The hormonal form of vitamin D, calcitriol, acts through the vitamin D receptor (VDR) to regulate calcium homeostasis, cell proliferation, differentiation, and various immune functions. Defects in the target actions of calcitriol thus have a broad spectrum of manifestations ranging from hyperreactivity to a lack of effects. Hereditary resistance to calcitriol (HVDRR) usually results from a mutation in the VDR gene and manifests as rickets. A collaborative study with Constantine Stratakis, NICHD, investigating the mutations in a family with HVDRR lead to the isolation of a bacterial artificial chromosome containing the VDR gene, and physical mapping of the VDR gene by fluorescent in situ hybridization and radiation hybrid analysis to chromosome 12cen-q12, flanked by markers SHGC 30216 and SHGC 9798. Furthermore, we used skin fibroblasts from subjects with HVDRR to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway. We used these natural """"""""knockout"""""""" cells to study the functional domains of VDR involved in the control of receptor localization. Fluorescent labeling techniques proved useful in investigating the mechanisms of VDR activation in living cells, and led us to further elucidate the pathophysiology of the vitamin D effector system. First, we developed fluorescent derivatives of calcitriol and characterized their biological activities. One of these labeled hormones, BODIPY-calcitriol, was used to study hormone binding to receptors in single living cells by confocal microscopy. These studies showed that hormone binds VDR both in the cytoplasm and in the nucleus, and indicated that calcitriol exposure induces translocation of cytoplasmic VDR into the nucleus. We identified a docking site for VDR in the endoplasmic reticulum, bound to calreticulin. We also showed that translocating VDR binds to microtubules both in living cells and in vitro, by copolymerization and coimmunoprecipitation experiments. Recently, we cloned green and blue fluorescent protein VDR chimeras to study translocation and intranuclear distribution of VDR. Transient expression of this GFP-VDR in mouse adenocarcinoma, rat osteosarcoma, and COS-7 cells showed that the chimeric protein is competent for hormone dependent transactivation and confirmed that hormone induces translocation of cytoplasmic VDR into the nucleus. Green fluorescent protein chimeras of mutant VDR variants are being used to gain further understanding of the mechanisms of vitamin D resistant rickets.
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