The Polycystic Kidney Disease-1 Promoter Is a Target of the β-Catenin/T-cell Factor Pathway*

Polycystic kidney disease (PKD) results from loss-of-function mutations in the PKD1 gene. There are also reports showing abnormally high levels of PKD1 expression in cystic epithelial cells. At present, nothing is known about the molecular mechanisms regulating the normal expression of thePKD1 gene or whether transcriptional disregulation of the PKD1 gene has a role in cyst formation. We have analyzed a 3.3-kb 5′-proximal portion of the human PKD1gene. Sequence analysis revealed the presence of consensus sequences for numerous transactivating factors, including four T-cell factor (TCF) binding elements (TBEs). Transcriptional activity of the 3.3-kb fragment and a series of deletion constructs was assayed in HEK293T cells. A 2.0-kb proximal promoter region containing one of the four TBEs (TBE1) was inducible up to 6-fold by cotransfection with β-catenin. β-catenin-mediated induction was inhibited by dominant-negative TCF and by deletion of the TBE1 sequence. 15- or 109-bp sequences containing the TBE1 site, when cloned upstream of a minimal promoter, were shown to respond to β-catenin induction. Gel shift assays confirmed that the TBE1 site is capable of forming complexes with TCF and β-catenin. To determine whether expression of the endogenous PKD1 gene responds to β-catenin, HT1080 cells were treated with LiCl, and HeLa cells were stably transfected with β-catenin. In both cases, endogenous PKD1 mRNA levels were elevated in response to these treatments. Taken together, these studies define an active PKD1 promoter region and suggest that the PKD1 gene is a target of the β-catenin/TCF pathway.

Autosomal dominant polycystic kidney disease (ADPKD) 1 is a very common inherited disease worldwide, having a gene frequency of 1 in 200 -1,000 individuals and causing 6 -9% of all end-stage renal disease (1,2). Mutations in the PKD1 gene are responsible for the vast majority (85-90%) of ADPKD cases (3,4). PKD is characterized by the neoplastic growth of tubular epithelial cells, which gives rise to the formation of kidneys containing numerous fluid-filled cysts and ultimately to renal failure.
In situ and Northern hybridization and immunocytochemical analyses have shown that the PKD1 gene is widely expressed in a number of embryonic and adult tissues and organs, including the kidney (5)(6)(7)(8)(9)(10). Targeted deletion of the PKD1 gene has shown that it is important for renal, pancreatic, cardiovascular, and skeletal development (9,(11)(12)(13)(14). However, there are conflicting observations as to whether PKD results from abnormally decreased or increased expression of the PKD1 gene (15). On the one hand, there is evidence that loss-of-function germ line mutations can lead to haploinsufficiency at the PKD1 locus (16,17) and additional evidence for a two-hit gene inactivation mechanism as a cause of PKD (18 -20). On the other hand, there is immunocytochemical evidence for overexpression of the protein product of the PKD1 gene, polycystin-1, in cystlining epithelial cells of polycystic kidneys (15). Furthermore, transgenic overexpression of a functional PKD1 gene can cause PKD (21). At the present time, there is no information concerning the mechanisms regulating transcription of the PKD1 gene.
␤-catenin is a major component of adherens junctions, linking the actin cytoskeleton to members of the cadherin family of transmembrane cell-cell adhesion receptors (22)(23)(24). Together with ␣and ␥-catenin, ␤-catenin binds to the cytosolic domain of E-cadherin to form a complex that is necessary for adhesion to occur. In addition, soluble ␤-catenin can translocate to the nucleus, where it associates with members of the TCF/LEF family of high mobility group (HMG)-box factors to alter the expression of certain target genes (25). By playing a dual role, a structural role at cell-cell junctions and a regulatory role in the nucleus, ␤-catenin can transduce changes in cell adhesion and junction formation to control transmembrane signaling and gene expression. The level of soluble ␤-catenin in the cell is regulated by its association with the tumor suppressor molecule adenomatous polyposis coli (APC), axin, and glycogen synthase kinase-3␤ (GSK-3␤). Phosphorylation of ␤-catenin by the APC-axin-GSK-3␤ complex leads to its degradation by the ubiquitin-proteasome system. The failure of this degradation in cells expressing mutated ␤-catenin leads to the accumulation of soluble ␤-catenin that can result in activation of oncogenic ␤-catenin-responsive genes (23)(24)(25).
