Extracellular ATP and downstream purinergic

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 1080 6 [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.
Methods
Results
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 in vitro. We found that this effect was mediated by the activation of P2 receptors [15]. In the present study, we first verified that ARs activation could enhance the ATP-induced odontoblastic differentiation of HDPCs. However, ARs activation by exogenous adenosine could not induce the odontoblastic differentiation of HDPCs independently, with the absence of ATP. The results suggested that the odontoblastic differentiation of HDPCs induced by ATP was probably due to the combined regulation of ARs and P2 receptors, and between these two receptors, P2 receptors might play a main role. In the present study, we found that the expression of AR, AR, and AR increased after ATP treatment, indicating that the three ARs in HDPCs were possibly activated after ATP exposure. It has been established that ARs are primarily activated by adenosine [10], which is generated by the hydrolysis of ATP [23]. Therefore, we speculated that the activation of ARs identified in this study may be a consequence of increased levels of adenosine generated by the hydrolysis of extracellular ATP. Then, we verified that 600 μM ATP could induce the odontoblastic differentiation of HDPCs, and we thus chose 600 μM ATP in this study rather than 800 μM used in the earlier study because of the comparatively small impact on cell proliferation [15]. In our previous study, the maximum expression of DSPP mRNA was seen at 24 h [15], whereas it was 48 h in this study; this might be due to the hysteresis effect of a relatively low concentration (600 μM) of ATP, compared to 800 μM. However, this difference in the level of DSPP mRNA at the times tested did not affect the differentiation of HDPCs. To determine whether the observed activation of ARs was directly related to odontoblastic differentiation of HDPCs, cells were pretreated with selective antagonists of A1R, A2BR, and A3R 1 h prior to ATP treatment. We found that inhibition of A1R and A2BR attenuated the ATP-induced up-regulation of DMP1 and mineralization, whereas inhibition of A2BR and A3R reduced DSPP; however, A3R inhibition had no effect on the mineralization. The difference in the chosen time points (48 h vs. 7 days) could be one reason for the discrepancies between DSPP mRNA and protein expression at these time points. These results indicated that ARs might contribute to ATP-meditated induction of odontoblastic differentiation of HDPCs. To further verify this speculation, selective agonists of A1R, A2BR, and A3R were used to analyze the effects on the ATP-mediated odontoblastic differentiation of HDPCs. The results showed that the activation of A1R and A2BR enhanced the ATP-induced up-regulation of DMP1 and mineralization, whereas A2BR and A3R activation enhanced that of DSPP; however, A3R activation had no effect on the mineralization. Thus, we conclude that A1R, and especially A2BR, might enhance ATP-induced odontoblastic differentiation of HDPCs, although the two receptors activate cAMPs through opposite mechanisms. This could be attributed to other molecular pathways known to be coupled to these receptors, such as MAPK, PI3K/Akt, and Wnt signaling, which are also associated with the differentiation of MSCs into osteoblasts [16,24,25]. In addition, adenosine receptors have been demonstrated to regulate the levels of inflammatory cytokines (eg., IL-6) which could also affect osteogenic differentiation [23,26]. Thus, in our study, we speculated that A1R and A2BR could influence the ATP-induced odontoblastic differentiation of HDPCs through one or more of these pathways. However, further studies are required to confirm this speculation.