Modulation of mouse RANKL gene expression by Runx2 and PKA pathway.
Runx2 regulates the target genes characteristic of osteoblastic phenotypes, while exerting diverse and sometimes controversial effects on osteoblastic cells depending on their differentiation stage. Receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) is a membrane bound cytokine essential for osteo(chondro)clastogenesis. During endochondral ossification, while Runx2-positive hypertrophic chondrocytes express RANKL, the steady-state expression of the RANKL gene in osteoblastic cells is, at later stages, kept at a relatively low level to sustain the established bone. The aim of this study was to elucidate the mechanism whereby Runx2 and the protein kinase A (PKA) pathway modulate RANKL expression, especially from the viewpoint of their functions in RANKL basic promoter activity and in chromatin structural changes in osteoblastic/stromal cells. Osteoblastic/stromal cell lines derived from normal and Runx2-deficient mice were used to analyze endogenous RANKL gene expression by real-time reverse transcription (RT)-PCR, the acetylation status of the H3 and H4 histone proteins associated with the 5'-flanking region of the RANKL gene by chromatin immunoprecipitation, and the exogenously transfected RANKL gene promoter activity both in the steady-state and under PKA-activated conditions. Here, we demonstrate that Runx2 suppresses steady-state RANKL gene expression by condensing chromatin, while showing a slightly positive effect on RANKL basic promoter activity. Besides acting through the CRE-like region (-0.96 kb) of the RANKL gene promoter, forskolin (FK) treatment transactivates the RANKL gene by antagonizing the function of Runx2, by reducing Runx2 mRNA expression and by opening the chromatin conformation far upstream (more than 40 kb) of the RANKL gene.
Mori
Kiyoshi
K
Department of Bioinformatics, Division of Molecular Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Kitazawa
Riko
R
Kondo
Takeshi
T
Maeda
Sakan
S
Yamaguchi
Akira
A
Kitazawa
Sohei
S
D000107:Acetylation; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006657:Histones / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts; D053244:Osteoprotegerin / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000235:genetics* / Q000378:metabolism*; D017154:Stromal Cells
D050659:Core Binding Factor Alpha 1 Subunit; D006657:Histones; D053244:Osteoprotegerin; D053245:RANK Ligand; C493716:Runx2 protein, mouse; C505689:Tnfsf11 protein, mouse; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases