title: Functional role for heat shock factors in the transcriptional regulation of human RANK ligand gene expression in stromal/osteoblast cells.
abstract: RANK Ligand (RANKL) is a critical osteoclastogenic factor that is expressed on stromal cells and osteoblasts. Most resorption stimuli induce osteoclast formation by modulating RANKL gene expression in marrow stromal/osteoblast cells. However, it is unclear how these stimuli modulate RANKL gene expression in the bone microenvironment. To characterize the transcriptional control of human RANKL gene expression in stromal/osteoblast cells, we PCR-amplified and cloned a 2-kb 5'-flanking sequence of the RANKL gene, using normal human osteoblast derived genomic DNA as a template. Sequence analysis identified the presence of several potential Heat Shock Factor (HSF) responsive elements (HSE) in the human RANKL gene promoter region. Co-expression of HSF-1 or HSF-2 with the RANKL gene promoter-luciferase reporter plasmid in human osteoblastic cells (NOBC) demonstrated a 2-fold and 4.5-fold increase in promoter activity, respectively. RT-PCR analysis for HSF-1 and 2 mRNA expression in human bone marrow-derived stromal cells (SAKA-T) and osteoblast cells detected only HSF-2 expression. As evident from EMSA analysis, in contrast to 1,25(OH)(2)D(3) SAKA-T cells treated with b-FGF demonstrated increased levels of HSF-2 binding to the HSE present in the RANKL gene promoter region. Immunocytochemical staining further confirmed nuclear localization of HSF-2 in both SAKA-T transformed stromal cells and human bone marrow derived primary stromal/preosteoblastic cells in response to b-FGF treatment. Furthermore, b-FGF treatment of SAKA-T cells transfected with the luciferase reporter plasmid containing the hRANKL HSE region (-2 kb to -1275 bp) upstream to a heterologous promoter showed increased levels of transactivation. Western blot analysis further demonstrated enhanced levels of RANKL expression and HSP-27 phosphorylation in SAKA-T cells treated with b-FGF. In addition, overexpression of HSF-2 in SAKA-T cells resulted in a 5-fold increase in the levels of RANKL expression in these cells. These data further suggest that HSF-2 is a downstream target of b-FGF to induce RANKL expression in stromal/osteoblast cells, and that HSF may play an important role in modulating RANKL gene expression in the bone microenvironment.
authors.affiliation: Department of Medicine/Division of Hematology, The University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Roccisana
authors.forename: Noriaki
authors.initials: N
authors.lastname: Kawanabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kajiya
authors.forename: Masanori
authors.initials: M
authors.lastname: Koide
authors.forename: G David
authors.initials: GD
authors.lastname: Roodman
authors.forename: Sakamuri V
authors.initials: SV
authors.lastname: Reddy
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; C070389:HSF2 protein, human; D008156:Luciferases; D002117:Calcitriol