Vitamin D Resistance And Related Disorders

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate calcium homeostasis, maintenance of skeletal integrity, immune defense, hormone secretion, hair follicle cycling, and mammary gland development. The long-term objective of our studies is to elucidate the pathophysiology of diseases caused by excess or insufficient calcitriol actions. We apply modern imaging, biochemical, and genetic approaches to study the regulation of calcitriol metabolism and actions at the molecular level. Abnormal hormonal effects may result from insufficient or excess vitamin D supplementation or from abnormal VDR functions. One of the diseases caused by aberrant VDR functions is hereditary resistance to calcitriol (HVDDR). This disease is usually results from a mutation in the VDR gene and manifests as rickets. We have used skin fibroblasts from subjects with HVDDR to explore the molecular mechanisms of VDR activation. These fibroblasts either lack VDR or express mutant VDRs, which display abnormalities in discrete steps of the receptor activation pathway. We found that several mutations cause defects in VDR subcellular localization and dimerization with retinoid X receptor (RXR). Our first objective was to characterize these defects. We cloned functional fluorescent protein chimeras of VDR and RXR and explored the impact of VDR mutations on VDR functions by site-directed mutagenesis, dynamic microscopy experiments, and nuclear export assays. Microscopy showed that calcitriol and synthetic calcitriol analogues induce rapid receptor redistribution from the cytoplasm into the nucleus. Defects in DNA binding, hormone binding, coactivator binding, and dimerization selectively influenced intranuclear compartmentalization and nucleocytoplasmic trafficking of VDR. We identified sequences of the VDR and the RXR that define interactions with the nuclear import and export machinery and showed that the effect of mutations impairing VDR import or export correlated with the ability of VDR to regulate transcription of target genes. Recently we visualized changes in coactivator and corepressor intracellular trafficking in response to calcitriol. These studies lead to a better understanding of the growth regulatory effects of calcitriol. Our studies in rat osteosarcoma cells demonstrated that accelerated degradation of RXR and the expression of a dominant negative truncated RXR could cause resistance to the growth inhibitory effects of calcitriol. We then synthesized and purified calcitriol antagonists (BCA11 and BCA21), and found that similarly to VDR agonists they inhibited proliferation of cancer cell lines and the growth of human breast cancer xenografts in nude mice (US provisional patent No. 60/300,909; filed June 22, 2001; NIH reference No. E-213/01/0). Using GST pull-down experiments and microscopy, we found that both VDR agonists and antagonists induce the release of the corepressor NCoR from VDR, and this leads to the export of factors from the nucleus to the mitochondria, where they cause apoptosis. The presence or absence of these factors defines, at least in part, the sensitivity of cells to the growth inhibitory effects of vitamin D derivatives. Our second objective was to elucidate mechanisms that regulate the subcellular targeting and metabolism of the hormone, calcitriol. First, we studied subcellular targeting of calcitriol with microscopy. These experiments relied on using our biologically active fluorescing derivatives of calcitriol together with green fluorescent protein chimeras of heat shock proteins (hsp), which were generated by our collaborators Dr. Adams and colleagues at Cedar-Sinai Medical Center. We found that overexpression of hsp70 family of proteins increased cellular uptake of calcitriol. Moreover, mutational analysis demonstrated that members of the hsp70 family of proteins play important roles in calcitriol endocytosis, targeting to the endoplasmic reticulum and the mitochondria. Our ongoing studies explore the roles of Klotho protein, a protein that prevents several age-related diseases in mice, in the regulation of calcitriol targeting and metabolism. We generated GFP tagged Klotho and demonstrated that osteoblasts, which do not express Klotho, internalize Klotho protein from the cell culture media and this may be the mechanism by which Kloto influences mitochondrial conversion of vitamin D metabolites in osteoblasts. We continue to explore novel factors that regulate mitochondrial synthesis of calcitriol, such as histamine, and expand our findings to the level of the intact animal. In conclusion, we made major progress in all three areas of our research. Our findings lead to a better understanding of diseases associated with insufficient or excess calcitriol actions.