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


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/β-catenin signaling pathway offers an exciting opportunity to develop novel anabolic bone agents to treat osteoporosis and disorders of bone mass.
Butler
Joseph S
JS
Clinical Research Centre, UCD School of Medicine & Medical Science, Mater Misericordiae University Hospital, Dublin, Ireland. josephsbutler@hotmail.com
Queally
Joseph M
JM
Devitt
Brian M
BM
Murray
David W
DW
Doran
Peter P
PP
O'Byrne
John M
JM
D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects*; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*
C501126:DKK1 protein, human; D006131:Growth Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D003907:Dexamethasone