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

Primary human osteoblasts from normal human hip samples were (Promocell, Heidelberg, Germany). Cells were cultured in Osteoblast Growth Medium (Promocell, Heidelberg, Germany) containing 10% FCS and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin) at 37°C, 5% CO₂. For all experiments primary human osteoblasts used were between 2^th and 5^th passage. Cells were treated over a 48 h time course with 10^-8 M Dexamethasone (Dex) (Sigma-Aldrich, Poole, UK). Institutional Review Board approval for this study was obtained from the Mater Misericordiae University Hospital Research Ethics Committee.

Alkaline phosphatase (ALP) activity was assayed by the measurement of released p-nitrophenol from p-nitrophenolphosphate (pNPP). Post-treatment cell lysates were collected by Cell Lytic MT (Sigma-Aldrich, Poole, UK) treatment and the extracted protein was frozen at -80°C for at least 2 h prior to ALP activity assay to disrupt cellular membranes. 10 μl of thawed cell lysates were incubated with 200 μl p NPP reagent (Sigma-Aldrich, Poole, UK) for 30 min at room temperature. ALP activity was measured at 405 nm and normalized to total protein extracted, which was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rochford, Illinois). One unit of ALP activity was defined as the amount required for the liberation of 1.0 μmol p-nitrophenol/min/mg of cellular protein.

Beta-catenin was visualized by indirect immunocytochemistry using a mouse anti β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the primary antibody. Primary human osteoblasts were plated on coverslips and treated with Dex-containing media. Cells were fixed in ice-cold methanol and permeabilized for ten minutes. Cells were then blocked with 10% goat serum for ten minutes at room temperature. Samples were incubated for 1 hr with primary antibody followed by a 30 min incubation with a goat anti-mouse TRITC-conjugate. Cells were viewed with a Zeiss Axioskop 40 fluorescence microscope using 20× objectives. Digital images were captured with a ProgRes C10^plus research digital camera (Jenoptik Laser Optik Systeme GmbH, Jena, Germany). The digital images were processed using the ProgRes CapturePro 2.1 image analysis softwear package (Jenoptik Laser Optik Systeme GmbH, Jena, Germany).

Mouse Wnt3a overexpressing cells (L-Wnt3a) and control non-transfected L-cells were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in DMEM with glutamax, 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. 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 to the manufacturer's instructions. Briefly, cells were passaged 1:10 in 8 ml medium without geneticin and left to grow for 10 days. Medium was collected from each cell line and replaced with 8 ml fresh medium for a further 3 days. This second batch of medium was then collected and the cells discarded. First and second batches were combined, sterile filtered (0.2 μm) and stored at -20°C until required.

Primary human osteoblasts plated in 24-well plates at 2 × 10⁴/cm² were transiently transfected with the Wnt-luciferase reporter construct pBAR (1 μg total; Dr. RT. Moon, University of Washington, Seattle, WA, USA) and the control reporter pfuBAR (1 μg total; Dr. RT. Moon, University of Washington, Seattle, WA, USA) using GeneJuice (Novagen, Madison, WI, USA). The β-catenin activated reporter (pBAR) contains 12 TCF binding sites (5'-AGATCAAAGG-3') separated by distinct 5 base linkers. These elements are directly upstream of a minimal thymidine kinase promoter which then drives the expression of firefly luciferase. The control reporter pfuBAR is identical to its sister reporter with the exception of a two base substitution in each TCF binding site. Both reporters contain a separate PGK promoter that constitutively drives the expression of a puromycin resistance gene, and both are in a lentiviral platform. Co-transfection with 0.5 μg of the internal control reporter pSL9EF1a(P)RLUC (Dr. RT. Moon, University of Washington, Seattle, WA, USA), driving constitutive expression of Renilla luciferase, was systematically performed to normalize for transfection efficiency. Sixteen hours after transfection, cells were washed and cultured for 48 hr in Dex-containing media with 2% FCS, in the presence or absence Wnt3a-conditioned media. Cells were lysated, and luciferase assays were performed with the Dual Luciferase Assay Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. 10 μl of cell lysates were first assayed for firefly luciferase and then Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.

