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

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 (1μg/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'.

Keywords: Cancer; Gene expression; Hyperplasia; Vitamin D.

Figure 1

1,25(OH)₂D decreases mRNA levels for β-catenin target genes in Caco-2 and SW480-ADH cells. Proliferating Caco-2 and SW480-ADH cells were treated with 100 nM 1,25(OH)₂D for 24 h. RNA was isolated and analyzed by RT-PCR. Gene expression was normalized to RPLP0 expression. Bars = mean ± SEM (n=3) (A) CYP24A1 mRNA. * Significantly different than EtOH treatment (p≤0.05). (B) β-catenin target gene mRNA levels. Bars represent the fold-change in expression following 1,25(OH)₂D treatment. * Significantly reduced by vitamin D treatment (p<0.05).

Fig. 2

Impact of 1,25(OH)₂ D treatment on body weight and β-catenin gene targets in CAC; APC^Δ580/Δ580 mice. Four week old mice were given three daily injections with vehicle or 1,25(OH)₂ D (1 μg/kg body weight) and killed 6 h after the final injection. Bars = mean ± SEM (A) Body weight (n= 6-15 mice/group). * = p<0.05 vs vehicle treated wild-type mice. (B) β-catenin target gene mRNA levels in the distal colon of CAC; APC^Δ580/Δ580 mice. (n=12-15/group). * p < 0.05 vs control mice.

Fig 3

Impact of 1,25(OH)₂ D treatment on serum 1,25(OH)₂ D and colonic expression of vitamin D target genes in CAC; APC^Δ580/Δ580 mice. Four week old mice were given three daily injections with vehicle or 1,25(OH)₂ D (1 μg/kg body weight) and killed six h after the final injection. Data are expressed as mean ± SEM. Values with different letter superscripts are significantly different from one another (p < 0.05, Tukey's HSD) (A) Serum 1,25(OH)₂D. (n = 10/group). (B) CYP24A1 mRNA and (C) TRPV6 mRNA (n=14-20 mice/group).

Fig. 4

Impact of 1,25(OH)₂ D treatment on β-catenin gene targets and cellular proliferation in CAC; APC^Δ580/Δ580 mice. Four week old mice were given three daily injections with vehicle or 1,25(OH)₂ D (1 μg/kg body weight) and killed 6 h after the final injection. RNA was isolated from the distal colonic mucosa of CAC; APC^Δ580/Δ580 mice and RT-PCR was used to measure β-catenin gene targets. Data are expressed as mean ± SEM (n=14 mice/group).

Fig. 5

VDR mRNA and protein expression in colonic lesions from CAC; APC^Δ580/Δ580 or CAC; APC^Δ580/WT mice. (A, B) Four week old CAC; APC^Δ580/Δ580 mice were given three daily injections with vehicle or 1,25(OH)₂ D (1 μg/kg body weight) and killed six h after the final injection. RNA was isolated from the distal colonic mucosa and RT-PCR was used to measure VDR mRNA (A). Data are expressed as mean ± SEM (n=5-20 mice/group). Values with different letter superscripts are significantly different from one another (p≤0.05, Tukey's HSD). (B) Whole protein extracts from colon were prepared and pooled. Western blot analysis was conducted for VDR protein levels. β-actin was used as a loading control. (Veh = vehicle; 1,25D = 1,25(OH)₂D) (C) Tumors from the distal colon of 20 wk-old CAC; APC^Δ580/WT mice were saved and RNA was isolated and analyzed by RT-PCR for VDR mRNA levels. Bars represent the mean + SEM (n = 24/group), * Significantly different from normal mucosa of wild-type mice (p<0.05).