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

Objective In this study, we measured the expression of R‐spondin 2 ( RSPO 2) in periodontal ligament ( PDL ) tissue and cells. Further, we examined the effects of RSPO 2 on osteoblastic differentiation of immature human PDL cells ( HPDLC s). 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. RSPO 2, a member of the RSPO family, has been reported to enhance osteogenesis in mice. However, little is known regarding the roles of RSPO 2 in PDL tissues. Methods Expression of RSPO 2 in rat PDL tissue and primary HPDLC s was examined by immunohistochemical and immunofluorescence staining, as well as by semiquantitative RT ‐ PCR . The effects of stretch loading on the expression of RSPO 2 and Dickkopf‐related protein 1 ( DKK 1) were assessed by quantitative RT ‐ PCR . Expression of receptors for RSPO s, such as Leucine‐rich repeat‐containing G‐protein‐coupled receptors ( LGR s) 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 RSPO 2 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 RSPO 2 were assessed by immunofluorescence staining and Western blotting analysis, respectively. In addition, the effect of Dickkopf‐related protein 1 ( DKK 1), an inhibitor of Wnt/β‐catenin signaling, was also examined. Results Rat PDL tissue and HPDLC s expressed RSPO 2, and HPDLC s also expressed RSPO 2 , while little was found in 2‐14 cells. Expression of RSPO 2 as well as DKK 1 in HPDLC s was significantly upregulated by exposure to stretch loading. LGR 4 was predominantly expressed in 2‐14 cells, which expressed low levels of LGR 5 and LGR 6 . RSPO 2 enhanced the Alizarin Red S and von Kossa‐positive reactions in 2‐14 cells. In addition, DKK 1 suppressed nuclear translocation of β‐catenin, activation of β‐catenin, and increases of Alizarin Red S and von Kossa‐positive reactions in 2‐14 cells, all of which were induced by RSPO 2 treatment. Conclusion RSPO 2, which is expressed in PDL tissue and cells, might play an important role in regulating the osteoblastic differentiation of immature human PDL cells through the Wnt/β‐catenin signaling pathway.