Article |
Address correspondence to Alberto Muñoz, Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain. Tel.: 34-91-5854640. Fax: 34-91-5854587. E-mail: amunoz{at}iib.uam.es
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Abstract |
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Key Words: vitamin D; vitamin D receptor; ß-catenin; E-cadherin; colon cancer
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Introduction |
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E-cadherin is a transmembrane linker protein of the intercellular adheren junctions that play a key role in the maintenance of the adhesive and polarized phenotype of epithelial cells (Takeichi, 1995; Gumbiner, 1996). Loss of E-cadherin expression is a common event during the transition from adenoma to carcinoma, which involves the alteration of the normal epithelial phenotype and the acquisition of invasive capacity (Birchmeier and Behrens, 1994; Perl et al., 1998; Christofori and Semb, 1999). E-cadherin is regarded as a tumor suppressor gene and its loss as a predictor of poor prognosis (Vleminckx, et al., 1991; Takeichi, 1993; Mareel et al., 1994; Guilford et al., 1998). ß-Catenin is a protooncogene encoding a cytoskeleton-associated protein, which in normal epithelial cells is bound mostly to the cytoplasmic tail of E-cadherin at the adherens junctions (for review see Morin, 1999). Free cytosolic ß-catenin levels are strictly controlled by phosphorylation of the NH2-terminal region of the protein by glycogen synthase kinase (GSK)* 3ß. This reaction requires association with axin/conductin and the product of the adenomatous polyposis coli (APC) tumor suppressor gene, and targets ß-catenin for ubiquitin-mediated degradation by the proteasome (Rubinfeld et al., 1996; Behrens et al., 1998; Kishida et al., 1998). Interaction of Wnt ligands with their membrane receptors blocks GSK-3ß, leading to the accumulation of free ß-catenin (for reviews see Eastman and Grosschedl, 1999; Peifer and Polakis, 2000). In the cell nucleus, ß-catenin binds members of the T cell transcription factor (TCF)/lymphoid enhancer-binding factor (LEF)-1 family and thus regulates gene expression (Behrens et al., 1996; Huber et al., 1996; Billin et al., 2000). Nearly all colon tumors present a deregulated ß-catenin signaling pathway by mutation of either APC or ß-catenin, which leads to the blockade of phosphorylation by GSK-3ß, resulting in ß-catenin stabilization (Inomata et al., 1996; Korinek et al., 1997; Morin et al., 1997), reduced APC-regulated nuclear export (Henderson, 2000; Rosin-Arbesfeld et al., 2000), and perhaps higher specific activity (Guger and Gumbiner, 2000). As a result, ß-catenin accumulates in the nucleus, leading to both the activation of genes involved in the control of cell proliferation and invasiveness such as c-myc, cyclin D1, peroxisome proliferator-activated receptor (PPAR) , matrilysin, c-jun, fra1, uPA receptor, fibronectin, CD44, Tcf-1, Cdx-1 and gastrin, and the loss of expression of DRCTNNB1A and differentiated epithelial markers such as Zonula occludens (ZO)-1 (He et al., 1998, 1999; Crawford et al., 1999; Gradl et al., 1999; Mann et al., 1999; Tetsu and McCormick, 1999; Roose et al., 1999; Vera et al., 1999; Kawasoe et al., 2000; Koh et al., 2000; Lickert et al., 2000). Mutations in the TCF-4 gene may also contribute to this process (Duval et al., 2000). In addition, APC mutations may also be responsible at least in part for chromosomal instability in colon cancer cells (Fodde et al., 2001; Kaplan et al., 2001).
