An AP-1 Site in the Nerve Growth Factor Promoter Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Nerve Growth Factor Expression in Osteoblasts†
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1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, induces nerve growth factor (NGF) synthesis in a variety of different cell lines. The mechanism by which 1,25(OH)2D3 induces NGF, however, is poorly understood. We used a series of full-length and truncated NGF promoter-human growth hormone (hGH) reporter gene plasmids to investigate the mechanism of 1,25(OH)2D3-induced NGF expression in osteoblasts. Untransfected rat osteosarcoma cells (ROS 17/2.8) treated with 1,25(OH)2D3 showed a 2-fold increase in NGF expression compared to control cells. ROS 17/2.8 osteosarcoma cells were transfected with the NGF-hGH reporter plasmids and treated with 10(-)8 M 1,25(OH)2D3. The full-length NGF promoter (-1800 to +120)-hGH reporter construct showed an approximately 2-fold increase in hGH release. Plasmids with successive 5'-deletions showed enhanced hGH expression in treated cells and control cells. A similar series of NGF promoter-hGH reporter gene constructs, lacking the AP-1 site located within the first intron of the NGF gene, were also transiently transfected into ROS 17/2.8 cells. When these cells were treated with the same dose of 1,25(OH)2D3, no increase in hGH expression was seen compared to control cells, demonstrating that this AP-1 site is essential for 1,25(OH)2D3-mediated NGF up-regulation. Since 1,25(OH)2D3 is known to activate the transcription of several genes through its interaction with the vitamin D receptor (VDR), we performed a series of gel electrophoretic mobility shift assays to determine if the VDR binds directly to the AP-1 sequence. No evidence of VDR binding, either as a homodimer or as a heterodimer, to the AP-1 sequence was observed. Treatment of ROS 17/2.8 cells with 1,25(OH)2D3, however, resulted in an increase in AP-1 binding activity; however, no significant changes in c-jun and c-fos levels were observed. Our data show that in osteoblasts, 1,25(OH)2D3 induces NGF expression indirectly by increasing AP-1 binding activity.
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