3.1 KY19382 induces ALP activity by activating Wnt/β-catenin signaling in human DP cells
KY19382, a newly synthesized small molecule derived from I3O, was screened by its capability to interfere with the CXXC5-Dvl protein-protein interaction, followed by activation of Wnt/β-catenin signaling (S. Choi et al., 2019). KY19382 showed significantly higher Wnt activity than I3O in a dose-dependent manner in HEK293T cells (Figure 1a). To determine the highest concentration of KY19382 without toxic effects in rat DP cells, cell viability was measured in multiple doses. Cell viability was not significantly affected at 1 and 5 μM KY19382 but decreased at 10 μM KY19382 for rat DP cells (Supplementary figure 1a). In addition, 1 and 5 μM KY19382 did not show any toxicity in human DP cells (Figure 1b). Thus, 1 and 5 μM KY19382 was used for subsequent in vitro studies.
To determine whether KY19382 activates Wnt/β-catenin signaling via GSK-3β inhibition, we examined the levels of β-catenin and p-GSK-3β (S9), an inactive form of GSK-3β, in DP cells. When treated with KY19382, the expression levels of β-catenin, p-GSK-3β (S9), and proliferation marker, PCNA were increased in DP cells (Figure 1c and supplementary figure 1b). The upregulation of Wnt/β-catenin signaling was further confirmed by cytochemical anaylsis of DP cells treated with KY19382 (Figure 1d and supplementary figure 1c). To assess the effect of KY19382 on ALP activity induction, we performed ALP staining and ALP activity assays. KY19382 treatment resulted in higher ALP staining intensity and activity compared to those in the non-treated control (Figure 1e, 1f and supplementary figure 1d, 1e).
The effect of Wnt/β-catenin signaling on KY19382-induced ALP activity was confirmed by using human β-catenin siRNA transfection on human DP cells. The knock-down effect of β-catenin siRNA transfection was verified using immunoblotting analysis (Supplementary figure 2a). KY19382-induced ALP expression was abolished by transfection with β-catenin siRNA in human DP cells, however this inhibitory effect was not shown in cells transfected with a control siRNA (Figure 1g). The quantification of ALP activity was also confirmed that KY19382 induces ALP activity via the Wnt/β-catenin signaling (Figure 1h).
Overall, these data showed that KY19382 significantly activated Wnt/β-catenin signaling in both human and rat DP cells without cytotoxicity. In addition, KY19382 treatment elevated ALP activity by activating Wnt/β-catenin signaling.