Epidemiological data suggest an inverse correlation between vitamin D dietary intake or sunlight exposure and human colorectal cancer (Garland et al., 1989; Newmark and Lipkin, 1992). Vitamin D, especially its most active metabolite 1,25-dihydroxyvitamin D3 (1
,25[OH]2D3), not only contributes to calcium homeostasis but also regulates cell proliferation and differentiation (Saez et al., 1993; Xi and Feldman, 1993; Buras et al., 1994; Kane et al., 1996). 1
,25(OH)2D3 and several synthetic vitamin D derivatives (deltanoids), which show reduced calcemic activity such as EB1089, MC903, and KH1060, inhibit the growth of epithelial, melanoma, soft tissue sarcoma, and leukemic cells by inducing cell cycle arrest or apoptosis (Diaz et al., 2000; Park et al., 2000). Furthermore, they inhibit the invasive capacity in vitro, the synthesis of several invasion-associated proteins (Hansen et al., 1994; González-Sancho et al., 1998; Koli and Keski-Oja, 2000), and the tumor-induced angiogenesis (Majewski et al., 1993) of breast cancer cells, and they show a chemopreventive activity in animal models of colorectal and breast cancer (Akhter et al., 1997; van Weelden et al., 1998).
Vitamin D and its analogues regulate gene expression by binding to specific vitamin D receptors (VDRs) of the nuclear receptor superfamily, which are ligand-modulated transcription factors (for review see McDonald et al., 2001). Upon ligand activation, VDR binds specific nucleotide sequences (vitamin D response elements, VDREs) in target genes to activate or repress their expression through multiple but ill-defined interactions with coactivator complexes and components of the basal transcription machinery (for review see McDonald et al., 2001). Several vitamin D target genes have been characterized in several tumor cell types such as c-myc, p21Waf, p27Kip1, tenascin-C, fibronectin, laminin and its receptor, apolipoprotein D, insulin-like growth factor binding protein 3, cyclin C, and several members of the transforming growth factor family and their receptors (Freedman, 1999). Two studies have reported the regulation of epidermal growth factor receptor expression (Tong et al., 1998) or the cross-talk between vitamin D and tumor growth factor ß signaling pathways (Yanagisawa et al., 1999), although the physiological relevance of these mechanisms remains unclear.
VDR is expressed in the normal colonic mucosa, slightly increases in the hyperplasic condition, and is clearly lower in late stages of carcinogenesis (Vandewalle et al., 1994). Accordingly, high VDR expression is associated with favorable prognosis in colorectal cancer patients, suggesting that these receptors are involved in this pathogenesis and their potential role as a predictive marker (Shabahang et al., 1993; Evans et al., 1998). Several clinical trials are underway to assess the activity of several vitamin D derivatives in patients with colorectal carcinoma and other neoplasias (Gross et al., 1998; Gulliford et al., 1998; Smith et al., 1999). However, their molecular and cellular mechanisms of action in colon carcinoma cells remain mostly unknown.
The SW480 cell line is one of the best characterized human colorectal cancer lines, and it has been widely used as a model system for the study of this neoplasia. It was established in 1976 from a primary human Duke's B colon adenocarcinoma of a 50-yr-old patient (Leibovitz et al., 1976). SW480 cells harbor most of the genetic abnormalities that characterize advanced colon cancers such as activation of K-ras oncogene, c-myc amplification, deletion of chromosome 18, and mutation of APC and p53 tumor suppressor genes (Tomita et al., 1992; Schwarte-Waldhoff et al., 1999). In addition, these cells are defective for E-cadherin and express high levels of nuclear ß-catenin, transforming growth factor ß, and epidermal growth factor receptors (Tomita et al., 1992). We used the SW480 cell line to examine the mechanism of action of 1,25(OH)2D3 and several nonhypercalcemic analogues in colon cancer cells. Our results show that these compounds have a prodifferentiation phenotypic effect on VDR-positive SW480 cells parallel to the induction of E-cadherin, induce ß-catenin nuclear export, and inhibit ß-catenin gene regulatory activity. Moreover, 1
,25(OH)2D3 promotes a direct VDRß-catenin interaction, which may decrease TCF-4ß-catenin complexes and may thus constitute another mechanism of inhibition of ß-catenin signaling.
