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, cell proliferation, differentiation, and various immune functions. Defects in calcitriol actions thus have a broad spectrum of manifestations ranging from a lack of effects to hyperactivity. Lack of hormone effects may result from insufficient vitamin D supplementation or from defects in VDR functions. Hereditary resistance to calcitriol (HVDDR) usually results from a mutation in the VDR gene and manifests as rickets. We have used skin fibroblasts from subjects with HVDDR to explore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptor activation pathway, including defects in nuclear hormone uptake and in VDR subcellular targeting. We developed new technologies to explore these abnormalities at the cellular level. We cloned functional GFP chimeras of VDR and stably expressed them in kidney and osteoblast cells. The naturally occurring mutations in the VDR were then generated by site-directed mutagenesis and expressed in kidney-derived cell lines such as 293, CV-1, and COS-7 cells. Microscopy showed that calcitriol and synthetic calcitriol analogues induce rapid receptor redistribution from the cytoplasm into the nucleus. Defects in DNA binding, hormone binding, and dimerization selectively influenced subcellular trafficking of VDR. Calcitriol antagonists (BCA11 and BCA21), which we synthesized and purified recently, also inhibited VDR subcellular trafficking. During the characterization of these antagonists we realized that they are highly potent inhibitors of cancer cell proliferation (US provisional patent No. 60/300,909; filed June 22, 2001; NIH reference No. E-213/01/0). Studies in cultured cancer cell lines (breast, melanoma, glioma, prostate, colon, and osteosarcoma) showed that they counteract the growth stimulatory effect of calcitriol, which is induced by physiological hormone concentration. Both BCA11 and BCA21 still acted as calcitriol to inhibit proliferation at high, pharmacological concentrations. BCA11 not only inhibited breast cancer growth, but also induced differentiation. BCA11 was as potent as the commonly used estrogen antagonist tamoxifen in inhibiting the growth of human breast cancer xenografts in nude mice without causing any noticeable side effects. BCA11 was also effective to inhibit the growth of estrogen receptor negative breast cancers. Because VDR is expressed in 80% of breast cancers while only 40% express estrogen receptors, antagonists of VDR could have important potential for drug development. Hormone resistance, however, may develop after prolonged treatment and further studies are needed to overcome this problem. Accelerated degradation of VDR or degradation of its heterodimerization partner, RXR, also influence calcitriol effects. We found that the antiproliferative effect of calcitriol is more sensitive to changes in RXR degradation than other VDR functions. Such an accelerated proteasomal degradation of RXR causes calcitriol resistance in rat osteosarcoma cells (ROS). Stable expression of YFP-RXR in ROS cells restored the responsiveness to the antiproliferative effects of calcitriol and retinoids, as did treatments with proteasomal inhibitors. We started to explore calcitriol storage and release in the adipose tissue. Ongoing studies are directed at understanding uptake mechanisms, hormonal regulation and physiological importance of calcitriol release from the adipose tissue. We continue to use GFP chimeras of wild-type and mutant VDR and intracellular vitamin D binding proteins together with our fluorescent labeled calcitriol to gain further understanding of the disorders of the vitamin D endocrine system.