The human PKP2/plakophilin-2 gene is induced by Wnt/β-catenin in normal and colon cancer-associated fibroblasts

Abstract

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/β-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/β-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/β-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by β-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/β-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

Keywords: PKP2/Plakophilin-2; Wnt/β-catenin signalling; colon cancer; gene regulation; normal and cancer-associated fibroblasts.

Figure 1

Wnt3A regulates the expression of multiple genes in CCD‐18Co human colon myofibroblasts. (a) Validation using RT‐qPCR of a representative sample of six genes (PTGER3, ODZ3, ILDR2, AMIGO2, WNT16 and TRPA1) that were up‐ or downregulated by Wnt3A in RNA‐seq experiments. Cells were treated with either Wnt3A (+) or vehicle (–) for 24 hr. The results shown are means ± S.D. of three independent biological samples, each one determined in triplicate (***p ≤ 0.001). (b) Wnt3A triggers the expression of a set of target genes encoding inhibitors of the Wnt/β‐catenin pathway. Cells were stimulated with either Wnt3A or vehicle for the indicated times and NKD1, NKD2, DKK2, and APCDD1 expression was analysed by RT‐qPCR. Results are shown as fold over control at the indicated times and are means ± S.D. of three independent biological samples, each one determined in triplicate (n.s., non‐significant; *** p ≤ 0.001).

Figure 2

Wnt3A induces the expression of Plakophilin‐2 in CCD‐18Co human colon myofibroblasts. (a) RT‐qPCR analysis showing the upregulation of PKP2 mRNA by Wnt3A. Results are shown as fold over control at the indicated times and are means ± S.D. of three independent biological samples, each one determined in triplicate (n.s., non‐significant; *** p ≤ 0.001). (b) Western blot analysis of Plakophilin‐2 upregulation by Wnt3A in a time course experiment. β‐Actin was used as a loading control. Fold increases over vehicle‐treated controls are indicated. A representative experiment is shown. (c) The Wnt/β‐catenin pathway/tankyrase inhibitor XAV‐939 reduces Wnt3A‐dependent Plakophilin‐2 upregulation. Cells were pre‐treated for 4 hr with either 1 µM XAV‐939 (+) or vehicle (–) and then stimulated with Wnt3A (+) for 24 hr. β‐Actin was used as a loading control. A representative experiment is shown. Bar plot represents means ± S.D. of four independent experiments (n.s., non‐significant; ** p ≤ 0.01). (d) Non‐canonical Wnt5A cannot induce Plakophilin‐2 accumulation. Myofibroblasts were stimulated with either Wnt3A, Wnt5A or both for 24 hr, and Plakophilin‐2 expression was analysed by Western blot. β‐Actin was used as a loading control. A representative experiment is shown. Bar plot represents means ± S.D. of four independent experiments (n.s., non‐significant; ** p ≤ 0.01). (e) RT‐qPCR analysis of the expression of PKP family members in response to Wnt3A stimulation. CCD‐18Co human colon myofibroblasts were treated with either Wnt3A (+) or vehicle (–) for 24 hr. Results are shown as fold over control and are means ± S.D. of three independent biological samples, each one determined in triplicate (n.s., non‐significant; *** p ≤ 0.001).

Figure 3

Wnt3A induces PKP2/Plakophilin‐2 expression in normal fibroblasts (NF) and in cancer‐associated fibroblasts (CAF). NF and CAF were obtained from CRC patients as described in the Materials and Methods and treated with either Wnt3A (+) or vehicle (–) for 24 hr. Total RNA and protein was purified and PKP2/Plakophilin‐2 mRNA and protein levels were analysed by RT‐qPCR (a) and western blot (b), respectively. Results in (a) are shown as fold over the NF control and are means ± S.D. of three independent biological samples, each one determined in triplicate (**p ≤ 0.01; *** p ≤ 0.001). β‐Actin was used as a loading control in (b) and fold increases over vehicle‐treated controls are indicated.