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Results |
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Next, we analyzed the regulation of endogenous ß-cateninTCF/LEF-1 target genes. 1,25(OH)2D3 reduced the cellular RNA levels of c-myc, Tcf-1, CD44, and PPAR
genes induced by ß-cateninTCF/LEF-1 (He et al., 1998, 1999; Roose et al., 1999; Vera et al., 1999) (Fig. 6, C and D). They were downregulated with biphasic kinetics: a rapid (48 h) and slight (2530%) reduction followed (after 48 h) by an additional diminution (to 5080%) when ß-catenin was no longer present into the nucleus. Though in untreated cells c-myc and CD44 RNA levels increased during incubation probably due to the proliferation status, 1
,25(OH)2D3 always had an inhibitory effect. In addition, 1
,25(OH)2D3 also rapidly induced the expression of ZO-1 (Fig. 6, C and D), which is repressed by ß-cateninTCF/LEF-1 (Mann et al., 1999). Like ß-catenin, ZO-1 is involved in intercellular adhesion and perhaps signaling (Gottardi et al., 1996). This increase was confirmed at the protein level by immunofluorescence and confocal microscopy analyses (Fig. 6 E). In untreated SW480-ADH cells, ZO-1 showed a diffuse cytoplasmic and punctate nuclear localization. 16 h after 1
,25(OH)2D3 addition, the cellular content of ZO-1 clearly increased and was also located in cell-to-cell contact areas. At 48-h after treatment, the subcellular distribution of ZO-1 was drastically altered, since it was almost completely peripheral. Thus, 1
,25(OH)2D3 has a dual effect on ZO-1: it relieves the repression by ß-cateninTCF/LEF-1 at the transcriptional level, and it induces the redistribution of ZO-1 protein to the plasma membrane. This relocation was concomitant with that of ß-catenin in accordance with the nuclear coexport of ZO-1 and catenins that takes place during the differentiation of MDCK epithelial cells (Rajasekaran et al., 1996). Although cyclin D1 has been proposed as a ß-cateninregulated gene (Tetsu and McCormick, 1999), neither cyclin D1 RNA nor protein expression were modified upon 1
,25(OH)2D3 addition to SW480-ADH cells (unpublished data). These data indicate that in SW480-ADH cells 1
,25(OH)2D3 downregulates ß-cateninTCF/LEF-1 target genes.
To examine how general the effects of 1,25(OH)2D3 observed in SW480-ADH cells were, we extended the study to other human colon carcinoma cell lines such as Caco-2, SW1417, HT-29 M6, and LS-147T. As in SW480-ADH cells, 1
,25(OH)2D3 induced a parallel activation (two- to fourfold) of a VDREreporter construct and an inhibition of ß-cateninTCF-4 transcriptional activity (2550%) (Fig. 7
A). SW480-R and SW620 cells were used as negative control. In addition, E-cadherin expression was increased (two- to threefold after 48 h) by 1
,25(OH)2D3 in these cell lines but in LS-174T, which were totally defective for this protein (Fig. 7 B).
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Nonhypercalcemic vitamin D3 analogues are potent inhibitors of the ß-catenin signaling pathway
Three synthetic vitamin D analogues with low calcemic properties, EB1089, KH1060, and MC903, used currently in clinical trials to treat several neoplasias (see Introduction for references) were studied for their effects on ß-catenin signaling in SW480-ADH cells. Like 1,25(OH)2D3, all three analogues displayed antiproliferative activity (unpublished data). We then studied whether these compounds induce the expression of E-cadherin mRNA and protein by Northern and Western blotting. As 1
,25(OH)2D3, doses in the 10-1110-7 M range of each compound induced E-cadherin expression (Fig. 10
A). All three compounds reduced the expression of a ß-cateninTCF-4 reporter construct in SW480-ADH cells (Fig. 10 B). The effects observed in the assays show that EB1089 and KH1060 are more potent than 1
,25(OH)2D3, whereas MC903 is less active. In agreement with our previous results, all three analogues promoted the morphological differentiation and nuclear export of ß-catenin in SW480-ADH cells but had no effect in VDR-defective SW480-R or SW620 cells as revealed by immunofluorescence (Fig. 10 C).