A number of observations have implicated ␤-catenin in the pathogenesis of PKD. Transgenic overexpression of either ␤-catenin or c-Myc, which is a downstream target of ␤-catenin, has been shown to give rise to PKD (26,27). Also, high levels of expression of c-Myc mRNA and protein are found in the cysts of both human ADPKD (28) and the cpk mouse model of PKD (29 -31), and the half-life of soluble ␤-catenin is increased in cell lines from cpk mice (32). Transient overexpression of the C-terminal tail of polycystin-1 has been shown to stabilize ␤-catenin and to activate transcription of the ␤-catenin target gene, siamois (33). Finally, polycystin-1 has been shown to be in a complex with E-cadherin and ␣and ␤-catenin in normal cells (34), and E-cadherin has been shown to be mistargeted in ADPKD cells (35), possibly because of abnormal polycystin-1 function. In this study, we have investigated the transcriptional regulation of the PKD1 promoter and have determined that the PKD1 gene is a target for the ␤-catenin/TCF complex.

MATERIALS AND METHODS
DNA Clones-The cosmid clone 2H2 (LANL) (GenBank accession no. AC005346) was used to amplify a 3,414-bp sequence (29,490 -32,904 of AC005346 or 77-3,490 of L39891) from Ϫ3,363 to ϩ51, relative to the established transcription start site of the PKD1 gene (36). The DNA product was cloned in pGEM-T (Promega). A KpnI site was introduced near the 5Ј-end of the 3.3-kb fragment during the initial PCR reaction, and a HindIII site was introduced near the 3Ј-end. The fragment resulting from KpnI/HindIII digestion (Ϫ3,346 to ϩ33; Fig. 1 Fig. 1, 0.20 kb), and ApaI (Ϫ1) (not shown). A 109-bp promoter fragment (Ϫ2018 to Ϫ1910) containing TBE1 was cloned in pTiLuc, which is pGL2-Basic with the adenovirus major late promoter TATA-box and the TdT initiator region (37). Site-directed deletion mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene). All constructs, mutations, and deletions were checked by sequencing. A Myc epitope-tagged wild-type human ␤-catenin pcDNA3 expression vector was a gift from P. Polakis. Cell Lines, Transfection, Immunofluorescence, and Reporter Assays-Human embryonic kidney 293T (HEK293T), human colon carcinoma HCT116 (HCT), and human fibrosarcoma HT1080 cells were obtained from the American Type Culture Collection. COS-1 and HeLa cells were obtained from R. Padmanabhan. HCT116 and COS-1 cells were maintained in Dulbecco's modified Eagle's medium/F12 (CellGro) with 10% (v/v) fetal bovine serum. HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose and 10% heat-inactivated fetal bovine serum. Transient transfections were performed by the calcium phosphate method with HEK293T cells and by Lipo-fectAMINE with COS-1 cells. A ␤-galactosidase expression plasmid was included in each transfection to monitor transfection efficiency. After 48 h the cells were lysed, and activities were determined using enzyme assay kits for luciferase (Packard Bio Science) and ␤-galactosidase (Promega). Relative light units are the luciferase units normalized to ␤-galactosidase. All experiments were carried out in triplicate and repeated at least twice. For stable transfections, HeLa cells were transfected with pcDNA3 as a control, and Myc-tagged CA ␤-catenin in pcDNA3 using FuGENE6 (Roche Molecular Biochemicals) and were selected with 400 g/ml G418. For immunofluorescence assays cells were grown to confluency, fixed in methanol at Ϫ20°C, and incubated with primary and secondary antibodies for 1 h at 4°C.
RT-PCR-Total cellular RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's protocol. The samples were then treated with DNase I (Ambion). 3 g of RNA from each sample was incubated with random primers and Superscript reverse transcriptase (Invitrogen) to yield cDNA as per the manufacturer's instructions. cDNA was amplified in 50-l reactions containing 2 l of the cDNA reaction mix, 1ϫ PCR buffer, 1 mM MgCl 2 for PKD1 or 2 mM MgCl 2 for ␤-catenin and for the ribosomal protein L7, 200 M of each dNTP, 25 pmol of each primer, and 2.5 units of Taq DNA polymerase (Invitrogen). For each gene, PCR was performed to determine the linear range of amplification that would permit a quantitative assessment of expression levels. Primers specific for PKD1 (36) were: forward, 5Ј-CGC CGC TTC ACT AGC TTC GAC-3Ј and reverse, 5Ј-ACG CTC CAG AGG GAG TCC AC-3Ј, giving a 260-bp product; primers specific for ␤-catenin were: forward, 5Ј-TTC GCC TTC ACT ATG GAC TACC-3Ј and reverse, 5Ј-ATC AGC AGT CTC ATT CCA AGCC-3Ј, giving a 559-bp product; and primers specific for the ribosomal protein L7 were: forward, 5Ј-GGG GGA AGC TTC GAA AGG CAA GGA GGA AGC-3Ј and reverse, 5Ј-GGG GGG TCG ACT CCT CCA TGC AGA TGA TGC-3Ј, giving a 475-bp product. Each primer set was amplified at 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s for the indicated number of cycles followed by a 10-min extension at 72°C. Amplified PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide (0.5 g/ml). Bands were analyzed by NIH Image software. Net band intensity (background-subtracted intensity) was normalized to values for L7 and plotted as relative units.

