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TGF-?-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals.

ABSTRACT: The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-? (TGF-?) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-? signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-?1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-?1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-?-induced Smad-dependent signaling, suppressed the TGF-?1-induced growth inhibition and SMC markers expression, but did not the TGF-?1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-?1-induced downregulation of EC marker expression. In addition, the TGF-?1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-?1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-?1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.

SUBMITTER: Yoshida M

PROVIDER: S-EPMC3432854 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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TGF-β-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals.

Yoshida Mariko M Okubo Naoto N Chosa Naoyuki N Hasegawa Tomokazu T Ibi Miho M Kamo Masaharu M Kyakumoto Seiko S Ishisaki Akira A

International journal of biological sciences 20120822 7

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissu ...[more]

PMID: 22949889

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Project description:BackgroundPeriodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs.MethodsThe expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining.ResultsWe observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3' untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis.ConclusionsOur results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Project description:BackgroundMechanotransduction mechanisms whereby periodontal ligament stem cells (PDLSCs) translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt transmembrane receptor, has been qualified as a key monitor for mechanical cues. However, the role of LRP6 in the mechanotransduction of mechanically induced PDLSCs remains obscure.MethodsThe Tension System and tooth movement model were established to determine the expression profile of LRP6. The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs. The ability of osteogenic differentiation and proliferation was estimated by alkaline phosphatase (ALP) staining, ALP activity assay, western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. Crystalline violet staining was used to visualize cell morphological change. Western blotting, qRT-PCR, and phalloidin staining were adopted to affirm filamentous actin (F-actin) alteration. YAP nucleoplasmic localization was assessed by immunofluorescence and western blotting. YAP transcriptional response was evaluated by qRT-PCR. Cytochalasin D was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs.ResultsLRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues. LRP6 deficiency impeded force-dependent osteogenic differentiation and proliferation in PDLSCs. Intriguingly, LRP6 loss caused cell morphological aberration, F-actin dynamics disruption, YAP nucleoplasmic relocation, and subsequent YAP inactivation. Moreover, disrupted F-actin dynamics inhibited osteogenic differentiation, proliferation, YAP nuclear translocation, and YAP activation in mechanically induced PDLSCs.ConclusionsWe identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade. Targeting LRP6 for controlling alveolar bone remodeling may be a prospective therapy to attenuate relapse of orthodontic treatment.

Dataset Information

TGF-?-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals.

Publications

TGF-β-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals.

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