An AP-1 site in the nerve growth factor promoter is essential for 1, 25-dihydroxyvitamin D3-mediated nerve growth factor expression in osteoblasts.
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.
Veenstra
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TD
Nephrology Research Unit, Department of Medicine, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Fahnestock
M
M
Kumar
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D000818:Animals; D002117:Calcitriol / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D016762:Genes, fos; D016758:Genes, jun; D006367:HeLa Cells; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D012516:Osteosarcoma; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D018808:Transcription Factor AP-1; D019382:Human Growth Hormone; D002117:Calcitriol