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Discussion |
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1,25(OH)2D3 inhibits the transcriptional activity of ß-catenin by two mechanisms. On the one hand, it rapidly increases the amount of VDR bound to ß-catenin, blocking the interaction of this catenin to TCF-4. Therefore, 1
,25(OH)2D3 modulates TCF/LEF-1 target genes in a manner opposite to ß-catenin. In some cells, such as Pam212 and LS-174T, this effect is independent of changes in E-cadherin expression. Secondly, in SW480-ADH cells changes in ß-catenin transcriptional activity caused by 1
,25(OH)2D3 are accompanied by the nuclear export of ß-catenin and its relocalization to the plasma membrane that happens concomitantly to E-cadherin protein expression. 1
,25(OH)2D3 might enhance the sequestration of ß-catenin in the plasma membrane compartment by E-cadherin or alternatively might stimulate ß-catenin nuclear export, a process mediated by APC (Henderson, 2000; Rosin-Arbesfeld et al., 2000). Alternatively, 1
,25(OH)2D3 may stimulate the APC-independent ß-catenin export described recently (Eleftheriou et al., 2001). Although an estimation of the respective contribution of these activities to the inhibition of ß-catenin signaling is very difficult, their respective timing supports the hypothesis that the primary effect is due to the formation of VDRß-catenin complexes. By favoring this, 1
,25(OH)2D3 may indirectly regulate the transcription of ß-cateninTCF/LEF-1 target genes such as Tcf-1, CD44, PPAR
, and ZO-1. The changes in RNA content for these genes after 1
,25(OH)2D3 treatment occur earlier than those in ß-catenin localization, which suggests that they are initiated as a result of the VDRTCF-4 competition for ß-catenin and later strengthened by the nuclear export of ß-catenin.
The physical interaction of VDR with ß-catenin adds to that reported previously for retinoic acid receptor (RAR) (Easwaran et al., 1999). However, the interaction of ß-catenin with these two nuclear receptors differs. RAR strictly depends on ligand binding, whereas a certain amount of VDRß-catenin complexes were found in vitro in the absence of 1,25(OH)2D3. In contrast, this basal interaction augments in the presence of 1
,25(OH)2D3 in vivo, suggesting the participation of ligand-dependent nuclear mediator(s) or mechanism(s). However, given the high activity of 1
,25 (OH)2D3 in our cell system, low amounts of metabolically active vitamin D derivatives in the culture medium may be sufficient to activate VDR in the absence of added agent. Likewise, the stimulation by ß-catenin of the effect of 1
,25 (OH)2D3 on a VDRE-dependent promoter agrees with that reported for RAR-responsive promoters (Easwaran et al., 1999). In vivo, the interaction between VDR and ß-catenin may ameliorate ß-cateninTCF-4 signaling. Upon ß-catenin stabilization due to its mutation or that of APC, binding to VDR may buffer its stimulatory action on TCF-4 target genes, a protective effect which can be lost along with VDR expression during malignant progression. Additionally, our data suggest that nuclear ß-catenin might transiently potentiate VDR transcriptional activity before ß-catenin moves out of the nucleus and/or VDR is extinguished.
The ß-catenin homologue -catenin/plakoglobin is also regulated by APC and functions as an oncogene (Kolligs et al., 2000). Like ß-catenin, it activates c-myc expression in APC-mutated cells, which together with mutations in its NH2-region is thought to be critical for its oncogenic activity (Kolligs et al., 2000).
-Catenin/plakoglobin indirectly activates TCF/LEF-1regulated genes by increasing the levels of ß-catenin and by inducing its nuclear translocation (Zhurinsky et al., 2000). These data show similarities but also differences in the mechanism of action of ß-catenin and
-catenin/plakoglobin. 1
,25(OH)2D3 induces the nuclear export of
-catenin/plakoglobin to the plasma membrane, which may also contribute to the phenotypic change described in this study.
Our results show the competition between VDR, TCF-4, and E-cadherin for binding to ß-catenin. Since the E-cadherin gene has been proposed to be down-regulated by ß-catenin upon binding of ß-cateninTCF/LEF-1 to its promoter (Huber et al., 1996) and 1,25(OH)2D3 induces a concomitant increase in VDRß-catenin binding and E-cadherin mRNA, an effect on the E-cadherin promoter is plausible. The formation of VDRß-catenin complexes may reduce the ß-catenin binding to TCF-4 and so lead to a relief of promoter repression and/or have a direct activating effect. In disagreement with this hypothesis, VDR overexpression in SW480-ADH cells did not increase the basal activation of the 1.1-kb E-cadherin promoter construct, although potential regulatory sequences outside this region cannot be ruled out. The moderate induction by 1
,25(OH)2D3 of this E-cadherin gene promoter construct does not unambiguously demonstrate that E-cadherin gene is regulated transcriptionally by this agent. However, this effect is consistently observed, and together with the inhibitory action of actinomycin and the lack of regulation of E-cadherin RNA half-life suggests an effect at the transcription level.
