Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT–mTOR signaling in the presence of CD44

In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation. Therefore, we searched for a compound that induces odontoblastic differentiation of dental pulp stem cells, regardless of the chemical structure and function of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic differentiation by the compound. Because vitamin K analogues can promote bone formation and tissue calcification, we focused on derivatives of naphthoquinone, the skeleton of vitamin K; we verified whether those compounds could induce odontoblastic differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a marker of odontoblasts, was expressed in dental pulp stem cells after treatment with shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected with siRNA against CD44, the shikonin-induced expression of dentin sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT–mTOR signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo CD44-mediated odontoblastic differentiation in our previous study; the present study indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic differentiation. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT–mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggested that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of dental pulp.


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In our previous study, we demonstrated that hyaluronan, a large molecule, induces 66 odontoblastic differentiation of dental pulp stem cells (DPSCs) via interactions with 67 CD44 [1]. However, the underlying cellular signaling mechanism has not yet been 68 elucidated, and it has been unclear whether small molecules can induce odontoblastic 69 differentiation of DPSCs. Therefore, we explored whether small molecules could induce 70 odontoblastic differentiation of DPSCs and attempted to identify the differentiation 71 induction mechanism. In addition, we assessed whether CD44 was essential for this 72 mechanism. 73 In the field of DPSC research, several research groups have shown that collagenase-74 treated cells isolated from human dental pulp showed more rapid proliferation, compared 75 with hematopoietic stem cells, and a similar degree of multipotency [2,3]. Subsequently, 76 various groups have described possible DPSCs. There have been reports of the induction 77 of odontogenic differentiation from DPSCs in vitro [4][5][6]. Moreover, human DPSCs can 78 differentiate into hepatocytes [7], and the differentiation of DPSCs into pancreatic β cells 79 can be induced by specific culture conditions [8]. Some in vivo experiments in rats have 80 demonstrated that DPSC transplantation can regenerate corneal epithelium [9], 81 myocardium [10], spinal cord [11,12], muscle (in a model of muscular dystrophy) [13], 82 and craniofacial bone (using collagen gel as a scaffold) [14]. 83 Notably, there are reports that cells expressing CD44, a marker of DPSCs, are present 84 in dental pulp tissue around the tooth root, and that these CD44-positive cells are involved 85 in mineralization [15,16]. Furthermore, our previous study revealed that DPSCs express 86 high levels of CD44; it also showed that hyaluronan, a ligand of CD44, can induce 87 odontoblastic differentiation of DPSCs. These findings suggest that dental pulp cells, 88 including DPSCs, differentiate into odontoblasts through a CD44-mediated mechanism 89 and participate in calcification.

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Although the intracellular signaling of DPSCs stimulated with hyaluronan involves 91 activation of various signals (e.g., MAPK and AKT) in a CD44-mediated manner, the 92 causal relationship between odontoblastic differentiation of DPSCs and these activating 93 signals is not clear. To the best of our knowledge, there have been few reports of inducers 94 or factors (e.g., hyaluronan) that efficiently induce odontoblastic differentiation of DPSCs.

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Furthermore, it remains unclear whether CD44 is required to induce odontoblastic 96 differentiation of DPSCs. Notably, menatetrenone (a vitamin K 2 analogue) is used 97 clinically as an osteoporosis treatment and hemostatic agent; it is known to be involved 98 in bone formation, tissue calcification, and blood coagulation [17,18]. However, the 99 clinical significance of menatetrenone in the dental field has not been fully investigated.

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In this study, we focused on vitamin K analogues containing naphthoquinone derivatives, 101 to determine whether these analogues can induce odontoblastic differentiation of DPSCs.

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(Tokyo, Japan). Vitamin K 2 and shikonin standard sample (99%) were purchased from      First, we examined whether naphthoquinone derivatives, which constitute the skeleton 167 of vitamin K, could induce odontoblastic differentiation of DPSCs. We found that, 168 among the vitamin K analogues we examined, shikonin prominently induced expression 169 of DSPP, a marker of odontoblastic differentiation (Fig 1). This result suggested that 170 shikonin could induce differentiation of DPSCs into odontoblasts.  Next, we examined the 50% inhibitory concentrations of shikonin and its enantiomer, 179 alkannin, against DPSCs (Fig 2A). The 50% inhibitory concentrations did not 180 significantly differ between shikonin (4.48 ± 0.51 μM) and alkannin (5.08 ± 1.21 μM) 181 (Table 1). Furthermore, DSPP was expressed by DPSCs stimulated with shikonin 182 concentrations greater than 0.1 μM (Fig 2B). Conversely, stimulation with alkannin did 183 not induce expression of DSPP by DPSCs (Fig 2C). These results suggested that shikonin 184 could induce differentiation of DPSCs to odontoblasts in an enantioselective manner.   191 We investigated the shikonin-related intracellular signal transduction that could induce 192 odontoblastic differentiation of DPSCs. AKT phosphorylation peaked at 30 min after 193 shikonin treatment ( Fig 3A); mTOR phosphorylation was also observed between 30 and 194 90 min after shikonin treatment (Fig 3B). Based on these results, we hypothesized that 195 AKT and mTOR play important roles in the mechanism by which shikonin induces 196 odontoblastic differentiation of DPSCs. Thus, we investigated whether DSPP expression 197 was inhibited upon administration of inhibitors for these proteins. When the 198 phosphorylation of AKT was suppressed by treatment with the PI3K inhibitor, Ly294002, 199 the expression of DSPP was suppressed (Fig 3C, D). In addition, when AKT was 200 inhibited with the pan-AKT kinase inhibitor, GSK690693, the expression of DSPP was 201 suppressed (Fig 3E, F). Furthermore, when phosphorylation of mTOR was inhibited by 202 the mTOR inhibitor, rapamycin, the expression of DSPP was suppressed (Fig 3G, H). DPSCs, despite shikonin treatment (Fig 4A). To investigate the relationships between 224 shikonin and CD44 with respect to the odontoblastic differentiation of DPSCs via AKT-225 mTOR signaling, we examined changes in AKT-mTOR signaling after shikonin 226 treatment in DPSCs that had been treated with CD44 siRNA. When CD44 was inhibited, 227 both AKT and mTOR signaling pathways were induced by shikonin treatment (Fig 4B).

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This result indicated in the absence of CD44, shikonin does not induce odontoblastic 229 differentiation of DPSCs, despite its ability to induce AKT-mTOR intracellular signaling.

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Our study showed that shikonin could induce odontoblastic differentiation of DPSCs.

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The AKT-mTOR signaling pathway was the main intracellular mechanism involved; 283 moreover, CD44 was important for the induction of odontoblastic differentiation.

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However, it remains unclear how the AKT-mTOR signaling pathway and CD44 are 285 specifically related to the shikonin-induced odontoblastic differentiation of DPSCs. In 286 addition, it remains unknown whether this effect of shikonin is present in vivo.

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Our previous study indicated that the hyaluronan-induced odontoblastic differentiation 288 of DPSCs was mediated by CD44. In the present study, the expression of CD44 was found