Adenosine receptors enhance the ATP-induced odontoblastic differentiation of human dental pulp cells

https://doi.org/10.1016/j.bbrc.2018.02.125Get rights and content

Highlights

  • ATP (600 μM) could induce the odontoblastic differentiation of HDPCs in vitro.

  • All four adenosine receptor subtypes were expressed in HDPCs.

  • Adenosine receptors were activated when HDPCs were treated with ATP.

  • A1R and A2BR played a positive role in ATP-induced HDPC odontoblastic differentiation.

Abstract

Purinergic signaling regulates various biological processes through the activation of adenosine receptors (ARs) and P2 receptors. ATP induces the odontoblastic differentiation of human dental pulp cells (HDPCs) via P2 receptors. However, there is no information available about the roles of ARs in HDPC odontoblastic differentiation induced by ATP. Here, we found that HDPCs treated with ATP showed higher activity of ADORA1 (A1R), ADORA2B (A2BR), and ADORA3 (A3R). Inhibition of A1R and A2BR attenuated ATP-induced odontoblastic differentiation of HDPCs, whereas activation of the two receptors enhanced the odontoblastic differentiation induced by ATP. However, activation of ARs by adenosine did not induce the odontoblastic differentiation of HDPCs independently without induction of ATP. Our study indicates a positive role for ARs in ATP-induced odontoblastic differentiation of HDPCs, and demonstrates that ATP-induced odontoblastic differentiation of HDPCs may be due to the combined administration of ARs and P2 receptors. This study provides new insights into the molecular mechanisms of pulpal injury repair induced by ATP.

Introduction

Human dental pulp cells (HDPCs) have attracted the interest of researchers in the field of tissue regeneration because of their accessibility and abundance of stem/progenitor cells [1,2]. Recent studies have reported that HDPCs can differentiate not only into odontoblasts for dentin regeneration, but also into osteoblasts, which can repair bone defects under appropriate conditions [3,4]. The differentiation of mesenchymal cells in HDPCs into odontoblasts is induced by multiple cytokines such as four and a half LIM domains 2 (FHL2) [5], bone morphogenetic protein (BMP2) [6], and ID1 (a downstream target of BMP2 signaling) [7].

Purinergic signaling can regulate the proliferation [8], differentiation [9], and death [8] of different types of stem cells. As critical signaling molecules in this pathway, adenosine triphosphate (ATP) and its hydrolysates act through purinergic receptors, which are classified into P1 (adenosine receptors [ARs], e.g. A1R, A2AR, A2BR, and A3R) and P2 (P2XR, e.g. P2X1–7R; P2YR, e.g. P2Y1,2,4,6,11–14R) receptors. P1 receptors are primarily activated by adenosine, whereas those in the P2 category are mainly regulated by purines such as ATP and adenosine diphosphate (ADP) [10]. Cutarelli et al. [11] were the first to report the biphasic effects of ATP on differentiation and mineralization in human osteoblasts, showing an increase in these processes in response to low concentrations (<100 μM), whereas high concentrations (>100 μM) led to a decrease in these processes, which they suggested was due to the combined activation of P2 receptors and ATP hydrolysis products (e.g. ADP, AMP, adenosine, the mineralization inhibitor PPi, and the mineralization promoter inorganic phosphate [Pi]). In contrast, the osteogenic effects of ATP on human bone marrow mesenchymal stem cells was due to adenosine stimulation of the AR subtype, A2BR [12].

Extracellular ATP and downstream purinergic signaling have also been proposed to contribute to dental pulp tissue healing and dentin regeneration. Mechanical and thermal stimulation of external dentin can induce ATP release in dental pulp through pannexins [13]. Cold stimulation was also reported to induce ATP release from human odontoblast-like cells [14]. Our previous study demonstrated that high concentrations of ATP (800 μM) can induce odontoblastic differentiation of HDPCs, whereas low concentrations (<400 μM) promoted cell proliferation, and that P2 receptors and the ERK/MAPK signaling pathway were involved in this ATP-induced odontoblastic differentiation [15]. These investigations indicated positive roles for ATP and P2 receptors in dental pulp wound healing and dentin formation. However, the specific role of ARs in ATP-induced odontoblastic differentiation of HDPCs, and the effects of adenosine, the hydrolysate of ATP, on HDPC odontoblastic differentiation remain unknown.

All four AR subtypes were demonstrated to be expressed in dental pulp stem cells (DPSCs), and stimulation of A1R might enhance the osteogenic differentiation of DPSCs, moreover, the progress of dentinogenesis is similar to that of osteogenesis, to some extent [16]. We conducted this study to identify the role of ARs in ATP-induced odontoblastic differentiation of HDPCs, and to determine whether the ARs activation by adenosine can induce HDPC odontoblastic differentiation independently, without the induction of ATP.

Section snippets

Cell culture

This study was approved by the Ethics Committee of Peking University School and Hospital of Stomatology (IRB-2013055), and informed consent was obtained. HDPCs were isolated according to a previous report [15]. Cells were cultured in proliferation medium (PM) containing α-minimum essential medium (α-MEM) (Gibco, Grand Island, NY) with 10% fetal bovine serum (Corning Cellgro, NY), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO), and 1% penicillin/streptomycin (Gibco, BRL). For ATP-induced

Expression of adenosine receptor subtypes in HDPCs

To study the expression of ARs in HDPCs, RT-PCR and real-time PCR were performed. The results showed that all four AR subtypes, A1R, A2AR, A2BR, and A3R, were expressed in HDPCs (Fig. 1A). However, the levels of A2AR and A2BR were much higher than those of A1R and A3R. A2BR exhibited the highest and A3R the lowest expression levels (Fig. 1B). These results were consistent with those of a previous study [16].

Adenosine receptor subtypes were activated in HDPCs exposed to ATP

To investigate the response of ARs to ATP exposure, the mRNA and protein expression

Discussion

Purinergic signaling affects various physiological processes, including osteogenesis [11], neurogenesis [9], inflammation [21], and pain [22]. Receptors involved in this signaling are classified into two main groups: P1 (adenosine receptors [ARs]) and P2 receptors. In our previous study, we had demonstrated that ATP could induce odontoblastic differentiation of HDPCs by analyzing the key events in this process, including DMP1 and DSPP expression, as well as the mineralization capacity of HDPCs

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We would like to thank Editage [www.editage.cn] for English language editing.

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