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

Background: The Wnt/beta-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/beta-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 beta-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 beta-catenin localization demonstrated a significant reduction in intracytosolic and intranuclear beta-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/beta-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/beta-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass.