis through activation of p21.


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

This work was supported by the Pharmacia/Washington University Biomedical Research Program.

Address for reprint requests and other correspondence: T. C. Simon, Washington Univ. School of Medicine, Dept. of Pediatrics, Campus Box 8208, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: simon_t{at}kids.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 10, 2002;10.1152/ajprenal.00343.2002

Received 24 September 2002; accepted in final form 3 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acuff, CG, Huang H, and Steinhelper ME. Estradiol induces C-type natriuretic peptide gene expression in mouse uterus. Am J Physiol Heart Circ Physiol 273: H2672-H2677, 1997[Abstract/Free Full Text].

2.   Barker, N, Morin PJ, and Clevers H. The Yin-Yang of TCF/beta -catenin signaling. Adv Cancer Res 77: 1-24, 2000[ISI][Medline].

3.   Behrens, J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, and Birchmeier W. Functional interaction of beta -catenin with the transcription factor LEF-1. Nature 382: 638-642, 1996[ISI][Medline].

4.   Canaan-Kuhl, S, Ostendorf T, Zander K, Koch KM, and Floege J. C-type natriuretic peptide inhibits mesangial cell proliferation and matrix accumulation in vivo. Kidney Int 53: 1143-1151, 1998[ISI][Medline].

5.   Chen, HH, and Burnett JC, Jr. C-type natriuretic peptide: the endothelial component of the natriuretic peptide system. J Cardiovasc Pharmacol 32: S22-S28, 1998[ISI][Medline].

6.   Chrisman, TD, and Garbers DL. Reciprocal antagonism coordinates C-type natriuretic peptide and mitogen-signaling pathways in fibroblasts. J Biol Chem 274: 4293-4299, 1999[Abstract/Free Full Text].

7.   Chusho, H, Tamura N, Ogawa Y, Yasoda A, Suda M, Miyazawa T, Nakamura K, Nakao K, Kurihara T, Komatsu Y, Itoh H, Tanaka K, Saito Y, and Katsuki M. Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc Nat Acad Sci USA 98: 4016-4021, 2001[Abstract/Free Full Text].

8.   De Larco, J, and Todaro G. Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation. J Cell Physiol 94: 335-342, 1978[ISI][Medline].

9.   Diamond, JR. Macrophages and progressive renal disease in experimental hydronephrosis. Am J Kid Dis 26: 133-140, 1995[ISI][Medline].

10.   Diamond, JR, van Goor H, Ding G, and Engelmyer E. Myofibroblasts in experimental hydronephrosis. Am J Pathol 146: 121-129, 1995[Abstract].

11.   Dignam, JD, Lebovitz RM, and Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nuc Acids Res 11: 1475-1489, 1983[Abstract].

12.   Doi, K, Ikeda T, Itoh H, Ueyama K, Hosoda K, Ogawa Y, Yamashita J, Chun TH, Inoue M, Masatsugu K, Sawada N, Fukunaga Y, Saito T, Sone M, Yamahara K, Kook H, Komeda M, Ueda M, and Nakao K. C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization. Arteriosclerosis Thromb Vasc Biol 21: 930-936, 2001[Abstract/Free Full Text].

13.   Easwaran, V, Song V, Polakis P, and Byers S. The ubiquitin-proteasome pathway and serine kinase activity modulate adenomatous polyposis coli protein-mediated regulation of beta -catenin-lymphocyte enhancer-binding factor signaling. J Biol Chem 274: 16641-16645, 1999[Abstract/Free Full Text].

14.   Edwards, PA. Control of the three-dimensional growth pattern of mammary epithelium: role of genes of the Wnt and erbB families studied using reconstituted epithelium. Biochem Soc Symp 63: 21-34, 1998[Medline].

15.   Furuya, M, Miyazaki T, Honbou N, Kawashima K, Ohno T, Tanaka S, Kangawa K, and Matsuo H. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Ann NY Acac Sci 748: 517-523, 1995[Abstract].

16.   Gabbiani, G. The cellular derivation and the life span of the myofibroblast. Pathol Res Prac 192: 708-711, 1996[ISI].

