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

Abstract

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 11,25-(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 μM), 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.