Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1alpha-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D(3), the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-gamma or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D(3) and the VDR in the control of adipocyte metabolism and lipid storage in vivo.

The vitamin D-3 receptor (VDR) is a nuclear receptor that modulates gene expression when complexed with its ligand 1-alpha,25-dihydroxycholecalciferol [1,25(OH)(2)-D(3)], which is the biologically active form of vitamin D-3. The cellular effects of VDR signaling include growth arrest, differentiation and/or induction of apoptosis, which indicate that the vitamin D pathway participates in negative-growth regulation. Although much attention has been directed in recent years toward the development of synthetic vitamin D analogs as therapeutic agents for a variety of human cancers including those derived from the mammary gland, studies on vitamin D as a chemopreventive agent for breast cancer have been quite limited. The VDR is expressed in normal mammary gland, where it functions to oppose estrogen-driven proliferation and maintain differentiation; this suggests that 1,25(OH)(2)-D(3) participates in negative-growth regulation of mammary epithelial cells. Furthermore, preclinical studies show that vitamin D compounds can reduce breast cancer development in animals, and human data indicate that both vitamin D status and genetic variations in the VDR may affect breast cancer risk. Collectively, findings from cellular, molecular and population studies suggest that the VDR is a nutritionally modulated growth-regulatory gene that may represent a molecular target for chemoprevention of breast cancer.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D2 inhibits breast cancer cell growth both in vivo and in vitro. In addition to its anti-proliferative effects, 1,25(OH)2D3 induces morphological and biochemical markers of apoptosis in MCF-7 cells. In the studies reported here, we compared the effects of 1,25(OH)2D3 and EB1089, a low calcemic vitamin D analog, on cell cycle kinetics and apoptosis in MCF-7 cells. Both vitamin D compounds reduced viable MCF-7 cell number in a time and dose dependent manner, with EB1089 approximately 50 fold more potent than 1,25(OH)2D3. Flow cytometric analysis indicated that both agents induced cell cycle arrest in G, G1 which was associated with accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein. MCF-7 cells treated with either 1,25(OH)2D3 or EB1089 for 48 h exhibited characteristics of apoptosis, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and DNA fragmentation. Cells treated with either agent exhibited up regulation of proteins associated with mammary gland regression (clusterin and cathepsin B) and down regulation of the anti-apoptotic protein bcl-2. These studies demonstrate that, despite its lower calcemic activity in vivo, the vitamin D analog EB1089 induces effects that are indistinguishable from those of 1,25(OH)2D3 on cell number, cell cycle and indices of apoptosis in MCF-7 cells in vitro. In addition, since both agents rapidly down regulate estrogen receptor, disruption of estrogen dependent signalling may play a role in the induction of apoptosis by vitamin D compounds in MCF-7 cells.

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. Our laboratory has shown that 1␣,25(OH) 2 D 3 induces morphological and biochemical markers of apoptosis (chromatin and nuclear matrix condensation, and DNA fragmentation) in breast cancer cells (MCF-7) (4, 5); however, the precise mechanism by which 1␣,25(OH) 2 D 3 and the VDR mediate apoptosis is poorly understood.

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Nuclear receptors exert profound effects on mammary gland physiology and have complex roles in the etiology of breast cancer. In addition to receptors for classic steroid hormones such as estrogen and progesterone, the nuclear vitamin D receptor (VDR) interacts with its ligand 1α,25(OH)2D3 to modulate the normal mammary epithelial cell genome and subsequent phenotype. Observational studies suggest that vitamin D deficiency is common in breast cancer patients and that low vitamin D status enhances the risk for disease development or progression. Genomic profiling has characterized many 1α,25(OH)2D3 responsive targets in normal mammary cells and in breast cancers, providing insight into the molecular actions of 1α,25(OH)2D3 and the VDR in regulation of cell cycle, apoptosis, and differentiation. New areas of emphasis include regulation of tumor metabolism and innate immune responses. However, the role of VDR in individual cell types (i.e., epithelial, adipose, fibroblast, endothelial, immune) of normal and tumor tissues remains to be clarified. Furthermore, the mechanisms by which VDR integrates signaling between diverse cell types and controls soluble signals and paracrine pathways in the tissue/tumor microenvironment remain to be defined. Model systems of carcinogenesis have provided evidence that both VDR expression and 1α,25(OH)2D3 actions change with transformation but clinical data regarding vitamin D responsiveness of established tumors is limited and inconclusive. Because breast cancer is heterogeneous, analysis of VDR actions in specific molecular subtypes of the disease may help to clarify the conflicting data. The expanded use of genomic, proteomic and metabolomic approaches on a diverse array of in vitro and in vivo model systems is clearly warranted to comprehensively understand the network of vitamin D regulated pathways in the context of breast cancer.

The vitamin D receptor (VDR) is expressed in human adipocytes and is transiently induced during early adipogenesis in mesenchymal progenitor cell models. VDR null mice exhibit enhanced energy expenditure and reduced adiposity even when fed high fat diets. Adipocyte-specific transgenic-expression of human VDR in mice enhances adipose tissue mass, indicating that VDR activation in adipocytes enhances lipid storage in vivo. In these studies, we conducted genomic profiling and differentiation assays in primary cultures of human adipose-derived mesenchymal progenitor cells to define the role of the VDR and its ligand 1,25-dihydroxyvitamin D3 (1,25D) in adipogenesis. In the presence of adipogenic media, 1,25D promoted lipid accumulation and enhanced the expression of FABP4, FASN, and PPARγ. Mesenchymal cells derived from 6-month old VDR null mice exhibited impaired adipogenesis ex vivo but differentiation was restored by stable expression of human VDR. STEAP4, a gene that encodes a metalloreductase linked to obesity, insulin sensitivity, metabolic homeostasis and inflammation, was highly induced in human adipose cells differentiated in the presence of 1,25D but was minimally affected by 1,25D in undifferentiated precursors. These studies provide a molecular basis for recent epidemiological associations between vitamin D status, body weight and insulin resistance which may have relevance for prevention or treatment of metabolic syndrome and obesity.