Our data show that 1,25(OH)2D3 increases E-cadherin gene transcription by a mechanism that is dependent on the AF-2 domain of VDR and mediated by the novo synthesis of short-lived proteins, although additional posttranscriptional routes cannot be discarded. E-cadherin expression is inhibited by the product of Snail gene, which is a transcriptional repressor on the E-boxes located near the transcription start site (Batlle et al., 2000; Cano et al., 2000). We show that 1
,25(OH)2D3 regulates the human E-cadherin promoter when a region of
1.1 kb upstream of the +92 site is studied, but it has no effect on the activity of the proximal promoter region that contains the E-boxes bound by Snail. These data suggest that 1
,25(OH)2D3 and Snail have opposite effects on distinct sequences of the E-cadherin promoter. Though no changes in Snail mRNA expression were detected upon 1
,25(OH)2D3 treatment, further research is required to determine a putative interplay between these agents.
Whether the increase observed in the cellular content of other intercellular adhesion proteins such as occludin, ZO-1, ZO-2, and vinculin is a direct effect of 1,25(OH)2D3 or a corollary of its primary effect on E-cadherin and/or ß-catenin is unknown. Since the ZO-1 gene is repressed by ß-cateninTCF-4 (Mann et al., 1999) and ZO-1 mRNA increases before E-cadherin protein levels are significantly elevated, that is, when ß-catenin is abundant in the nucleus, the effect of 1
,25(OH)2D3 on this gene is probably due to the rapid formation of VDRß-catenin complexes and the subsequent reduction of ß-cateninTCF-4 complexes. Additionally, ZO-1 protein forms complexes with
-, ß- and
-catenins (Rajasekaran et al., 1996), and like ß-catenin it can locate in the cell nucleus where it may function as a signaling molecule (Gottardi et al., 1996). In untreated SW480-ADH cells, ZO-1 is distributed diffusely in the cytoplasm and in intranuclear foci. Upon 1
,25(OH)2D3 treatment, ZO-1 is first increased and then redistributes to the cell surface as in MDCK cells after E-cadherin expression (Rajasekaran et al., 1996). The relevance of the effects of 1
,25(OH)2D3 on ZO-1 reported here is supported by a recent report describing the regulation of c-ErbB2/Neu expression by ZO-1 (Balda and Matter, 2000). It is also significant that other genes regulated by 1
,25(OH)2D3 in SW480-ADH cells play important roles: occludin expression has been associated with tumor differentiation (Kimura et al., 1997), whereas vinculin is involved in the organization of tight junctions (Watabe-Uchida et al., 1998). These changes are consistent with the key role of E-cadherin in the maintenance of epithelial characteristics and account for the drastic change induced by 1
,25(OH)2D3 in the differentiation status of SW480-ADH cells.
The rapid inhibitory effect of 1,25(OH)2D3 on c-myc expression, another TCF/LEF-1ß-catenin target gene, probably results from the combined effects of activities at various levels, which take place before nuclear export of ß-catenin. First, 1
,25(OH)2D3 may induce the binding of regulatory proteins to the first intron of the gene (Pan and Simpson, 1999). Second, the formation of VDRß-catenin complexes may inhibit the activation by ß-cateninTCF/LEF-1, as may the alteration of
-catenin/plakoglobin localization (Kolligs et al., 2000).
