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

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.

Sclerostin and Dickkopf-1 as Therapeutic Targets in Bone Diseases

“…RANKL has been identified as a key regulator of the increased bone resorption; however, the specific regulators responsible for the decreased bone formation have not yet been identified. Several studies have demonstrated that glucocorticoids induce DKK1 expression in osteoblasts both in vitro (118) and in vivo (33), and that inhibition of DKK1 using small interfering RNA (siRNA) prevented the dexamethasone-induced suppression of primary human osteoblast differentiation (119). A significant induction of both DKK1 and sclerostin expression was observed in bones of mice treated with prednisolone for 56 d, which also resulted in significant negative effects on trabecular bone.…”

Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling

“…DKK1 also acts within the bone to specifically block Wnt/β-catenin signaling in osteoblasts, thereby inhibiting osteoblast development and activity [19]. DKK1 exerts this function by binding to either of the single-pass transmembrane receptor proteins Kremen 1 or Kremen 2 and also within the carboxyl-terminal propeller domains of either the LRP5 or LRP6 co-receptors [20].…”

HIV‐1 protein induced modulation of primary human osteoblast differentiation and function via a Wnt/β‐catenin‐dependent mechanism

“…HOBs were treated with 100ng/mL of HIV-1 gp120 (NIH AIDS Research and Reference Reagent Program) over a 48h time course. The HIV-1 gp120 used in this line of experiments was one of a number of agents being assessed simultaneously for osteogenic effects in HOBs and the same experimental controls were used for all agents analyzed 20 .…”

“…L-Wnt3a culture medium was supplemented with 400 µg/ml geneticin to maintain selective pressure. Conditioned medium from L-Wnt3a and control L-cells was collected according as per manufacturer’s protocols and as previously reported 20 . Cells were passaged 1:10 in 8 ml medium without geneticin and left to grow for 10 days.…”

“…Moon, University of Washington, Seattle, WA, USA) and the control reporter pfuBAR (1µg total; Dr. RT. Moon, University of Washington, Seattle, WA, USA) using GeneJuice (Novagen, Madison, WI, USA), as previously reported 20 . The β-catenin activated reporter (pBAR) used to transfect HOBs contains 12 TCF binding sites (5’-AGATCAAAGG-3’) separated by distinct 5 base linkers.…”