R‐spondin 2 promotes osteoblastic differentiation of immature human periodontal ligament cells through the Wnt/β‐catenin signaling pathway

Abstract

Objective: In this study, we measured the expression of R-spondin 2 (RSPO2) in periodontal ligament (PDL) tissue and cells. Further, we examined the effects of RSPO2 on osteoblastic differentiation of immature human PDL cells (HPDLCs). Background: R-spondin (RSPO) family proteins are secreted glycoproteins that play important roles in embryonic development and tissue homeostasis through activation of the Wnt/β-catenin signaling pathway. RSPO2, a member of the RSPO family, has been reported to enhance osteogenesis in mice. However, little is known regarding the roles of RSPO2 in PDL tissues. Methods: Expression of RSPO2 in rat PDL tissue and primary HPDLCs was examined by immunohistochemical and immunofluorescence staining, as well as by semiquantitative RT-PCR. The effects of stretch loading on the expression of RSPO2 and Dickkopf-related protein 1 (DKK1) were assessed by quantitative RT-PCR. Expression of receptors for RSPOs, such as Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4, 5, and 6 in immature human PDL cells (cell line 2-14, or 2-14 cells), was investigated by semiquantitative RT-PCR. Mineralized nodule formation in 2-14 cells treated with RSPO2 under osteoblastic inductive condition was examined by Alizarin Red S and von Kossa stainings. Nuclear translocation of β-catenin and expression of active β-catenin in 2-14 cells treated with RSPO2 were assessed by immunofluorescence staining and Western blotting analysis, respectively. In addition, the effect of Dickkopf-related protein 1 (DKK1), an inhibitor of Wnt/β-catenin signaling, was also examined. Results: Rat PDL tissue and HPDLCs expressed RSPO2, and HPDLCs also expressed RSPO2, while little was found in 2-14 cells. Expression of RSPO2 as well as DKK1 inHPDLCs was significantly upregulated by exposure to stretch loading. LGR4 was predominantly expressed in 2-14 cells, which expressed low levels of LGR5 and LGR6. RSPO2 enhanced the Alizarin Red S and von Kossa-positive reactions in 2-14 cells. In addition, DKK1 suppressed nuclear translocation of β-catenin, activation of βcatenin, and increases of Alizarin Red S and von Kossa-positive reactions in 2-14 cells, all of which were induced by RSPO2 treatment.

LGRs in Skeletal Tissues: An Emerging Role for Wnt‐Associated Adult Stem Cell Markers in Bone

“…(92,93) Addition of RSPO2 to an undifferentiated human PDL cell line expressing LGR4 induces more robust osteogenesis, although it is still unproven as this is mediated through a RSPO-LGR interaction. (89) An LGR-mediated mechanism may be accelerating osteogenic differentiation of tooth-associated progenitor cells, similar to in vitro results seen in studies with bone marrowderived cells and established cell lines. (65,66)…”

“…(85)(86)(87)(88) Expression of LGR4 in particular was reported in an immature human PDL cell line (2 to 14 cells). (89) Under orthodontic strain in a rat model, LGR5 expression in vivo localizes with Runx2+ cells, specifically in the periapical region of the PDL during tooth movement and alveolar bone remodeling under the influence of these cyclic strain techniques. (90) Further,…”

“…(79) In parallel, siRNA knockdown of LGR4 in stem cells of the apical papilla (SCAPs) isolated from developing tooth roots was shown to inhibit the process of in vitro odontogenic differentiation, and this was correlated with decreased amounts of stabilized β-catenin. (80) Arima et al (89) LGR4 Adipose-derived stem cells…”

SOX9‐activated PXN‐AS1 promotes the tumorigenesis of glioblastoma by EZH2‐mediated methylation of DKK1

“…Among which, its functional contribution to lncRNAs‐mediated cancer development has attracted people's attention 28. Through researches of numerous years, it was confirmed that enhanced nuclear translocation of β‐catenin indicates the activation of Wnt/β‐catenin pathway 29. The transcription factor SOX9 has been reported to activate Wnt/β‐catenin pathway in non‐small‐cell lung cancer 19.…”

R-Spondin 2 Induces Odontogenic Differentiation of Dental Pulp Stem/Progenitor Cells via Regulation of Wnt/β-Catenin Signaling

“…Several studies have demonstrated that R-spondin proteins contribute to the regulation of tissue patterning and differentiation (Zhou et al, 2012;Storm et al, 2016;Zhu et al, 2016). Rspo2 has been reported to have the capacity of promoting osteoblast differentiation within MC3T3 E1 cells (Friedman et al, 2009;Zhu et al, 2016), mouse bone marrow stem cells (Knight et al, 2018), and human periodontal ligament cells (Arima et al, 2019) by modulating Wnt/β-catenin signaling. Bone marrow stem cells (BMSCs) isolated from Rspo2 Ftl mice (deficient in Rspo2) showed reduced osteogenesis compared with BMSCs from wild-type litter mates (Knight et al, 2018).…”