Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell‐like cells via Ror2/JNK signaling

Hasegawa, Daigaku; Wada, Naohisa; Yoshida, Shinichiro; Mitarai, Hiromi; Arima, Mai; Tomokiyo, Atsushi; Hamano, Sayuri; Sugii, Hideki; Maeda, Hidefumi

doi:10.1002/jcp.26086

Cited by 49 publications

(38 citation statements)

References 38 publications

(93 reference statements)

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“…PCR was performed using KAPA Express Extract (Kapa Biosystems, Woburn, MA, USA) in a Thermal Cycler Dice Real Time System (Takara) under the following conditions: 95°C for 10 seconds (initial denaturation), then 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds, followed by a dissociation protocol at 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds, in accordance with our recent study . Primer sequences, annealing temperatures, cycle numbers, and product sizes for DKK1 , RSPO2 , CD49d , CD146, N‐cadhelin , p75NTR, and β‐actin are shown in Table .…”

Section: Methodsmentioning

confidence: 99%

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

Arima

Hasegawa

Yoshida

et al. 2018

J of Periodontal Research

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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.

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Section: Methodsmentioning

confidence: 99%

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

Arima

Hasegawa

Yoshida

et al. 2018

J of Periodontal Research

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“…We also found one possible mechanism underlying the increase of ALP expression in Anxa5 deletion by upregulation of BMP4, which is known to activate ALP in various tissues . Downregulation of Wnt5a in primary chondrocytes may also be responsible because it has been reported to decrease ALP expression in cultured chondrocytes, enthesis explant, and periodontal ligament (PDL) cells . The increased Bmp4 and decreased Wnt5a levels may be due to the enhanced chondrogenic differentiation.…”

Section: Discussionmentioning

confidence: 68%

Annexin A5 Involvement in Bone Overgrowth at the Enthesis

Shimada

Ideno

Arai

et al. 2018

Journal of Bone and Mineral Research

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Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5 ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5 mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5 mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5 mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5 and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5 mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.

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“…Cell line 1-17 was also an immortalized human PDL cell line that we established previously [ 20 ]. 2-14, 2-23, and 1-17 cells were reported as human PDLSC-like cells in our previous studies [ 9 , 10 , 20 ]. Cells maintained in alpha-minimum essential medium ( α -MEM; Gibco-BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) (10% FBS/ α -MEM) at 37°C in a humidified atmosphere with 5% CO 2 were used in all experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Half of the medium was exchanged every 2 or 3 days. After 3 weeks of culture, the cells were fixed with 4% paraformaldehyde (PFA; Merck Millipore) and then washed with distilled water and stained with Alizarin red S as described previously [ 9 ]. The area of each Alizarin red S-positive region was imaged under a Biozero digital microscope (Keyence, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…Cells seeded at 2 × 10 4 cells per well in 24-well plates (Becton Dickinson Labware) were cultivated in 10% FBS/ α -MEM in the presence of 0.5 mM methylisobutylxanthine (Sigma), 0.5 μ M hydrocortisone (Sigma), 60 μ M indomethacin (Sigma), and 100 μ M ascorbic acid (Nacalai) for 3 weeks as described previously [ 9 ]. Lipid-containing fat cells were identified by Oil Red O staining.…”

Section: Methodsmentioning

confidence: 99%

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MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells

Hasegawa

Kaneko

et al. 2020

Stem Cells International

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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.

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Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell‐like cells via Ror2/JNK signaling

Cited by 49 publications

References 38 publications

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

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

Annexin A5 Involvement in Bone Overgrowth at the Enthesis

MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells

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