RESULTS
The PKD1 gene lies in a complex region of DNA on chromosome 16 (36). More than two-thirds of the 5Ј-end of the gene is duplicated several times in an area proximal to the PKD1 gene on chromosome 16. This PKD1 homologous region appears to give rise to multiple transcripts (39). A 38,849-bp cosmid clone (LANL) 2H2 has in its 3Ј-end, the 5Ј-portion of the PKD1 gene and a long 5Ј-upstream region. Based on homology with available PKD1 sequence and differences with PKD1 homologous regions, 2 we can conclude that this cosmid clone contains the genuine PKD1 gene. This cosmid DNA was used to amplify a 3,414-bp sequence from Ϫ3,363 to ϩ51, which was analyzed for putative cis-acting elements utilizing the OMIGA sequence analysis software (Oxford Molecular) and TESS (www.cbil. upenn.edu/tess/index.html).
Typical TATA-and CAAT-boxes are absent from this proximal 5Ј-flanking region; however, a GC-rich region was identified within close proximity to the transcription start site. Numerous transcription factor consensus binding motifs were found in the PKD1 promoter, including AP-1, AP-2, Sp1, Ets, and four TCF binding elements (CTTTGA/TA/T). To test for promoter activity, a number of deletion constructs were made, and their activities were determined following transfection into human (HEK293T) and monkey (COS-1) kidney cell lines (Fig.  1). The constructs appeared to have similar promoter activities in the two cell lines. The 2.0-kb construct containing the Ϫ2,018 to ϩ33 region supported the highest level of transcription in both cell lines, suggesting that there may be a negative element in the distal 1.3 kb-region.
To determine whether ␤-catenin is potentially capable of regulating the transcription of the PKD1 gene, we transfected PKD1 promoter-luciferase reporters into HEK293T cells and measured their responses to cotransfected ␤-catenin expression vectors encoding wild-type or mutant CA ␤-catenin. HEK293T cells had been shown previously to express TCF-4 and to support ␤-catenin-dependent activation of the cyclin D1 promoter (40). ␤-catenin induction of the 3.3-kb construct (data not shown) was less significant when compared with that of the 2.0-kb fragment. Therefore, we concentrated our studies on the 2.0-kb fragment, which contains the most proximal TCF site (TBE1). The transcriptional activity of the 2.0-kb fragment showed a dose-dependent response to increasing amounts of cotransfected wild-type ␤-catenin ( Fig. 2A). The response of the 2.0-kb fragment to a CA ␤-catenin (Fig. 2B, left panel) was similar to that of the ␤-catenin target, cyclin D1 (right panel) under the same transfection and assay conditions. To further confirm the role of ␤-catenin/TCF-4 in the activation of the PKD1 promoter, we attempted to inhibit its activity in HEK293T cells with a dominant-negative TCF-4 mutant (DN TCF). As shown in Fig. 2C, cotransfected DN TCF suppressed the activity of the 2.0-kb promoter fragment in the presence or absence of exogenous ␤-catenin.
To determine whether the response of the 2.0-kb construct to induction by ␤-catenin depends specifically on the TBE1 site, we tested the activities of several deletions in the TBE1 region. An extended deletion of 109-bp at the 5Ј-end of the 2.0-kb fragment (gatctctggc acattttatt tgctctgtct caccacatgg attttgtttt tttgtttCTT TGTTttttga gatggagtct cactcttgtt gcccaggctg gagtgccat) completely abrogated ␤-catenin induction (Fig. 3A). Deletion of 15 bp (underlined) containing the TCF consensus motif (capital letters) did not abolish induction (data not shown), possibly because there are other AT-rich sequences in this region that could substitute as TCF binding sites. A 28-bp deletion (bold face) removing this AT-rich DNA and the TCF motif strongly reduced the effect of transfected ␤-catenin (Fig.  3A). To further examine the 109-bp sequence, its functional response was tested when cloned upstream of a minimal promoter. As shown, the 109-bp sequence was capable of being induced by ␤-catenin (Fig. 3B). These results, taken together, suggest that there might be interactions between ␤-catenin and/or TCF with other transcription factors within this region that are involved in conferring the observed ␤-catenin responsiveness.
To determine whether the TBE1 sequence is capable of binding TCF, we carried out electrophoretic mobility shift assays (EMSAs) using a synthetic double-stranded DNA, gtttCTTT-GTTtttt, containing the consensus TCF binding motif (capital letters). The oligo was able to form a DNA-protein complex with a GST-fusion protein containing the DNA-binding domain of human TCF-4 (Fig. 4A, GST-TCF). As a control, a DNA-protein complex was formed with GST-TCF and an optimal TCF-4 site,  TOP (T), but failed to form with the mutated binding site, FOP (F). The TBE1 complex with GST-TCF could be competed with an excess of unlabeled TBE1. The 15-bp TBE1 oligo also formed DNA-protein complexes with nuclear extract from HEK293T cells (Fig. 4B, arrows), suggesting that the PKD1 TBE1 site can bind native TCF and ␤-catenin. The TBE1 complexes were completely eliminated by competition with excess unlabeled TBE1 and partially eliminated by competition with excess unlabeled TOP (the asterisk indicates a remaining DNA-protein complex). EMSA using the TOP oligo also resulted in the formation of two DNA-protein complexes, which were completely eliminated by competition with excess TBE1 or TOP. As shown in Fig. 4C, the wild-type 15-bp oligo was able to form a complex with GST-TCF (but not GST alone) and with nuclear extracts from HCT, which have a ␤-catenin-stabilizing mutation (41), and from HEK293T cells transfected with mutated constitutively active ␤-catenin (293T). Mutation of this 15-bp oligo (gcgcCTTTGggtttt), leaving only 5 bp of the core of the TCF motif (capital letters), abrogated the binding both with GST-TCF and with the HCT and 293T nuclear extracts (Fig. 4C).  Although we were unable to supershift the wild-type 15-bp oligo using an anti-␤-catenin antibody, possibly because of other protein factors preventing an interaction with the antibody, we did supershift a probe containing the TBE1 core site (cgggCTTTGTTgggg) and nuclear extract from HCT cells (Fig.  4C, right panel). As expected, no such complex was formed using an anti-c-Myc antibody. The functional response of this 15-bp TBE1 TCF site was also tested in isolation. A single copy of the wild-type 15-bp TBE1 sequence in pGL3-Basic was able to confer ␤-catenin responsiveness in a dose-dependent manner, which was strongly inhibited by cotransfection with DN TCF (Fig. 5A), whereas mutation of the core TCF motif strongly reduced ␤-catenin responsiveness (Fig. 5B), consistent with the wild-type sequence acting as a TCF binding site.
To determine whether expression of the endogenous PKD1 gene responds to ␤-catenin, human fibrosarcoma HT1080 cells were treated with 20 mM LiCl, a known inhibitor of GSK-3␤ activity that results in the stabilization of cytosolic ␤-catenin and elevation of nuclear ␤-catenin. PKD1 gene expression was assayed by RT-PCR and compared with the expression of a control that should not respond to ␤-catenin, the ribosomal protein L7 gene. As shown in Fig. 6A, PKD1 mRNA levels increased ϳ1.6-fold in response to LiCl, whereas L7 mRNA did not increase. We also stably transfected HeLa cells with CA ␤-catenin and determined the effect of overexpression of this mutant form of ␤-catenin, which cannot be targeted for degra-dation. ␤-catenin levels were shown to be increased in these transfected cells by RT-PCR (Fig. 6B) and by immunofluorescence (Fig. 6C). Cells transfected with ␤-catenin showed ϳ3fold increase in PKD1 mRNA levels, whereas L7 mRNA levels were unaffected (Fig. 6B). These results are consistent with the endogenous PKD1 gene being a target of the ␤-catenin pathway.

DISCUSSION
The 5Ј-flanking 3.3-kb region of the human PKD1 gene was isolated and analyzed for promoter activity. Functional analysis of deletion constructs fused to a luciferase reporter in FIG. 5. Response of the 15-bp TBE1 sequence to ␤-catenin. A, a single copy of the wild-type 15-bp TBE1 sequence containing the consensus TCF binding motif (underlined) was able to confer ␤-catenin responsiveness in a dose-dependent manner in HEK293T cells, which was strongly inhibited by cotransfection with DN TCF. B, mutation of the core TCF motif in the 15-bp TBE1 sequence (gg) strongly reduced the responsiveness to CA ␤-catenin. HEK293T and COS-1 cells revealed strong transcriptional activity within this 3.3-kb region, with the highest activity localized to the 2.0-kb proximal fragment. Analysis of the 5Ј-flanking region by TESS predicted a cluster of Sp-1 binding sites just upstream of the transcription start site. Sp-1 plays a key role in regulating the transcription initiation of TATA-less promoters (42) and is temporally and spatially regulated during nephrogenesis in a pattern that suggests that it may be important in kidney development (43).
