DKK1 adenovirus

Adult bone regeneration is orchestrated by the precise actions of osteoprogenitor cells (OPCs). However, the link and regulatory mechanisms between OPC proliferation and differentiation remain to be determined. Here, researchers present evidence that during intramembranous bone formation, OPC proliferation is controlled by Notch signaling, whereas differentiation is initiated by activation of canonical Wnt signaling. Spatiotemporal segregation of Notch and Wnt signaling activation during the early stages of bone regeneration suggests a crosstalk between these two pathways. Manipulation of these two essential pathways in vitro and in vivo revealed that Wnt activation leads to the initiation of osteogenic differentiation, while inhibition of Notch signaling results in termination of the proliferative phase. These studies suggest that canonical Wnt signaling is a key regulator of the crosstalk between OPC proliferation and differentiation during intramembranous primary bone healing.

Here, using adenovirus expressing the Wnt antagonist Dkk1, the researchers aimed to demonstrate that inhibition of Wnt signaling abolishes the inhibitory effect on Notch, thereby maintaining Notch activation. iAxin2/GFP mice were treated locally with either Ad-null or Ad-Dkk1 at the time of tibial defect surgery (Figure 1a). Dkk1 treatment resulted in a significant decrease in Axin2 expression in regenerated tissue, confirming successful Wnt inhibition (Figure 1a). On postoperative day 7, immunostaining for NICD2 (activated Notch2 intracellular domain) was increased in the injury site treated with the Wnt antagonist Dkk1 (Figure 1b), indicating prolonged activated Notch signaling in the presence of the Wnt antagonist. In addition, on postoperative day 7, Hey1 and Hes1 expression was increased in the Ad-Dkk1 treated group (Figure 1c, d), indicating that Wnt inhibition resulted in a sustained increase in Notch signaling activation. The researchers performed qRT-PCR for proliferating cell nuclear antigen (Pcna) and detected a significant increase in expression levels in Ad-Dkk1 treated lesions, indicating that Wnt inhibition is sufficient to increase OPC proliferation by activating Notch signaling, which acts as a proliferation activator (Figure 1e). FACS analysis of regeneration after Ad-Dkk1 treatment was performed at days 3, 7, and 10 after surgery, and the number of SSCs was quantified (Figure 1f). On day 3, there was no difference between the control and Ad-Dkk1 lesions, as proliferation is about to begin. However, on days 7 and 10, the number of SSCs in Ad-Dkk1 treated lesions was significantly increased, indicating that Wnt inhibition leads to Notch activation, which in turn leads to increased and prolonged SSC proliferation at the lesion site (Figure 1f). Hey1 immunofluorescence staining showed increased nuclear Hey1 staining in Dkk1 treated OPCs (Figure 1g). qRT-PCR confirmed that Dkk1 treatment resulted in Wnt inhibition, as indicated by Axin2 downregulation (Figure 1h). In response to Wnt inhibition, Hey1 expression increased (Figure 1i), confirming the hypothesis that Wnt signaling regulates Notch signaling in OPCs.

Figure 1. Wnt inhibition sustains Notch activation and lengthens the proliferative phase. (Lee S, et al., 2021)