Activation of canonical Wnt/β-catenin signaling inhibits H₂O₂-induced decreases in proliferation and differentiation of human periodontal ligament fibroblasts

Human periodontal ligament fibroblasts (hPLFs) are exposed to oxidative stress during periodontal inflammation and dental treatments. It is hypothesized that hydrogen peroxide (H₂O₂)-mediated oxidative stress decreases survival and osteogenic differentiation of hPLFs, whereas these decreases are prevented by activation of the Wnt pathway. However, there has been a lack of reports that define the exact roles of canonical Wnt/β-catenin signaling in H₂O₂-exposed hPLFs. Treatment with H₂O₂ reduced viability and proliferation in hPLFs in a dose- and time-dependent manner and led to mitochondria-mediated apoptosis. Pretreatment with lithium chloride (LiCl) or Wnt1 inhibited the oxidative damage that occurred in H₂O₂-exposed hPLFs. However, knockout of β-catenin or treatment with DKK1 facilitated the H₂O₂-induced decreases in viability, mitochondrial membrane potential, and Bcl-2 induction. Osteoblastic differentiation of hPLFs was also inhibited by combined treatment with 100 μM H₂O₂, as evidenced by the decreases in alkaline phosphatase (ALP) activity and mineralization. H₂O₂-mediated inhibition of osteoblast differentiation in hPLFs was significantly attenuated in the presence of 500 ng/ml Wnt1 or 20 mM LiCl. In particular, H₂O₂ stimulated the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) at protein and mRNA levels in hPLFs, whereas the induction was almost completely suppressed in the presence of Wnt1 or LiCl. Furthermore, siRNA-mediated silencing of Nrf2 blocked H₂O₂-induced decreases in ALP activity and mineralization of hPLFs with the concomitant restoration of runt-related transcription factor 2 and osteocalcin mRNA expression and ALP activity. Collectively, these results suggest that activation of the Wnt/β-catenin pathway improves proliferation and mineralization in H₂O₂-exposed hPLFs by downregulating Nrf2.