Abstract
BackgroundThe 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.MethodsPrimary 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 β-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).ResultsPrimary 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.ConclusionsWnt/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/β-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass.
Highlights
The Wnt/b-catenin pathway is a major signaling cascade in bone biology, playing a key role in bone development and remodeling
We examined the phenotypic effects of attenuating the expression of Dkk1, a Wnt antagonist, in primary human osteoblasts exposed to dexamethasone
Dexamethasone exposure in vitro reduces alkaline phosphatase activity deposition in primary human osteoblasts To study the effects of dexamethasone, on markers of osteoblast differentiation, we exposed primary human osteoblasts to 10-8 M dexamethasone over a 72 h time course
Summary
The Wnt/b-catenin pathway is a major signaling cascade in bone biology, playing a key role in bone development and remodeling. There has been an increasing interest in elucidating the role played by developmental pathways, such as Wnt/ b-catenin cascade, in bone mass regulation [2]. The role of canonical Wnt signaling in bone development and remodeling was first identified when the lossof-function mutation of the LDL receptor-related protein (LRP5) was associated with osteoporosis psuedoglioma syndrome, a recessive disorder characterized by osteogenesis imperfecta with low bone mass and a predisposition to fractures as well as ocular defects [7,8]. We determined the effects of dexamethasone exposure on Wnt/b-catenin signaling in primary human osteoblasts. We examined the phenotypic effects of attenuating the expression of Dkk, a Wnt antagonist, in primary human osteoblasts exposed to dexamethasone
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