Silencing Dkk1 expression rescues dexamethasone-induced suppression of primary human osteoblast differentiation

Abstract

Background. The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in bone development and remodeling. The objectives of this study were firstly, to determine the effects of dexamethasone exposure on Wnt/β-catenin signaling at an intracellular and transcriptional level, and secondly, to assess the phenotypic effects of silencing the Wnt antagonist, Dickkopf-1 (Dkk1) in the setting of dexamethasone exposure. Methods. Primary human osteoblasts were exposed in vitro to 10 -8 M dexamethasone over a 72 h time course. The phenotypic marker of osteoblast differentiation was analyzed was alkaline phosphatase activity. Intracellular -catenin trafficking was assessed using immunoflourescence staining and TCF/LEF mediated transcription was analyzed using a Wnt luciferase reporter assay. Dkk1 expression was silenced using small interfering RNA (siRNA). Results. Primary human osteoblasts exposed to dexamethasone displayed a significant reductions in alkaline phosphatase activity over a 72 h time course. Immunoflourescence analaysis of β-catenin localization demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to dexamethasone exposure. These changes were associated with a reduction of TCF/LEF mediated transcription. Silencing Dkk1 expression in primary human osteoblasts exposed to dexamethasone resulted in an increase in alkaline phosphatase activity when compared to scrambled control. Conclusions. Wnt/β-catenin signaling plays a key role in regulating glucocorticoid- induced osteoporosis in vitro. Silencing Dkk1 expression rescues dexamethasone-induced suppression of primary human osteoblast differentiation. Targeting of the Wnt/-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass. © 2010 Butler et al; licensee BioMed Central Ltd.