Repression of Wnt/β-catenin response elements by p63 (TP63)

Abstract

Submitted: TP63 (p63), a member of the tumor suppressor TP53 (p53) gene family, is expressed in keratinocyte stem cells and well-differentiated squamous cell carcinomas to maintain cellular potential for growth and differentiation. Controversially, activation of the Wnt/β-catenin signaling by p63 (Patturajan M. et al., 2002, Cancer Cells) and inhibition of the target gene expression (Drewelus I. et al., 2010, Cell Cycle) have been reported. Upon p63 RNA-silencing in squamous cell carcinoma (SCC) lines, a few Wnt target gene expression substantially increased, while several target genes moderately decreased. Although ΔNp63α, the most abundant isoform of p63, appeared to interact with protein phosphatase PP2A, neither GSK-3β phosphorylation nor β-catenin nuclear localization was altered by the loss of p63. As reported earlier, ΔNp63α enhanced β-catenin-dependent luc gene expression from pGL3-OT having 3 artificial Wnt response elements (WREs). However, this activation was detectable only in HEK293 cells examined so far, and involved a p53 family-related sequence 5' to the WREs. In Wnt3-expressing SAOS-2 cells, ΔNp63α rather strongly inhibited transcription of pGL3-OT. Importantly, ΔNp63α repressed WREs isolated from the regulatory regions of MMP7. ΔNp63α-TCF4 association occurred in their soluble forms in the nucleus. Furthermore, p63 and TCF4 coexisted at a WRE of MMP7 on the chromatin, where β-catenin recruitment was attenuated. The combined results indicate that ΔNp63α serves as a repressor that regulates β-catenin-mediated gene expression.

Keywords: TCF4; TP63; Wnt; p63; β-catenin.

Figure 1.

Alteration of the Wnt/β-catenin target gene expression by p63-silencing.(A) Expression of indicated genes was quantified by RT-qPCR. (B) Western blot analyses for the proteins. Positions of p63 isoforms and smolylated (SUMO) p63 are also shown. Molecular masses of the standard proteins are marked in parentheses (in k_D).

Figure 2.

Analyses of the interactions of PP2A, p63 and Wnt/β-catenin signaling proteins. (A) Csi and p63si-transfected FaDu cells were fractionated to the cytosol (Cyt) and nuclear (Nuc) extracts. Cytosol and nuclear samples were loaded on the gel in a ratio of 1:2. Proteins detected in each fraction (Input) are shown. Immunoprecipitates (IP) by a PP2A-C antibody (mouse IgG) were subjected to Western blotting (WB) for proteins indicated. ΔNp63α detected by a p63 monoclonal antibody, 4A4, is marked by arrow. IgG heavy chain (HC) and light chain (LC) are shown. (B) PP2A-C gene silencing and immunoprecipitation. Nuclear extracts from FaDu cells transfected with siRNA targeting PP2A-C (PPsi) and Csi were analyzed as in (A). Western blotting for detection of PP2A-C and p63 are shown. (C) PP2A phosphatase activity in Csi and p63si-transfected FaDu cells. Released phosphate amounts are shown in picomoles (pmol). (D) HeLa cells were transfected with Flag-β-catenin in combination with ΔNp63α or the vector plasmid, fractionated, and immunoprecipitated with an anti-Flag antibody (rabbit IgG). Obtained fractions (Input) and the immunoprecipitates (IP) were analyzed for indicated proteins. Arrowheads indicate the positions of 75 k_D and 50 k_D standard proteins. (E) HeLa cells were cotransfected with ΔNp63α, Myc-TCF4 and Flag-β-catenin as in (D). The nuclear extracts (Input) and immunoprecipitates (IP) with an anti-Myc antibody (rabbit IgG) were analyzed. Myc-tagged TCF4 appeared as a single band of ˜80 k_D. Control experiment was carried out by cotransfection of ΔNp63α and Myc-tagged β-catenin (Myc-β-cat) followed by nuclear fractionation and immunoprecipitation with the anti-Myc antibody (right panels)

Figure 3.

luc expression assay with pGL3-OT in HEK293. (A) Proposed WRE consensus sequences and WREs in pGL3-OT and pGL3-OF are aligned. (B) p53 binding consensus sequence and p53FM are aligned. (C) Enhancer structures of pGL3-OT and the mutants are shown. Blue boxes represent WREs and related stretches, and orange boxes p53 consensus half-sites. Letter X indicates the mutated nucleotide in pGL3-OF. Nucleotide numbers between the elements are in parentheses. (D)-(G) Results of the luc assays with indicated plasmids at 48 hr of transfection. Amounts (ng) of regulator plasmids (p63, β-catenin) are indicated for each reaction. Luciferase activities are shown in relation to the control reaction with the empty vector (1.0).

Figure 4.

luc expression assay with pGL3-OT in SAOS-2, Huh7 and FaDu cells. Results of the luc assays with pGL3-OT (A) and del-p53FM (B) in SAOS-2 cells. After transfection of FaDu cells with Csi and p63si (for 24 hr), pGL3-OT was introduced. Luciferase activity was measured at 48 hr of the luc plasmid transfection (C). Huh7 cells were transfected with pGL3-OT (D) and MMP7-WRE1-rep-luc (E) in combination with ΔNp63α, β-catenin and TCF4. Luc activities are indicated as in Figure 3.

Figure 5.

Structural and functional analyses of WREs of MMP7 and CCND2.(A)-(C), Line drawings and nucleotide sequences for the WREs analyzed in this study. Endogenous promoter region containing TATA box (TATA) and initiator site (Inr), and the body of the gene are shown by white boxes. Bold lines mark the endogenous sequences, while the thin lines mark sequences in the plasmids. The light blue and green boxes signify the promoter and the luc gene in pGL3-promoter, respectively. Nucleotides matching the WRE consensus in the positive and negative strands are indicated by arrows. Nucleotides deviated from the consensus are in lower case. (D)-(G), Results of the luc assays with SAOS-2 cells using the reporter plasmids shown in (A)-(C). Transfected plasmids and the DNA amounts are shown for each experiment.

Figure 6.

ChIP analysis for the MMP7-WRE1 and CCND2-WREb sites in FaDu.Relative positions of the primers used in the PCR are shown for MMP7-WRE1 (A, left) and CCND2-WREb (B, left). Filled and open boxes indicate WREs and p53 consensus half-sites, respectively. The amount of DNA precipitated by each antibody was quantified by PCR. After subtraction of the background value obtained with control IgG, DNA copy numbers relative to the input DNA (1.0) were determined (A, B, right). Statistic significance: *, 0.01 < P < 0.05