FGF-2 stimulation of RANK ligand expression in Paget's disease of bone.
Receptor activator for nuclear factor-kappaB ligand (RANKL), a critical osteoclastogenic factor expressed in marrow stromal/preosteoblast cells is up-regulated in Paget's disease of bone (PDB). We previously demonstrated that heat-shock factor-2 (HSF-2) is a downstream target of fibroblast growth factor-2 (FGF-2) signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells. In this study, we identified a 2.5-fold increase in serum FGF-2 levels in patients (n = 8) with PDB compared with normal subjects (n = 10). We showed that HSF-2 co-immunoprecipitates with heat-shock protein-27 (HSP-27) and that FGF-2 stimulation significantly increased phospho-HSP-27 levels in marrow stromal cells. Confocal microscopy revealed HSF-2 colocalization with HSP-27 in unstimulated cells and HSF-2 nuclear translocation upon FGF-2 stimulation. We further show that FGF-2 stimulation significantly increased the levels of phosphorylated signal transducers and activators of the transcription (p-STAT-1) in these cells. Western blot analysis confirmed that small interfering RNA suppression of STAT-1 significantly decreased (3.2-fold) RANKL expression and promoter activity in FGF-2-stimulated cells. Chromatin immunoprecipitation assay revealed STAT-1 binding to a putative motif located far upstream (-8 kb) in the hRANKL gene promoter region. These results suggest STAT-1 is a downstream effector of FGF-2 signaling and that elevated levels of FGF-2 stimulates RANKL expression in PDB.
Sundaram
Kumaran
K
Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Senn
Joseph
J
Yuvaraj
Sambandam
S
Rao
D Sudhaker
DS
Reddy
Sakamuri V
SV
D001854:Bone Marrow Cells / Q000166:cytology; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005786:Gene Expression Regulation; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010001:Osteitis Deformans / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology; D014157:Transcription Factors / Q000378:metabolism
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D053245:RANK Ligand; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; C070389:HSF2 protein, human