NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells

doi:10.1016/j.bbrc.2013.11.120

Biochemical and Biophysical Research Communications

Volume 443, Issue 2, 10 January 2014, Pages 500-504

https://doi.org/10.1016/j.bbrc.2013.11.120 Get rights and content

Highlights

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BMP2/DLX3 pathway supports osteogenic differentiation of dental follicle cells (DFCs).
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NOTCH signaling is induced by DLX3 and during the osteogenic differentiation of DFCs.
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NOTCH signaling impairs the osteogenic differentiation and the expression of DLX3.
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A BMP2/DLX3/NOTCH feedback loop regulates the osteogenic differentiation of DFCs.

Abstract

Dental follicle cells (DFCs) are dental stem/progenitor cells and the genuine precursors of alveolar osteoblasts and dental cementoblasts. A previous study showed that the transcription factor DLX3 (distal less homeobox 3) supports the osteogenic differentiation in DFCs via a positive feedback loop with the bone morghogenetic protein (BMP) 2. Until today, however, the control of this BMP2/DLX3 pathway by additional signaling pathways remains elusive. Previous studies also suggested that the NOTCH signaling pathway plays a role in the osteogenic differentiation of DFCs. In this study we showed that DLX3 overexpression and the initiation of the osteogenic differentiation by BMP2 or dexamethasone induced the NOTCH signaling pathway in DFCs. However, the induction of NOTCH-signaling impaired not only the osteogenic differentiation (ALP activity and mineralized nodules) but also the expression of the transcription factor DLX3 and the activation of the BMP-signaling pathway. So, NOTCH signaling plays a regulatory role for the osteogenic differentiation of DFCs. In conclusion, results of our study suggest that the NOTCH-signaling pathway, which is activated during the osteogenic differentiation of DFCs, regulates the BMP2/DLX3 directed differentiation of DFCs via a negative feed-back loop.

Introduction

For more than 10 years dental stem/progenitor cells can be isolated from human dental tissues [1], [2], [3]. Since that time dental stem cells are highly suggested for cellular therapies in dentistry and a clinical pilot study with patients suffering from periodontitis have shown the feasibility of such a therapy [4]. However, undifferentiated dental cells are also very useful for studies about cellular and molecular processes under in vitro conditions. Dental follicle cells (DFCs) are a good example [5], [6], [7], [8], [9], because they are the genuine precursors of the periodontium including alveolar osteoblasts, dental cementoblasts and periodontal fibroblasts [5], [6], [10], [11]. Thus, DFCs were used for molecular studies about the osteogenic differentiation of periodontal precursor cells. Here, genome wide gene expression profiles were compared during different stages of the osteogenic differentiation and regulated genes were identified among others, which are linked to signaling pathways such as TGFβ/BMP, WNT or NOTCH [12], [13], [14]. Moreover, we have already shown that the transcription factor DLX3 (distal less homeobox 3) supports the osteogenic differentiation in DFCs via a BMP2 positive feedback loop [15]. Until today, however, the control of this BMP2/DLX3 pathway by further signaling pathways remain elusive.

Among other signaling pathways, which coordinate the proliferation and differentiation of mesenchymal stem cells, the NOTCH-signaling plays an important role. This pathway mediates the communication of adjacent cells that express either a NOTCH receptor or a membrane-bound NOTCH ligand such as Jagged1. The signaling of the NOTCH-pathway is activated by a receptor-ligand binding and a cleavage of NOTCH by γ secretase and translocation of the intracellular domain of NOTCH (NICD) into the nucleus, where it binds to transcription factors such as RBP-JK. The NOTCH signaling pathway regulates for example the odontogenic differentiation of dental pulp stem cells (DPSCs). Zhang et al. found that overexpression of the NOTCH ligand Jagged1 or the overexpression of the NICD inhibited the odontogenic differentiation of DPSCs [16]. Interestingly, NOTCH1 is a marker of DFCs and its expression is regulated during the osteogenic differentiation of DFCs [5], [17]. Moreover, the NOTCH target genes were up-regulated after overexpression of DLX3 in DFCs [15]. These previous studies suggested that the NOTCH signaling pathway plays also a decisive role for the regulation of the osteogenic differentiation in DFCs. In this study we investigated the relation between NOTCH and the osteogenic differentiation of DFCs that is supported by the BMP2/DLX3 pathway.

Section snippets

Cell culture

Impacted human third molars were surgically removed and collected from patients with informed consent. Dental follicle cells (DFCs) were isolated as described previously [17]. Briefly, the follicle tissues were digested in a solution of collagenase type I, hyaluronidase (Sigma–Aldrich, Munich, Germany), and DNAse I (Roche, Mannheim, Germany) and seeded in cell culture dishes. DFCs were initially cultivated in Mesenchym Stem Medium (PAA, Pasching, Austria) at 37 °C in 5% CO₂. The standard cell

DLX3 induces NOTCH signaling

NOTCH1 is a specific marker for DFCs and Chen et al. disclosed that NOTCH1 signaling regulates the proliferation and self-renewal of DFCs by modulating the G1/S phase transition and telomerase activity [22]. Previous studies also suggested that NOTCH1 plays a role during the osteogenic differentiation of DFCs [5], [17], but little is known about the role of this important signaling pathway. Therefore we investigated the NOTCH signaling pathway in DFCs during the osteogenic differentiation,

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NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells

Highlights

Abstract

Introduction

Section snippets

Cell culture

DLX3 induces NOTCH signaling

Matrix Biol.

Bone

Cell Biol. Int.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo

Proc. Natl. Acad. Sci. U.S.A.

Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine

J. Dent. Res.

Stem/progenitor cells of dental and gingival tissue origin

Utility of PDL progenitors for in vivo tissue regeneration: a report of 3 cases

Oral Dis.

Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro

Cell Tissue Res.