The effect of 1,25 dihydroxyvitamin D₃ treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles

Highlights

• Colon specific deletion of both APC alleles causes adenomatous colon lesions.

• These lesions have elevated colonic beta catenin target gene expression.

• 1,25 Dihydroxyvitamin D increase colonic expression of classical vitamin D target.

• 1,25 Dihydroxyvitamin D does not reduce expression of beta catenin target genes.

• APC deficient colon lesions have low expression of the vitamin D receptor.

Abstract

In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)₂D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)₂D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions of mice with colon-specific deletion of both APC gene alleles (CAC; APC^Δ580/Δ580). At four weeks of age, CAC; APC^Δ580/Δ580 and control mice were injected with vehicle or 1,25(OH)₂D (1 μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APC^Δ580/Δ580 mice. 1,25(OH)₂D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APC^Δ580/Δ580 and control mice indicating the treatments activated the VDR. However, 1,25(OH)₂D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APC^Δ580/Δ580 or control mice. VDR mRNA and protein levels were lower (−65% and −90%) in the colon of CAC; APC^Δ580/Δ580 mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of transient receptor potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APC^Δ580/Δ580 mice. Our data show that short term exposure to 1,25(OH)₂D does not suppress colonic β-catenin signaling in vivo.

This article is part of a special issue entitled ‘17th Vitamin D Workshop’.

Introduction

Studies show that lifestyle factors such as diet can decrease the development of colorectal cancer [1], [2]. One factor proposed to suppress colorectal cancer development is vitamin D. Epidemiological studies show an inverse association between high serum 25 hydroxyvitamin D (25(OH)D), the marker of vitamin D status, and colon cancer risk [3], [4], [5], [6], [7], [8]. The anti-cancer effects of vitamin D are mediated through the vitamin D receptor (VDR) which binds to the active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)₂D), and regulates the transcription of genes involved in anti-cancer events (e.g., growth arrest, metastasis, angiogenesis) in colon cancer cells [9]. However, an alternate hypothesis to explain vitamin D-mediated growth arrest is that VDR disrupts β-catenin signaling and blocks the Wnt-signaling pathway.

In normal colonic cells, β-catenin is retained in the cytoplasm by interacting with adenomatous polyposis coli (APC) or at the plasma membrane by binding to E-cadherin at adherens junctions. A destruction complex of Axin, APC, and glycogen synthase kinase-3 keeps β-catenin levels low in the nucleus by promoting its proteasomal degradation thereby reducing transcriptional regulation of genes by β-catenin [10], [11]. Activation of Wnt signaling releases β-catenin from the destruction complex, permits β-catenin nuclear accumulation, and increases binding of β-catenin to the TCF/LEF-1 transcriptional complex to regulate genes controlling cell proliferation (e.g., c-Myc) [12]. APC inactivating gene mutations that inhibit β-catenin-APC interactions are found in 85 percent of sporadic colorectal cancers [13].

Several in vitro studies have demonstrated that 1,25(OH)₂D inhibits β-catenin transcriptional activity in colon cancer cells [14], [15]. In addition, siRNA-mediated knockdown of VDR in SW480-ADH cells increased β-catenin-mediated reporter gene activity and inhibited 1,25(OH)₂D-mediated suppression of transcript levels from β-catenin target genes [15]. Consistent with this observation, downregulation of VDR gene expression by overexpression of Snails 1 or 2 blunted 1,25(OH)₂D-mediated suppression of β-catenin transcriptional activity [16], [17]. In SW480-ADH colon cancer cells, 1,25(OH)₂D treatment induced an interaction between VDR and β-catenin [14], [18] which reduced the interaction between β-catenin and TCF-4 necessary for gene transcription [14]. In addition, two-hybrid assays show that the VDR AF-2 domain directly interacts with the C terminus of β-catenin [19]. The interaction of VDR with β-catenin and the suppression of β-catenin-mediated gene transcription in the presence of 1,25(OH)₂D has also been shown in other colon cancer cell lines with mutant APC genes, such as Caco-2 and HT-29 [20] and in human embryonic kidney cells (HEK-293) [19]. However, direct testing of the ability of 1,25(OH)₂D to reduce β catenin-regulated transcript levels in vivo has not been reported.

Here we examined whether activating VDR with 1,25(OH)₂D can inhibit β-catenin transcriptional activity in the absence of APC function in vivo. To do this, we used the CAC; APC^Δ580/Δ580 mouse model we developed in which both APC alleles are inactivated specifically in the epithelial cells of the colon [21]. These mice develop dysplastic lesions in the distal colon that are characterized by high levels of nuclear and cytoplasmic β-catenin and increased cellular proliferation. Although β-catenin regulated transcripts are elevated in the distal colonic lesions from these mice, we found that a short course of three 1,25(OH)₂D injections did not decrease expression of known β-catenin targets in either wild type mice or in CAC; APC^Δ580/Δ580 mice in vivo.