17.   Giese, K, Amsterdam A, and Grosschedl R. DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1. Genes Dev 5: 2567-2578, 1991[Abstract].

18.   Hartmann, C, and Tabin CJ. Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development 127: 3141-3159, 2000[Abstract/Free Full Text].

19.   Hsieh, M, Johnson MA, Greenberg NM, and Richards JS. Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary. Endocrinology 143: 898-908, 2002[Abstract/Free Full Text].

20.   Huang, H, Acuff CG, and Steinhelper ME. Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide. Am J Physiol Heart Circ Physiol 271: H1565-H1575, 1996[Abstract/Free Full Text].

21.   Hughes, J, Brown P, and Shankland SJ. Cyclin kinase inhibitor p21CIP1/WAF1 limits interstitial cell proliferation following ureteric obstruction. Am J Physiol Renal Physiol 277: F948-F956, 1999[Abstract/Free Full Text].

22.   Igaki, T, Itoh H, Suga S, Hama N, Ogawa Y, Komatsu Y, Mukoyama M, Sugawara A, Yoshimasa T, Tanaka I, and Nakao K. C-type natriuretic peptide in chronic renal failure and its action in humans. Kidney Int 55: S144-S147, 1996.

23.   Iwano, M, Plieth D, Danoff TM, Xue C, Okada H, and Neilson EG. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110: 341-350, 2002[Abstract/Free Full Text].

24.   Jho, EH, Zhang T, Domon C, Joo CK, Freund JN, and Costantini F. Wnt/beta -catenin/Tcf signaling induces the transcription of axin2, a negative regulator of the signaling pathway. Mol Cell Biol 22: 1172-1183, 2002[Abstract/Free Full Text].

25.   Kim, K, Pang KM, Evans M, and Hay ED. Overexpression of beta -catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators. Mol Biol Cell 11: 3509-3523, 2000[Abstract/Free Full Text].

26.   Korinek, V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, and Clevers H. Constitutive transcriptional activation by a beta -catenin-Tcf complex in APC-/- colon carcinoma. Science 275: 1784-1787, 1997[Abstract/Free Full Text].

27.   Mann, B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, and Hanski C. Target genes of beta -catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Nat Acad Sci USA 96: 1603-1608, 1999[Abstract/Free Full Text].

28.   Miller, C, Pavlova A, and Sassoon DA. Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech Dev 76: 91-99, 1998[ISI][Medline].

29.   Miller, JR, Hocking AM, Brown JD, and Moon RT. Mechanism and function of signal transduction by the Wnt/beta -catenin and Wnt/Ca2+ pathways. Oncogene 18: 7860-7872, 1999[ISI][Medline].

30.   Nagle, RB, and Bulger RE. Unilateral obstructive nephropathy in the rabbit. II. Late morphologic changes. Lab Invest 38: 270-278, 1978[ISI][Medline].

31.   Ng, YY, Huang TP, Yang WC, Chen ZP, Yang AH, Mu W, Nikolic-Paterson DJ, Atkins RC, and Lan HY. Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats. Kidney Int 54: 864-876, 1998[ISI][Medline].

32.   Nguyen, HT, Thomson AA, Kogan BA, Baskin LS, and Cunha GR. Expression of the Wnt gene family during late nephrogenesis and complete ureteral obstruction. Lab Invest 79: 647-658, 1999[ISI][Medline].

33.   Ohta, S, Shimekake Y, and Nagata K. Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter. Eur J Biochem 242: 460-466, 1996[Abstract].

34.   Papkoff, J, and Schryver B. Secreted int-1 protein is associated with the cell surface. Mol Cell Biol 10: 2723-2730, 1990[ISI][Medline].

35.   Pfeifer, A, Aszodi A, Seidler U, Ruth P, Hofmann F, and Fassler R. Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science 274: 2082-2086, 1996[Abstract/Free Full Text].

