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.