Search

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 in HPDLCs 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 β‐catenin, and increases of Alizarin Red S and von Kossa‐positive reactions in 2‐14 cells, all of which were induced by RSPO2 treatment. Conclusion: RSPO2, 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. [ABSTRACT FROM AUTHOR]

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor‐α (TNF‐α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF‐α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence‐related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast‐inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF‐α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF‐α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF‐α‐treated sHDPCs, whereas restored the reduction in TNF‐α‐treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF‐α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF‐α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF‐α. These data could argue the possible involvement of different signaling pathways in tumor necrosis factor‐α‐inducing events depending on the cell aging stage, which might cause different cell responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]