36.   Porter, JG, Catalano R, McEnroe G, Lewicki JA, and Protter AA. C-type natriuretic peptide inhibits growth factor-dependent DNA synthesis in smooth muscle cells. Am J Physiol Cell Physiol 263: C1001-C1006, 1992[Abstract/Free Full Text].

37.   Reis, AM, Jankowski M, Mukaddam-Daher S, Tremblay J, Dam TV, and Gutkowska J. Regulation of the natriuretic peptide system in rat uterus during the estrous cycle. J Endocrinol 153: 345-355, 1997[Abstract].

38.   Riese, J, Yu X, Munnerlyn A, Eresh S, Hsu SC, Grosschedl R, and Bienz M. LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88: 777-787, 1997[ISI][Medline].

39.   Rosenzweig, A, and Seidman CE. Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 60: 229-255, 1991[ISI][Medline].

40.   Shin, SJ, Wen JD, Lee YJ, Chen IH, and Tsai JH. Increased C-type natriuretic peptide mRNA expression in the kidney of diabetic rats. J Endocrinol 158: 35-42, 1998[Abstract/Free Full Text].

41.   Simon, TC, Cho A, Tso P, and Gordon JI. Suppressor and activator functions mediated by a repeated heptad sequence in the liver fatty acid binding protein gene (Fabpl). J Biol Chem 272: 10652-10663, 1997[Abstract/Free Full Text].

42.   Smith, RC, Branellec D, Gorski DH, Guo K, Perlman H, Dedieu JF, Pastore C, Mahfoudi A, Denefle P, Isner JM, and Walsh K. p21CIP1-mediated inhibition of cell proliferation by overexpression of the Gax homeodomain gene. Genes Dev 11: 1674-1689, 1997[Abstract].

43.   Stark, K, Vainio S, Vassileva G, and McMahon AP. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature 372: 679-683, 1994[ISI][Medline].

44.   Surendran, K, McCaul SP, and Simon TC. A role for Wnt-4 in renal fibrosis. Am J Physiol Renal Physiol 282: F431-F441, 2002[Abstract/Free Full Text].

45.   Tao, J, Mallat A, Gallois C, Belmadani S, Mery PF, Nhieu JT, Pavoine C, and Lotersztajn S. Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells. J Biol Chem 274: 23761-23769, 1999[Abstract/Free Full Text].

46.   Tomasek, JJ, Gabbiani G, Hinz B, Chaponnier C, and Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3: 349-363, 2002[ISI][Medline].

47.   Ueno, H, Haruno A, Morisaki N, Furuya M, Kangawa K, Takeshita A, and Saito Y. Local expression of C-type natriuretic peptide markedly suppresses neointimal formation in rat injured arteries through an autocrine/paracrine loop. Circulation 96: 2272-2279, 1997[Abstract/Free Full Text].

48.   Vainio, S, Heikkila M, Kispert A, Chin N, and McMahon AP. Female development in mammals is regulated by Wnt-4 signaling. Nature 397: 405-409, 1999[ISI][Medline].

49.   Van de Wetering, M, Cavallo R, Dooijes D, van Beest M, van Es J, Loureiro J, Ypma A, Hursh D, Jones T, Bejsovec A, Peifer M, Mortin M, and Clevers H. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88: 789-799, 1997[ISI][Medline].

50.   Van de Wetering, M, and Clevers H. Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. EMBO J 11: 3039-3044, 1992[Abstract].

51.   Wodarz, A, and Nusse R. Mechanisms of Wnt signaling in development. Ann Rev Cell Devel Biol 14: 59-88, 1998[ISI][Medline].

52.   Yamashita, J, Itoh H, Ogawa Y, Tamura N, Takaya K, Igaki T, Doi K, Chun TH, Inoue M, Masatsugu K, and Nakao K. Opposite regulation of Gax homeobox expression by angiotensin II and C-type natriuretic peptide. Hypertension 29: 381-387, 1997[Abstract/Free Full Text].

53.   Young, CS, Kitamura M, Hardy S, and Kitajewski J. Wnt-1 induces growth, cytosolic beta -catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts. Mol Cell Biol 18: 2474-2485, 1998[Abstract/Free Full Text].


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