The role of sphingosine-1-phosphate signaling pathway in cementocyte mechanotransduction

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Highlights

  • A novel immortalized murine cementocyte cell line was employed to explore the mechanism of cementum mechanotransduction.

  • Compressive stress inhibited major S1P signaling components and promoted anabolic factors in IDG-CM6 cells.

  • S1P signaling played a vital role in mediating the mechanotransduction process of IDG-CM6 cells.

  • S1P signaling might be a novel therapeutic target to solve external root resorption, especially for orthodontic patients.

Abstract

Iatrogenic external root resorption can become a serious pathological condition with clinical tooth movement. Little is known regarding how cementum responds to mechanical loading in contrast to bone, especially under compressive stress. In the field of bone biology, several studies have established the contribution of sphingosine-1-phosphate (S1P) signaling in bone remodeling, mechanical transduction and homeostasis. As osteocytes and cementocytes share similar morphological and functional characteristics, this study aimed to investigate the mechanotransduction ability of cementocytes and to explore the contribution of S1P signaling under compressive stress induced mechanotransduction. We found that compressive stress inhibited major S1P signaling and promoted the expression of anabolic factors in IDG-CM6 cells, a novel immortalized murine cementocyte cell line. By inhibiting S1P signaling, we verified that S1P signaling played a vital role in regulating the expression of the mechanotransduction factors prostaglandin E2 (PGE2) and β-catenin, as well as factors responsible for cementogenesis and cementoclastogenesis in IDG-CM6 cells. These results support the hypothesis that cementocytes act as key mechanically responsive cells in cementum, responding to compressive stress and directing local cementum metabolism.

Introduction

In approximately 93% of adolescents receiving orthodontic treatment, external root resorption is a frequent iatrogenic effect [1,2]. If handled improperly, severe root resorption may compromise occlusal stability and even the longevity of the tooth. Cementum, as a specialized calcified substance covering the root surface, is fundamental in maintaining periodontal homeostasis and long-term stability of teeth [3,4]. Its ability to repair and fill resorbed root surfaces during tooth movement is considered as one of the biological foundations of orthodontic treatment [5]. Even so, external root resorption tends to begin with the resorption of cementum when subjected to undesirable local orthodontic force [6,7]. However, research addressing the mechanism of cementum mechanosensation, mechanotransduction and wound healing are limited.

During formation of the apically located cellular cementum, some cementoblasts become embedded in the cementoid matrix and become cementocytes. Like osteocytes, whose complex dendritic network enables intercellular communication and provides mechanical cues from bone loading and unloading [8,9], cementocytes also feature dendritic processes and a lacuno-canalicular network, making an osteocyte-like network of communication and mechanosensory function possible [10]. In addition, cementocytes express identical key markers known to be important in osteocyte differentiation, including dentin matrix protein 1 (Dmp1), E11/gp38/podoplanin, phosphate regulating endopeptidase homolog, X-linked (PHEX) and sclerostin (SOST) [10]. These biological similarities suggest similar physiological functions. Our recent preliminary study found significant changes of key regulatory factors in an immortalized murine cementocyte cell line, IDG-CM6, in response to fluid flow shear stress [11]. This, for the first time, provided direct in vitro evidence that cementocytes might act as mechanically responsive cells.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator involved in various critical cell processes [12,13]. Produced by the phosphorylation of sphingosine via sphingosine kinases (SPHKs, SPHK1 localized at the membrane and SPHK2 localized in cytosol), S1P can either act as a second messenger or bind to S1P receptors (S1PRs) 1–5, mainly S1PR1-3, on the cell membrane, to mediate intracellular signal transduction [[14], [15], [16]]. In the field of bone biology, several studies have recently pointed out the contribution of S1P signaling in bone remodeling, mechanical transduction and bone homeostasis. Through interaction with receptor activator of nuclear factor kappa B ligand (RANKL), Wnt, calcitonin and cathepsin K, S1P signaling controls the differentiation and proliferation of osteoclast precursors and it also regulates cellular activities of osteoblasts by mobilizing intracellular [Ca2+]i, BMP6, as well as activating ERK and Akt signaling [[17], [18], [19], [20], [21]]. A recent study revealed that S1P signaling modulates several steps of the osteocyte cellular response to continuous oscillatory fluid flow, including increased synthesis and release of prostaglandin E2 (PGE2), as well as down-regulation of RANKL/osteoprotegerin (OPG) ratio in response to continuous oscillatory fluid flow. This work showed that S1P signaling participated in mechanotransduction of osteocytes and can further regulate cell functions under mechanical stress [22]. In our study, we found IDG-CM6 cementocyte cells express all the necessary components of a functional S1P signaling cascade. Based on the similar characteristics shared by osteocytes and cementocytes, we hypothesized that S1P signaling might also play an important role in mediating mechanotransduction in cementocytes.

For the present study, IDG-CM6 cells, a novel immortalized murine cementocyte cell line was used to determine the contribution of S1P signaling in mechanotransduction under compressive stress. In contrast to osteocytes where S1P signaling has been shown to enhance the effects of mechanotransduction, we found that inhibition of this signaling pathway enhanced mechanotransduction in cementocytes.

Section snippets

Cell culture and application of compressive stress

The immortalized murine cementocyte cell line IDG-CM6 was created by crossing the 8-kb Dmp1-GFP (green fluorescent protein) transgenic mouse line with the Immortomouse. The Immortomouse has an interferon-gamma (IFN-γ)-inducible promoter driving expression of a thermolabile large T antigen (H-2Kb-tsA58) that enables conditional immortalization of cells derived from this mouse. The cells express the SV40 T antigen when cultured at 33 °C in the presence of IFN-γ and proliferate rapidly. In the

Compressive stress inhibited S1P signaling in IDG-CM6 cells

Expression of major components of the S1P signaling pathway were determined by RT-PCR and Western blot analysis. Both the mRNA and protein levels of the S1P receptors, S1PR1 and S1PR2 were significantly decreased after application of compressive stress, while no significant difference was found on the expression of S1PR3 (Fig. 1A1-A3 and B1-B3). The expression of the sphingosine kinase, SPHK2, remained stable in the stress group (Fig. 1A4 and B4), while SPHK1 was significantly increased under

Discussion

Cementocytes are very difficult to isolate because they are embedded in the thin layer of cellular cementum covering only the apical region of a root. This has been a significant barrier to the understanding of its biological and biomechanical functions. A lack of cell models also greatly limited in vitro investigation. Recently, Zhao and colleagues used a novel approach to isolate and characterize an immortalized cementocyte cell line, IDG-CM6, for the first time [11]. The application of this

Declaration of competing interest

Authors declare that there is no conflict of interest.

Acknowledgements

This work was funded by National Natural Science Foundation of China (Grant No. 81701005, 81801019) and Science and Technology Department, Sichuan, China (Grant No. 2018JY0139).

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