1 alpha,25 dihydroxyvitamin D3 rapidly regulates the mouse osteoprotegerin gene through dual pathways.
1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG gene expression both by accelerating the degradation of mRNA and by suppressing promoter activity. The latter process was mediated through the AP-1 binding site by a reduction in the proportion of phospho-c-Jun in a JNK-independent manner.


BACKGROUND
Osteoclastogenesis is regulated by an integrated network of numerous bone metabolic factors, among which 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] promotes osteoclastogenesis by reciprocally upregulating the expression of RANKL and downregulating that of osteoprotegerin (OPG).


METHODS
To analyze the mechanism by which 1 alpha,25(OH)(2)D(3) suppresses OPG, we characterized cis-acting elements of the mouse OPG gene and assessed the post-transcriptional modifications by actinomycin D assays.


RESULTS
1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG expression and shortened the half-life of OPG mRNA; additionally, the c-Jun homodimer bound to the AP-1 binding site (TGACTGA, -293/-287) and maintained steady-state transcription of the OPG gene. Furthermore, mutation of the AP-1 site negated 1 alpha,25(OH)(2)D(3)-driven OPG suppression. Moreover, 1 alpha,25(OH)(2)D(3) treatment of ST2 cells decreased the amount of phosphorylated c-Jun protein (phospho-c-Jun), while the total amount of c-Jun remained constant; however, the amount of phosphorylated Jun N-terminal kinase (JNK) was nearly unchanged by 1 alpha,25(OH)(2)D(3) treatment.


CONCLUSIONS
Taken together with the observation that the OPG promoter has no consensus negative vitamin D-responsive elements, these data suggest that 1 alpha,25(OH)(2)D(3) transrepresses mouse OPG by reducing the proportion of phospho-c-Jun in a JNK-independent manner. Our data indicated that short-term treatment with 1 alpha,25(OH)(2)D(3) effectively downregulated OPG expression both by accelerating the degradation of OPG mRNA and by transrepressing the OPG gene through its AP-1 binding site in the catabolic phase. The OPG gene became insensitive to 1 alpha,25(OH)(2)D(3) treatment, however, and reverted to its steady-state expression level over time, leading to the anabolic phase of the effect of 1 alpha,25(OH)(2)D(3) on bone.
Kondo
Takeshi
T
Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Kitazawa
Riko
R
Maeda
Sakan
S
Kitazawa
Sohei
S
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D018920:Coculture Techniques; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D053244:Osteoprotegerin; D011401:Promoter Regions, Genetic / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D020218:Response Elements / Q000235:genetics; D013997:Time Factors; D024363:Transcription Initiation Site; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology*
D006023:Glycoproteins; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C506031:Tnfrsf11b protein, mouse; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D003609:Dactinomycin