The Molecular Basis of Vitamin D Receptor and β-Catenin Crossregulation

The signaling/oncogenic activity of β-catenin can be repressed by activation of the vitamin D receptor (VDR). Conversely, high levels of β-catenin can potentiate the transcriptional activity of 1,25-dihydroxyvitamin D3 (1,25D). We show here that the effects of β-catenin on VDR activity are due to interaction between the activator function-2 (AF-2) domain of the VDR and C terminus of β-catenin. Acetylation of the β-catenin C terminus differentially regulates its ability to activate TCF or VDR-regulated promoters. Mutation of a specific residue in the AF-2 domain, which renders the VDR trancriptionally inactive in the context of classical coactivators, still allows interaction with β-catenin and ligand-dependent activation of VDRE-containing promoters. VDR antagonists, which block the VDRE-directed activity of the VDR and recruitment of classical coactivators, do allow VDR to interact with β-catenin, which suggests that these and perhaps other ligands would permit those functions of the VDR that involve β-catenin interaction.The authors wish to acknowledge the support of National Institutes of Health grants DK058196 and U54 CA100971 (S.W.B.), the AACR-Bristol Myers Squibb Translational Fellowship in Colon Cancer (S.S.), and the following LCCC Core Facilities: macromolecular analysis, microscopy, and tissue culture

Age-Related Changes in the Epithelial and Stromal Compartments of the Mammary Gland in Normocalcemic Mice Lacking the Vitamin D3 Receptor

The vitamin D3 receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation and involution. To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17β-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment

Effect of VDR ablation on serum calcium, estrogen, and estrogen responsive tissues in aging WT and VDR KO mice.

<p>A. Serum calcium levels in aging WT and VDR KO mice were equivalent prior to (2 and 8 months) and throughout the aging time course (12–16 months). B. Serum estrogen was measured by radioimmunoassay in WT and VDR KO mice at 2, 8, 12, 14, and 16 months of age. There was a significant reduction in serum estrogen by 12 months of age in the VDR KO mice that persisted through 16 months of age. *Statistically significant by Students t test, WT vs. VDR KO, n = 8–10 p<0.05. C. Representative hematoxylin and eosin Y stained sections of the ovary and the uterus, an estrogen responsive tissue. VDR KO tissues show signs of atrophy, likely due to incomplete follicle formation in the ovary and thus decreasing serum estrogen and inducing atrophy in the uterus compared to WT ovary supporting follicle formation and a responsive and robust uterus. Scale bar: 200 µm.</p

Morphological and quantitative assessment of inguinal mammary glands from 12, 14, and 16 month old WT and VDR KO mice.

<p>A. (Left Panels) Glands from VDR KO mice show decreased secondary and tertiary branching, signs of ductal thickening (ectasia), and large lymph nodes as the adipose tissue begins to atrophy with age. In contrast, WT glands show an extensive array of secondary and tertiary branches resting in a vast fat pad of plentiful adipose tissue. (Middle Panels) Glands at higher magnification from VDR KO mice show reduced secondary and tertiary branching and areas of darkly stained lesions along primary ducts (arrows). (Right Panels) Histological sections stained with hematoxylin and eosin Y show clusters of inflammatory cells along the primary ducts within glands of VDR KO mice compared to WT glands. B. Primary, secondary and tertiary branch points were quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016479#s4" target="_blank">materials and methods</a>. Primary branch counts showed a significant decrease between WT and KO glands at 16 months whereas secondary and tertiary branches were significantly decreased in glands from VDR KO mice at 14 and 16 months compared to WT control mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. C. Whole mounts from VDR WT and KO mice were utilized to measure the area of mammary fat pads using AxioVision software. Fat pad area was calculated by tracing around the exterior of the ductal branches of the mammary fat pads. At 14 and 16 months of age, there is a significant reduction in the mammary fat pads of VDR KO mice compared to WT controls. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. D. Whole mounts from VDR WT and KO mice were utilized to measure the mammary lymph nodes. Using AxioVision software, the exterior of the glandular lymph node was traced to calculate the area. At all time points, lymph nodes within glands from VDR KO mice were significantly enlarged compared to lymph nodes from WT mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05.</p