Predesigned short interfering RNA (siRNA) targeting human Dkk1 (Hs_DKK1_1) and a control scrambled RNA targeting a sequence not sharing homology with the human genome (AllStars Negative Control) were purchased (Qiagen, Crawley, UK). Primary human osteoblasts were transfected with siRNAs and control scrambled RNA using the RNAiFect transfection reagent (Qiagen, Crawley, UK) as per manufacturer's protocol. Briefly, siRNA or scrambled RNA solutions were prepared 15-25 min before the cell transfection. The ratio of siRNA to the RNAiFect reagent was 1 μg siRNA to 3 μl transfection reagent. The siRNA-RNAiFect transfection complexes were incubated for 15 min at room temperature (15-25°C). Osteoblast growth medium was replaced with fresh medium and the siRNA-RNAiFect suspension was added drop-wise onto the cells. The cells with adherent complexes were incubated for 24 h at 37°C, 5% CO₂, at which point the medium was changed and experimentation was commenced. Transfection efficiency was determined in three preliminary experiments in which a fluorescent control RNA-RNAiFect complex (Qiagen, Crawley, UK) was transfected into the cells instead of the siRNA-RNAiFect complex. The uptake of the fluorescent RNA as determined by fluorescence microscopy was in the range of 75-85%. The ratio of siRNA to the RNAiFect reagent was determined three preliminary experiments with a ratio of 1 μg siRNA:3 μl RNAiFect providing a maximal gene silencing of 75% knockdown as determined by qRT-PCR.

Dkk-1 mRNA regulation in primary human osteoblasts treated with dexamethasone was measured by quantitative Real Time PCR using human Dkk-1 QuantiTect assay (Qiagen, Crawley, UK). The QuantiProbe sequence for Dkk-1 was 5'-CACACCAAAGGACAAGA-3'. The forward primer sequence for Dkk-1 was 5'-GGGAATTACTGCAAAAATGGAATA-3', and the reverse primer sequence was 5'-ATGACCGGAGACAAACAGAAC-3'. Total RNA was extracted by TRI-reagent/chlorophorm method, then assayed in duplicate using a Rotorgene 3.0 Real Time PCR instrument (Corbett Research, Cambridge, UK) and the Real Time PCR amplification kit SYBR Green I (Qiagen, Crawley, UK). Using gene specific primer pairs, Dkk1 gene products were measured by Absolute Quantification and were reported as a function of crossing time (Ct), the cycle number at which PCR amplification becomes linear. mRNA expression was normalized to control and GAPDH expression resulting in Mean Fold Change values or ΔΔCt. Following cycling, to ensure specificity, melt curve analysis was carried out to verify the amplification of PCR products starting at 65°C and ramping to 95°C at 0.1°C/sec. One peak in the melt curve indicated no secondary, non-specific products were formed.

Human Dkk1 ELISA kit (R&D Systems Europe Ltd, Abingdon, UK) was obtained to analyse Dkk1 protein expression on cell supernatant. ELISA was performed in accordance with manufacturer's protocols.

All experimental data presented were obtained from three independent experiments, each in triplicate. Data were expressed as mean ± SE. Statistical differences were calculated using Student's t-test or ANOVA for multiple comparisons. p values < 0.05 was considered statistically significant.

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. The marker of osteoblast differentiation assessed was alkaline phosphatase activity.

Primary human osteoblasts exposed to dexamethasone in vitro displayed a reduction in alkaline phosphatase activity over a 72 h time course (Figure 1). Significant reductions in alkaline phosphatase activity were seen at 12 h (p < 0.001), 24 h (p < 0.001), 48 (p < 0.001), 72 h (p < 0.001; Student's t-test) of dexamethasone exposure when compared to control primary human osteoblasts (Figure 1).

In order to determine the effects of dexamethasone exposure on Wnt/β-catenin signaling in primary human osteoblasts, we firstly examined β-catenin trafficking using immunoflourescence analysis. Primary human osteoblasts were grown to confluency and exposed to 10^-8 M dexamethasone over a 48 h time course.

Control primary human osteoblasts, not exposed to dexamethasone treatment and cultured in osteoblast growth medium alone, demonstrated a strong perinuclear and intranuclear staining of β-catenin, representing activated Wnt/β-catenin signaling (Figure 2a). Primary human osteoblasts exposed to dexamethasone demonstrated a reduction in intracellular staining for β-catenin at 24 h (Figure 2b) and this reduction in β-catenin staining persisted at 48 h (Figure 2c), representing a significant inhibition of Wnt/β-catenin signaling.