Figure 4

The human PKP2 gene promoter is activated by β‐catenin/TCF. (a) Actinomycin D (ACTD) blocks Wnt3A‐induced accumulation of PKP2 mRNA. CCD‐18Co cells were pre‐treated for 1 hr with either 5 µg/ml ACTD or vehicle and then stimulated with Wnt3A for 8 hr. Total RNA was purified and PKP2 mRNA levels were analysed by RT‐qPCR. Results are shown as fold over control and are means ± S.D. of three independent biological samples, each one determined in triplicate (n.s., nonsignificant; *** p ≤ 0.001). (b) Diagram of the PKP2 promoter constructs used in this study showing the location of the putative wild‐type (5′‐CTTTG[A/T][A/T]‐3′, white boxes) or mutant (5′‐CTTTGGC‐3′, X‐crossed white boxes) β‐catenin/TCF binding sites. (c) HEK‐293T cells were co‐transfected with the indicated PKP2 promoter constructs together with TCF4‐VP16 (+; a constitutively active form of TCF‐4) or an empty vector (–). The β‐catenin/TCF responsive DKK1 promoter was used as a positive control. A Renilla luciferase plasmid (pRL‐TK) was used as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla. Error bars represent S.D. (***p ≤ 0.001). (d) HEK‐293T cells were co‐transfected with the indicated PKP2 promoter wild‐type or mutant constructs together with TCF4‐VP16 (+) or an empty vector (–) and the experiment was performed as in (c). Error bars represent S.D. (*p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 5

An enhancer sequence located 20 kb upstream from the PKP2 transcription start site harbours a functional β‐catenin/TCF responsive element. (a) Diagram of the PKP2 enhancer constructs used in this study showing the location of the putative wild‐type (5′‐CTTTG[A/T][A/T]‐3′, white boxes) or mutant (5′‐CTTTGGC‐3′, X‐crossed white boxes) β‐catenin/TCF binding sites. (b) HEK‐293T cells were co‐transfected with either the forward (–20666/–20091) or reverse (–20091/–20666) 575 bp PKP2 enhancer construct together with TCF4‐VP16 (+) or an empty vector (–). The DKK1 promoter was used as a positive control and pRL‐TK as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla. Error bars represent S.D. (***p ≤ 0.001). (c) HEK‐293T cells were co‐transfected with the indicated PKP2 enhancer wild‐type or mutant constructs together with TCF4‐VP16 (+) or an empty vector (–) and the experiment was performed as in (b). Error bars represent S.D. (n.s., nonsignificant; *** p ≤ 0.001). (d) A diagram summarizing the human PKP2 gene regulatory sequences found in this study. White boxes represent functional β‐catenin/TCF binding sites.

Figure 6

Plakophilin‐2 antagonizes β‐catenin/TCF transcriptional activity. (a) HEK‐293T cells were transfected with reporter plasmids (pTOPFLASH or pFOPFLASH) to evaluate the transcriptional activity of the Wnt/β‐catenin pathway, together with an expression vector for β‐catenin (+) and in the presence or absence of an expression plasmid for Plakophilin‐2. pRL‐TK was used as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla, related to those of the mutant pFOPFLASH construct, and represented as fold over control. Error bars represent S.D. (***p ≤ 0.001). (b) HEK‐293T cells were transfected with reporter plasmids (4xwtCBF1Luc or 4xmtCBF1Luc) to evaluate the transcriptional activity of the Notch pathway, together with an expression vector for IntraCellular Notch Domain (ICND, +) and in the presence or absence of an expression plasmid for Plakophilin‐2. The experiment was performed as in (a). (c) HEK‐293T cells were transfected with a reporter plasmid (NF3) to evaluate the transcriptional activity of the NF‐κB pathway, together with an expression vector for p65 (+) and in the presence or absence of an expression plasmid for Plakophilin‐2. Samples were processed as in (a), values were normalized to those of Renilla, and represented as fold over control. Error bars represent S.D. (*p ≤ 0.05). (d) HEK‐293T cells were transfected with the β‐catenin/TCF responsive DKK1 promoter, together with an expression vector for β‐catenin (+) and in the presence or absence of an expression plasmid for Plakophilin‐2. The experiment was performed as in (a) and values are represented as fold over control. Error bars represent S.D. (***p ≤ 0.001). (e) β‐catenin protein levels are not affected by Plakophilin‐2 expression. Extracts from the experiment in (d) were analysed by Western blot to rule out the possibility that the effects of Plakophilin‐2 on β‐catenin/TCF‐dependent transcription were due to changes in β‐catenin levels as a consequence of Plakofilin‐2 expression.