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Ciliary IFT80 regulates dental pulp stem cells differentiation by FGF/FGFR1 and Hh/BMP2 signaling.

ABSTRACT: Primary cilia and intraflagellar transport (IFT) proteins control a wide variety of processes during development and tissue homeostasis. However, their potential roles in the regulation of stem cell differentiation and tooth development remain elusive. Here, we uncovered the critical roles of ciliary IFT80 in cilia formation and differentiation of dental pulp stem cells (DPSCs). IFT80-deficient DPSCs showed reduced fibroblast growth factor receptor 1 (FGFR1) expression, leading to the disruption of FGF2-FGFR1 signaling. We found, during DPSC differentiation, FGF2-FGFR1 signaling induces stress fiber rearrangement to promote cilia elongation, meanwhile stimulates PI3K-AKT signaling to aid Hh/bone morphogenetic protein 2 (BMP2) signaling activation. These signaling pathways and their coupling were disrupted in IFT80-deficient DPSCs, causing impaired differentiation. Our findings revealed a novel mechanism that ciliary protein regulates the odontogenic differentiation of DPSCs through FGF/FGFR1 and Hh/BMP2 signaling.

SUBMITTER: Yuan X

PROVIDER: S-EPMC6775288 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Ciliary IFT80 regulates dental pulp stem cells differentiation by FGF/FGFR1 and Hh/BMP2 signaling.

Yuan Xue X Liu Min M Cao Xu X Yang Shuying S

International journal of biological sciences 20190806 10

Primary cilia and intraflagellar transport (IFT) proteins control a wide variety of processes during development and tissue homeostasis. However, their potential roles in the regulation of stem cell differentiation and tooth development remain elusive. Here, we uncovered the critical roles of ciliary IFT80 in cilia formation and differentiation of dental pulp stem cells (DPSCs). <i>IFT80</i>-deficient DPSCs showed reduced fibroblast growth factor receptor 1 (FGFR1) expression, leading to the dis ...[more]

PMID: 31592124

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Project description:Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/?catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/?-catenin pathway.SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/?-catenin pathway activation assessed by western blotting, ?-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of ?-catenin.WNT3A induced ?-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/?-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, ?-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous ?-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/?-catenin signaling.These data suggest that stabilization of ?-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/?-catenin signaling to promote cell maturation.

Project description:BackgroundHuman dental pulp stromal cells (hDPSCs) are promising sources of mesenchymal stem cells (MSCs) for bone tissue regeneration. Circular RNAs (circRNAs) have been demonstrated to play critical roles in stem cell osteogenic differentiation. Herein, we aimed to investigate the role of circAKT3 during osteogenesis of hDPSCs and the underlying mechanisms of its function.MethodsWe performed circRNA sequencing to investigate the expression profiles of circular RNAs during osteogenesis of hDPSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression pattern of circAKT3 and miR-206 in hDPSCs during osteogenesis. We knocked down circAKT3 and interfered the expression of miR-206 to verify their regulatory role in hDPSC osteogenesis. We detected hDPSCs mineralization by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining and used dual-luciferase reporter assay to validate the direct binding between circAKT3 and miR-206. To investigate in vivo mineralization, we performed subcutaneous transplantation in nude mice and used hematoxylin and eosin, Masson's trichrome, and immunohistochemistry staining.ResultsTotally, 86 circRNAs were differentially expressed during hDPSC osteogenesis, in which 29 were downregulated while 57 were upregulated. circAKT3 was upregulated while miR-206 was downregulated during hDPSC osteogenesis. Knockdown of circAKT3 inhibited ALP/ARS staining and expression levels of osteogenic genes. circAKT3 directly interacted with miR-206, and the latter one suppressed osteogenesis of hDPSCs. Silencing miR-206 partially reversed the inhibitory effect of circAKT3 knockdown on osteogenesis. Connexin 43 (CX43), which positively regulates osteogenesis of stem cells, was predicted as a target of miR-206, and overexpression or knockdown of miR-206 could correspondingly decrease and increase the expression of CX43. In vivo study showed knockdown of circAKT3 suppressed the formation of mineralized nodules and expression of osteogenic proteins.ConclusionDuring osteogenesis of hDPSCs, circAKT3 could function as a positive regulator by directly sponging miR-206 and arresting the inhibitive effect of miR-206 on CX43 expression.

Dataset Information

Ciliary IFT80 regulates dental pulp stem cells differentiation by FGF/FGFR1 and Hh/BMP2 signaling.

Publications

Ciliary IFT80 regulates dental pulp stem cells differentiation by FGF/FGFR1 and Hh/BMP2 signaling.

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