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J. Biol. Chem., Vol. 279, Issue 11, 10500-10507, March 12, 2004

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Functional Role for Heat Shock Factors in the Transcriptional Regulation of Human RANK Ligand Gene Expression in Stromal/Osteoblast Cells^*

Jennifer L. Roccisana, Noriaki Kawanabe, Hiroshi Kajiya, Masanori Koide, G. David Roodman, and Sakamuri V. Reddy, Recipient of a Department of Defense Medical Research Award¶

From the Department of Medicine/Division of Hematology, The University of Pittsburgh, Pittsburgh, Pennsylvania 15213 and the Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15213

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)₂D₃ 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.

Received for publication, April 9, 2003 , and in revised form, December 16, 2003.

^* This work was supported by National Institutes of Health Grants DE 12603 and AR 049363.

^¶ To whom correspondence should be addressed: Division of Hematology-Oncology, Dept. of Medicine, University of Pittsburgh, Liliane S. Kaufmann Bldg., Suite 601, 3471 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-4542; Fax: 412-692-4144; E-mail: reddysv{at}msx.upmc.edu.

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