Sequence analysis of the PKD1 promoter also revealed four core TCF/LEF binding element (TBE1-4) consensus sequences. We focused on the most proximal element, TBE1, which is the only TCF site contained within the 2.0-kb proximal fragment. The 2.0-kb fragment showed a dose-responsive induction by ␤-catenin, which was inhibited by cotransfection with a dominant-negative TCF construct. Deletion of a 109-bp sequence containing the TBE1 site completely abolished the ␤-catenin responsiveness of the 2.0-kb fragment. In addition, both this 109-bp fragment and a 15-bp fragment within it containing the TBE1 site were able to confer ␤-catenin responsiveness when placed upstream of a minimal promoter. The response of the 15-bp TBE1 site was reduced or eliminated by mutation of the core consensus TCF motif or by dominantnegative TCF. Furthermore, the wild-type 15-bp sequence, but not the mutated 15-bp sequence, was able to form DNA-protein complexes using nuclear extracts from cells known to contain ␤-catenin/TCF and with a GST-TCF fusion protein. We conclude from these experiments that the TBE1 site in the PKD1 promoter is a ␤-catenin/TCF response element. Although we did not focus on the three upstream TBE elements (TBEs 2-4), it is quite possible that they are also ␤-catenin/TCF response elements because we have shown that a 1.3-kb fragment containing all three (but lacking TBE1) is also induced by ␤-catenin (data not shown). Further analysis will be required to determine which (one or whether all three) has the potential to act as a TCF response element. Finally, to determine whether expression of the endogenous PKD1 gene is able to respond to increases in ␤-catenin, we treated human fibrosarcoma HT1080 cells with LiCl, which is a known inhibitor of GSK-3␤ activity and results in the stabilization of ␤-catenin. We also stably transfected HeLa cells with CA ␤-catenin. In both cases, we were able to demonstrate that endogenous PKD1 mRNA levels were elevated in response to the treatments, supporting the idea that the PKD1 gene is a ␤-catenin target. Genetic and biochemical studies have indicated that the secreted Wnt factors, through Frizzled receptors, activate Disheveled and inhibit GSK-3␤ to stabilize ␤-catenin (24,25). It is also possible that ␤-catenin levels are regulated by other mechanisms such as the PKB/akt pathway (44). The extent to which these pathways participate in the regulation of PKD1 expression will have to be determined in future studies.
A number of observations have suggested that the level of PKD1 expression may be important in PKD. Mice containing multiple copies of a human PKD1 transgene were found to have cystic kidneys, suggesting that PKD1 overexpression can play a role in cystic disease (21). Elevated polycystin-1 mRNA and protein levels have been reported in cystic epithelium (5,8,15,45). Recent in situ hybridization analyses have shown persistent expression of PKD1 mRNA in proximal tubules of adult ADPKD kidneys (46), and PKD1 mRNA and polycystin-1 protein have been reported in end-stage ADPKD kidneys (47). These later observations may be explained by the dedifferentiated state of cystic epithelial cells or by a loss of a negative feedback loop regulating PKD1 gene expression. Because this work has shown that the PKD1 gene is a target of ␤-catenin, it is interesting to note that several instances have been reported that may be relevant to PKD. ␤-catenin has been shown to regulate the reorganization of renal epithelial cell aggregates into long tubules (48) and therefore may be important in normal tubulogenesis. Increased stability of ␤-catenin was found in immortalized cpk cells (a mouse model of recessive PKD) (32) and in primary ADPKD cells. 3 Transgenic mice overexpressing either ␤-catenin (26) or c-Myc (a target of ␤-catenin) (27) have PKD, and high levels of expression of c-Myc mRNA and protein are found in the cysts of both human ADPKD (28) and of the cpk mouse model of PKD (29 -31) (however, see Ref. 49). Finally, Wnt-4 expression recently was reported to be induced in polycystic kidneys (50). Together, these results suggest that increased levels of soluble ␤-catenin may cause overexpression of the normal (and/or mutated) PKD1 gene, which in turn may contribute to cyst formation in PKD.