The effects of 1,25(OH)2D3 in SW480-ADH cells are transient and depend on its nuclear receptors. VDR content is low in normal colon epithelial cells, increases at the early stages of tumor progression, and is almost absent in the more malignant carcinoma cells (Vandewalle et al., 1994). This agrees with the finding that 1
,25(OH)2D3 inhibits proliferation in human rectal mucosa (Thomas et al., 1992) and with its activity in the weakly tumorigenic SW480-ADH cells and its inefficacy in the highly tumorigenic SW480-R cells. Our data are consistent with the proposed protective role of dietary vitamin D or sunlight exposure and with the predictive use of VDR expression in colon cancer biopsies. Our results suggest that liganded VDR may hinder the loss of differentiation and the increase in proliferation at the early stages of carcinogenesis. 1
,25(OH)2D3 has a pleiotropic biological activity with complex cell-specific antitumor properties that include induction of apoptosis, growth arrest, inhibition of invasiveness, and stimulation of differentiation. The pattern of expression of the coactivators (SRC-1, CBP, GRIP-1/TIF-2, and others) and corepressors (NCoR, SMRT, Alien) (Polly et al., 2000; for review see McDonald et al., 2001) that interact with VDR and the activation of other signaling pathways such as that of TGF-ß (Yanagisawa et al., 1999) may be responsible for the effects of 1
,25(OH)2D3 in a particular cell type. Our data reveal a complex network of interactions between cell junction proteins with signaling abilities such as ß-catenin,
-catenin/plakoglobin, and ZO-1 and nuclear hormone receptors such as VDR, which together with other signaling mediators regulate gene expression and the phenotype of epithelial cells in a combinatorial fashion.
In summary, we report here on a novel activity of 1,25(OH)2D3 in human colon carcinoma cells, consisting of the induction of E-cadherin and the inhibition of ß-catenin signaling, which has an antitumor effect in vivo. 1
,25(OH)2D3 exerts these protective effects in SW480-ADH cells carrying a panel of mutations in critical genes such as p53, ras, and APC and expressing negligible amounts of E-cadherin but overexpressing c-myc and in other human colon cell lines expressing functional VDR. These results point to the key role of VDR expression in colon carcinogenesis and support the use of nonhypercalcemic vitamin D derivatives for the treatment of this neoplasia.
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Materials and methods |
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Cloning of the human E-cadherin gene promoter
A -178/+92 human E-cadherin promoter fragment was amplified by PCR using Pfu DNA polymerase (Stratagene) and 5'-GACTACGCGTACTCCAGGCTAGAGGGTCAC-3' and 5'-GATCGATATCCGGGTGCGGTC-GGGTCGGGCCGGGCA-3' as sense and antisense oligonucleotides, respectively. The -987/+92 E-cadherin promoter fragment was amplified using the same antisense oligonucleotide and 5'-AGTCGGTACCGA-GAGTGCAGTGGCTCACGC-3' as sense oligonucleotide. Fragments were digested with MluI/EcoRV or SacI/NcoI restriction enzymes and inserted in pGL3 reporter vector (Promega). Cloned fragments were sequenced in order to rule out differences with respect to the published sequence (sequence data available from GenBank/EMBL/DDBJ under accession no. L34545).
Antibodies
The following antibodies were used: rabbit polyclonal anti-VDR (sc-1008; Santa Cruz Biotechnology, Inc.), mouse monoclonal antiß-catenin (C19220; Transduction Laboratories), rat monoclonal antimouse E-cadherin (ECCD-2; a gift from Dr. M. Takeichi, Kyoto University, Kyoto, Japan), rabbit polyclonal antioccludin (71-1500; Zymed Laboratories), rabbit polyclonal anti-ZO1 (61-7300; Zymed Laboratories), rabbit polyclonal antiZO-2 (71-1400; Zymed Laboratories), rabbit polyclonal antivinculin IgG (sc-7649; Santa Cruz Biotechnology, Inc.), goat antiTCF-4 (sc-8632; Santa Cruz Biotechnology, Inc.), FITC-conjugated goat antimouse IgG (115-095-003; Jackson ImmunoResearch Laboratories), TRICT-conjugated antirat IgG (112-025-003; Jackson ImmunoResearch Laboratories), TRICT-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories), goat antirabbit IgG (H + L) HRP-conjugated (67437; ICN Biomedicals), goat antimouse IgG (H + L) HRP-conjugated (67428; ICN Biomedicals), goat antirat IgG (H + L) HRP-conjugated (31472; Pierce Chemical Co.), and rabbit antigoat IgG (612762; ICN Biomedicals).
