Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue.

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.

Periodontal ligament (PDL) stem cells (PDLSCs) have been reported as a useful cell source for periodontal tissue regeneration. However, one of the issues is the difficulty of obtaining a sufficient number of PDLSCs for clinical application because very few PDLSCs can be isolated from PDL tissue of donors. Therefore, we aimed to identify a specific factor that converts human PDL cells into stem-like cells. In this study, microarray analysis comparing the gene profiles of human PDLSC lines (2-14 and 2-23) with those of a cell line with a low differentiation potential (2-52) identified the imprinted gene mesoderm-specific transcript (MEST). MEST was expressed in the cytoplasm of 2-23 cells. Knockdown of MEST by siRNA in 2-23 cells inhibited the expression of stem cell markers, such as CD105, CD146, p75NTR, N-cadherin, and NANOG; the proliferative potential; and multidifferentiation capacity for osteoblasts, adipocytes, and chondrocytes. On the other hand, overexpression of MEST in 2-52 cells enhanced the expression of stem cell markers and PDL-related markers and the multidifferentiation capacity. In addition, MEST-overexpressing 2-52 cells exhibited a change in morphology from a spindle shape to a stem cell-like round shape that was similar to 2-14 and 2-23 cell morphologies. These results suggest that MEST plays a critical role in the maintenance of stemness in PDLSCs and converts PDL cells into PDLSC-like cells. Therefore, this study indicates that MEST may be a therapeutic factor for periodontal tissue regeneration by inducing PDLSCs.