Since significant changes in intracellular β-catenin trafficking were observed, experiments were performed to elucidate the effects of dexamethasone exposure on Wnt/β-catenin signaling at a transcriptional level. We transiently transfected primary human osteoblasts with the highly sensitive Wnt-luciferase reporter construct pBAR and the control reporter pfuBAR, and exposed them to 10^-8 M dexamethasone over a 48 h time course. Significant reductions in luciferase activity were identified at 24 h (p < 0.01) and 48 h (p < 0.01; Student's t-test) of dexamethasone treatment in the pBAR reporter cells, and the luciferase activity in the pfuBAR reporter cells remained unchanged during treatment (Figure 3).

In order to validate these findings primary human osteoblasts transfected with pBAR were treated with Wnt 3a conditioned media (Wnt agonist) and L-cell conditioned media (control) over a 48 h time course. Significant reductions in luciferase activity were identified when 10^-8 M dexamethasone was added to both Wnt 3a conditioned media (p < 0.001) and control L-cell conditioned media (p < 0.05; Student's t-test) over the 48 h time course, representing a reduction in Wnt/β-catenin signaling through the inhibition of TCF/LEF mediated transcription (Figure 4).

In order to explore the phenotypic effects that aberrations in Wnt/β-catenin signaling have on primary human osteoblasts exposed to dexamethasone, we silenced the expression of Dickkopf-1 (Dkk1), a powerful Wnt antagonist, using siRNA. Primary human osteoblasts were transfected with siRNA targeting Dkk1 expression or scrambled control RNA and Dkk1 gene expression was assessed using quantitative RT-PCR (Figure 5) and ELISA (Figure 6) to confirm knockdown. Quantitative RT-PCR confirmed a 75% reduction in Dkk1 gene expression in the siRNA transfected cells relative to untransfected control cells. This represented a significant reduction when compared to both the scrambled control and transfection control cells (ie. cells treated with RNAiFect alone), which demonstrated no change in Dkk1 gene expression. This reduction in Dkk1 expression was confirmed at protein level when the Dkk1 concentration of cell supernatant was analysed using ELISA. An 85% reduction in Dkk1 concentration was seen in the cell supernatant of siRNA transfected cells relative to untransfected control cells. This also represented a significant reduction when compared to both scrambled control and transfection control cells.

In order to confirm that knockdown of Dkk1 was inhibiting Wnt/β-catenin signaling in primary human osteoblasts, we firstly examined β-catenin trafficking using immunoflourescence analysis. Primary human osteoblasts were grown to confluency and transfected with siRNA targeting Dkk1 expression or scrambled control RNA. siRNA transfected primary human osteoblasts (Figure 7a) demonstrated a strong perinuclear and intranuclear staining of β-catenin, in comparison to scrambled control primary human osteoblasts (Figure 7b). This represents activation of Wnt/β-catenin signaling in response to silencing Dkk1 expression.

These changes in intracellular β-catenin trafficking were accompanied by changes in Wnt/β-catenin signaling at a transcriptional level. Primary human osteoblasts transfected with Wnt-luciferase reporter construct pBAR and the control reporter pfuBAR, were subsequently treated with siRNA targeting Dkk1 expression or scrambled control RNA. A significant increase in luciferase activity was observed in the siRNA transfected cells when compared to scrambled control cells (p < 0.001; Student's t-test) in the pBAR reporter cells, whilst the luciferase activity in the pfuBAR reporter cells remained unchanged during treatment (Figure 8).

Primary human osteoblasts transfected with siRNA targeting Dkk1 expression or scrambled control RNA were subsequently exposed to 10^-8 M dexamethasone over a 72 h time course. Bone turnover was assessed by analyzing alkaline phosphatase activity. Control primary human osteoblasts exposed to dexamethasone over the 72 h time course displayed reductions in alkaline phosphatase activity at 12 h, 24 h, 48 h and 72 h. However, when primary human osteoblasts were transfected with siRNA attenuating Dkk1 expression, there was an increase in alkaline phosphatase activity at each individual time point when compared to scrambled control. Significant increases relative to scrambled control were identified at 12 h (p < 0.05), 24 h (p < 0.05), 48 h (p < 0.01) and 72 h (p < 0.01; Student's t-test) of dexamethasone exposure (Figure 9).