Immunostaining
Cells were rinsed four times in PBS, fixed in cold methanol for 30 s at -20°C and rinsed in PBS. The nonspecific sites were blocked by incubation with PBS containing 1% BSA for 1 h at room temperature. Cells were then washed four times in PBS and incubated with the primary antibodies diluted in PBS containing 1% BSA for 1 h at room temperature or overnight at 4°C. After four washes with PBS, cells were incubated with secondary antibodies for 45 min at room temperature, washed, and mounted in VectaShield (Vector Laboratories). Confocal microscopy was performed with a Bio-Rad Laboratories MRC-1024 laser scanning microscope equipped with an Axiovert 100 invert microscope (ZEISS) at excitation wavelengths of 488 nm (for FITC) and 543 nm (for TRICT). Each channel was recorded independently, and pseudocolor images were generated and superimposed. Images were processed by the Adobe Photoshop® 5.0 software (Adobe Systems, Inc.).
RNA preparation and Northern analysis
Purification of poly(A)+ RNA was carried out as reported elsewhere (Vennström and Bishop, 1982). Northern blots were performed on nylon membranes (Nytran; Schleicher & Schuell) following standard protocols (Sambrook et al., 1989). All probes were labeled by the random priming method (Feinberg and Vogelstein, 1983). Hybridizations were carried out overnight at 65°C in 7% SDS, 500 mM sodium phosphate buffer, pH 7.2, and 1 mM EDTA as described by Church and Gilbert (1984). Filters were washed twice for 30 min each in 1% SDS and 40 mM sodium phosphate buffer, pH 7.2, at 65°C. The sizes of respective mRNAs were calculated using RNA ladder markers (Bio-Rad Laboratories). Membranes were exposed to HyperfilmTM MP films (Amersham Pharmacia Biotech). The following probes were used: for VDR and RXR, the complete human cDNA; for E-cadherin, a fragment (nucleotides 22092649) of human cDNA; for ß-catenin, the whole mouse cDNA; for c-myc, the third exon of the human cDNA (donated by Dr. J. León, Facultad de Medicina, University of Cantabria, Santander, Spain); for CD44, the full-length human cDNA; for Tcf-1, the full-length
Tcf-1 (van de Wetering et al., 1991) (both donated by Drs. E. Sancho and H. Clevers, University Medical Center, Utrecht, Netherlands); for ZO-1, a 558-pb fragment of the human cDNA obtained by reverse transcriptase-PCR with the 5'-CTTAACTATGCCCAGTGG-3' and 5'-CTTGTGGTGAGTAAGGAG-3' oligonucleotides as sense and antisense primers, respectively; for PPAR
, a 407-bp fragment of the human cDNA obtained by reverse transcriptase-PCR with the 5'-CTACGGTGTTCATGCATGTGAG-3' and 5'-CACAATGTCTCGATGTCGTGG-3' oligonucleotides as sense and antisense primers, respectively; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the complete human cDNA.
Immunoprecipitation and Western blotting
Immunoprecipitation of whole cell extracts with specific antibodies was carried out as described elsewhere (Lozano and Cano, 1998). Whole cell extracts were prepared by washing the monolayers twice in PBS, and the cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1.5 mM MgCl2, 10 mM NaF, 10% glycerol, 4 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 50 mM Hepes, pH 7.4, plus phosphatase- and protease-inhibitor mixture [PPIM: 25 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin]) for 15 min on ice followed by centrifugation at 13,000 rpm for 10 min at 4°C. Immunoprecipitated proteins were analyzed in 7.5% or 12% SDS-PAGE gels. Immunoblotting of cell lysates or immunoprecipitates was performed by protein transfer to Immobilon-P membranes (Millipore Corp.) and incubation with the appropriate specific antibody. Blots were developed using the ECL detection system (Amersham Pharmacia Biotech).
In vitro proteinprotein interaction
100 ng each of bacterially produced GSTß-catenin protein and human VDR translated in vitro in rabbit reticulocyte lysates (TNT® T7 Quick Kit; Promega) were mixed with 500 µl of immunoprecipitation buffer (IB: 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1% NP-40, 1% Triton X-100) and incubated for 1 h at 4°C. GSTß-catenin complexes were collected by addition of glutathioneSepharose 4B (Amersham Pharmacia Biotech), washed twice with IB, and resuspended in Laemmli sample buffer. Proteins in the complexes were analyzed in 7.5% SDS-PAGE gels, which was followed by immunoblotting with the indicated antibodies.
