Vitamin D receptor gene polymorphisms: analysis of ligand binding and hormone responsiveness in cultured skin fibroblasts.

Recent reports have suggested that polymorphisms in the gene encoding the vitamin D receptor (VDR) determine a portion of the genetic contribution to bone mineral density (BMD). Individuals homozygous for the allele lacking the Bsm I restriction site in the intron between exons 8 and 9 (BB genotype) have been found to have lower BMD than individuals homozygous for the allele having the Bsm I site (bb genotype). Interestingly, this polymorphism has also been associated with prostate cancer risk. The observed changes in BMD and prostate cancer risk might be due to an alteration in the function or abundance of the VDR leading to differential responsiveness of target cells to the action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. To test this hypothesis, we cultured dermal fibroblasts from donors with BB, Bb, and bb genotypes and determined the level of VDR expression and the cellular responsiveness to 1,25(OH)2D3 treatment. VDR abundance, affinity for [3H]1,25(OH)2D3, and VDR mRNA levels were not detectably different in BB cells compared to bb cells. Moreover, equal expression of both VDR gene alleles was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA from Bb fibroblasts. Fibroblast responsiveness to 1,25(OH)2D3, assessed by induction of 24-hydroxylase mRNA, was similar between BB and bb cell types in dose-response experiments. Although there were individual variations in the parameters we measured, there were no detectable or consistent differences in mean values from our small sample of cultured dermal fibroblasts. In conclusion, we did not detect significant differences in VDR properties or cellular responsiveness to 1,25(OH)2D3 that correlated with VDR genotype. Our findings suggest that these polymorphisms do not affect VDR function, but rather may be a marker for a nearby gene that is responsible for the genotype-associated variation in osteoporosis and prostate cancer risk.

1,25-dihydroxyvitamin D3 induction of nerve growth factor in L929 mouse fibroblasts: effect of vitamin D receptor regulation and potency of vitamin D3 analogs.

Decreased nerve growth factor (NGF) synthesis in the hippocampus and reduced nerve growth factor receptor immunoreactivity in CH1-4 basal forebrain areas have been implicated in neurodegeneration. Vitamin D receptors (VDR) have been located in brain areas affected by neurodegenerative diseases. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the active form of vitamin D, has been shown to induce NGF in L929 mouse fibroblasts and rat hippocampus. In the present study we analyzed the VDR in L929 cells, which we used as a model system. We studied the regulation of VDR abundance and the ability of 1,25-(OH)2D3 to induce NGF synthesis. Scatchard analysis of [3H]1,25-(OH)2D3 binding showed the VDR concentration to be 173 fmol/mg protein and the affinity to be 0.12 nM. VDR was localized to nuclei of L929 cells by immunocytochemistry. Treatment of cells with forskolin (FSK; 50 microM), which activates the cAMP-protein kinase A pathway, resulted in an 8- to 10-fold up-regulation of VDR by 6 h, and VDR remained elevated at 24 h, as we have reported for other cells. NGF secretion was measured in serum-free conditioned medium using a double sided enzyme-linked immunosorbent assay. 1,25-(OH)2D3 treatment (0.1 pM to 10 nM) for 24 h increased the NGF concentration 2- to 3-fold, an effect that plateaued at 1 nM 1,25-(OH)2D3. VDR up-regulation by FSK pretreatment augmented the NGF response to 1,25-(OH)2D3 2-fold compared to that in vehicle-pretreated cells for a total 6-fold increase compared to basal NGF levels. The vitamin D analogs EB-1089 and 22-oxacalcitriol, which have been found to be less calcemic than 1,25-(OH)2D3, also induced NGF synthesis. The effects of these analogs were further enhanced by prior up-regulation of VDR with FSK. In conclusion, we have characterized the VDR in L929 cells and shown that 1,25-(OH)2D3 and its less calcemic analogs induce NGF. Furthermore, up-regulation of VDR abundance enhanced NGF induction. These effects of 1,25-(OH)2D3 and its analogs via VDR to regulate NGF synthesis may have significance for the eventual treatment of neurodegenerative diseases that are caused by decreased NGF production.

Vitamin D nuclear binding to neurons of the septal, substriatal and amygdaloid area in the Siberian hamster (Phodopus sungorus) brain.

Autoradiographic experiments were performed on brains of Siberian hamsters (Phodopus sungorus) injected with tritiated 1,25-dihydroxycholecalciferol. Nuclear labeling was prevented in the presence of excess unlabeled hormone. Strong nuclear concentration of radioactivity was observed in neurons of the nucleus basalis of Meynert, the medial septal nucleus, the nucleus of the diagonal band of Broca and the central amygdaloid group. The latter has been defined as consisting of the central nucleus of the amygdala, its extension into the sublenticular part of the substantia innominata of Reichert, and the lateral division of the bed nucleus of the stria terminalis. All these structures have been reported to be involved in memory and other cognitive processes, and to be affected by age-dependent neurodegenerative disorders such as Alzheimer's disease. Corresponding localization of 1,25-dihydroxycholecalciferol receptor sites in these select basal forebrain nuclei of the Siberian hamster may implicate vitamin D (soltriol), the steroid hormone of sunlight, in memory processing.

Immunohistochemical characterization of a widely spread Arg8-vasopressin-like neuroendocrine system in the cricket Teleogryllus commodus Walker (Orthoptera, Insecta).

Two distinct neurons located paramedially within the suboesophageal ganglion of female Australian crickets Teleogryllus commodus were shown to contain Arg8-vasopressin-like immunoreactivity. Immunostained processes project throughout the ganglion. They pass the circumoesophageal connectives, and finally they branch out and enter the neurohemal region of the deuterocerebrum. By means of radioimmunoassay in homogenized cricket brains and suboesophageal ganglia evidence could be found for the presence of a neurohypophyseal-peptide-like substance. Immunofluorescence findings indicate that immunoreactive axonal tracts extend backwards through the ventral nerve cord to the terminal ganglion. Numerous beaded axons with specific immunostaining were also detected within the lateral nerves.