Transactivation assays
Nearly confluent cells were transfected in triplicate P-60 dishes using LipofectAMINETM reagent (Life Technologies) following the manufacturer guidelines. The 4 x VDREDR3-Tk-Luc construct containing four copies in tandem of a consensus DR3 response element for vitamin D cloned upstream of the herpes virus simplex thymidine kinase gene promoter and the luciferase reporter gene was provided by Dr. C. Carlberg (Heinrich-Heine-Universität, Düsseldorf, Germany). To study ß-cateninTCF/LEF-1 transcriptional activity, we transfected either TOP-flash and FOP-flash containing multimerized wild-type (CCTTTGATC) or mutated (CCTTTGGCC) TCF/LEF-1binding sites upstream of a minimal c-fos promoter driving luciferase gene expression (Korinek et al., 1997) (a gift from Dr. H. Clevers). 1 µg of human VDR cDNA (cloned in pSG5) or human wild-type TCF-4 (cloned in pcDNA3.1) or mutant TCF-4 (N-TCF-4 lacking the NH2-terminal ß-catenininteracting region in pcDNA3) (Baulida et al., 1999) and ß-catenin S37A (cloned in pCGN; a gift from Dr. A. Ben-ZeÄev, The Weizman Institute, Rehovot, Israel) were used for exogenous expression together with 0.25 µg of the reporter luciferase plasmids. For exogenous E-cadherin expression, we used the complete murine cDNA (cloned in the pBATEM2 vector; a gift from Dr. M. Takeichi, Kyoto University). The expression vectors for the truncated VDR lacking the 11 COOH-terminal amino acids (
AF2) (Jiménez-Lara and Aranda, 1999) and for other point-mutated VDR (L417S and E420Q) (cloned in pSG5) were donated by Dr. Ana Aranda (Instituto de Investigaciones Biomédicas). As internal control of the transfection efficiency, 0.5 µg of the pRSV-LacZ containing a ß-galactosidase reporter gene was used. Luciferase and ß-galactosidase activities were measured 48 h after transfection.
Transepithelial electrical resistance
The functional integrity of tight junctions was assayed by measuring the electrical resistance towards ion flux of epithelial cell layers cultured on porous tissue culture inserts (3090; Falcon). We used the Millicell electrical resistance system (Millipore Corp.) connected to the electrode system Endohm-24 (World Precision Instruments) following the manufacturer guidelines. TERs were calculated after subtracting the background given by a blank culture insert.
Proliferation assays
50,000 cells were seeded in 24-well dishes (Falcon) and incubated in normal growth medium in the presence or absence of the indicated concentrations of 1,25(OH)2D3. 48-h later, cells were pulsed with 1 µCi/ml 3H-thymidine for 3 h. At the end of the labeling period, the medium was removed and the cells were rinsed twice with PBS and fixed with chilled 10% trichloroacetic acid for 10 min. Trichloroacetic acid was then removed and the monolayers were air-dried at room temperature for 20 min. Thereafter, precipitated cellular macromolecules were dissolved in 500 µl of 0.1 N NaOH-0.1% SDS, and 450 µl of each sample was diluted in 5 ml of scintillation solution OptiPhase HighSafe (Wallac Scintillation Products). Radioactivity was measured using a 1209 RackBeta counter (LKB Wallac).
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Footnotes |
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* Abbreviations used in this paper: APC, adenomatous polyposis coli; DR, direct repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK, glycogen synthase kinase; GST, glutathione S-transferase; LEF, lymphoid enhancer-binding factor; 1
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Acknowledgments |
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H.G. Pálmer and J.M. González-Sancho were recipients of fellowships from the Comunidad Autónoma de Madrid. This work was supported by a grant from the Plan Nacional de Investigación y Desarrollo (SAF98-0060), Ministerio de Ciencia y Tecnología of Spain.
Submitted: 5 February 2001
Revised: 30 May 2001
Accepted: 14 June 2001
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