Glycogen synthase kinase-3β inhibitor promotes the migration and osteogenic differentiation of rat dental pulp stem cells via the β-catenin/PI3K/Akt signaling pathway

Background/purpose Glycogen synthase kinase-3β (GSK3β) inhibitor enhances bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone defects. The present study aimed to investigate the effect of AR-A014418 (AR, a specific glycogen synthase kinase-3β inhibitor) on the migration and osteogenic differentiation of rat-derived dental pulp stem cells (rDPSCs), and further explore the underlying mechanism. Materials and methods rDPSCs were isolated from rats, and then cultured with different concentrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration, osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8 assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot, and RT-PCR, respectively. Results Our present study demonstrated that AR of various concentrations (1 μM, 2.5 μM, and 5 μM) not only promoted the rDPSC proliferation and migration, but also increased calcium deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers (RUNX2, OPN, OCN, and OSX) in rDPSCs. It was also found that the administration of AR resulted in an increase in the expression level of p-GSK3β (Ser), β-catenin, p-PI3K, and p-Akt, and a reduction in p-GSK3β (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced cell migration and osteogenic differentiation of rDPSCs induced by AR. Conclusion Our results provide evidence that AR significantly promotes migration and osteogenic differentiation of rDPSCs by activating β-catenin/PI3K/Akt signaling pathway.


Introduction
As all known, stem cells can self-renew and differentiate into other cell types. In the last decade, mesenchymal stem cells (MSCs) have shown great potential in the field of regenerative medicine due to their capacities of selfrenewal and multilineage differentiation. 1,2 MSCs could be isolated from numerous tissues including adipose tissues, 3 bone marrow, 4 skeletal muscle, 5 and dental tissues. 6 In 2004, Seo et al. 7 initially isolated and identified dental pulp stem cells (DPSCs) from the human pulp tissues. Since DPSCs not only display a MSC-like property but also can be conveniently harvested, they are considered promising stem cell sources for clinical use. 8 Extensive basic studies proved the potent potential of DPSCs in the regeneration of bone. 9,10 Additionally, a previous clinical study confirmed the safety and therapeutic effects of DPSCs in treating severe periodontal defects. 11 It is undoubted that the osteogenic differentiation capacity of DPSCs is crucial for bone regeneration. Besides, the migration of endogenous or exogenous DPSCs to bone injury or loss sites is also significant for bone regeneration. 12 Therefore, enhancement of osteogenic differentiation and migration of DPSCs is an important avenue in improving the therapeutic efficacy of DPSC-based therapy.
In recent years, increasing studies have found that some small molecules show osteogenic function via diverse signaling mechanisms. 13e15 Given that it is easy to synthesize small molecules, which are non-immunogenic and cost-effective, it seems an attractive choice for directing osteogenic differentiation and migration of PDLSCs. Glycogen synthase kinase-3 (GSK3) is a serineethreonine protein kinase containing two isoforms (GSK3a and GSK3b), which is an important component of various key signaling pathways, including insulin, mTOR, NF-kB, and Wnt/b-catenin. 16 Many studies have implicated the Wnt signaling pathway in the lineage differentiation and migration of MSCs. 17,18 It is well known that GSK3b inhibitors can activate the Wnt signaling pathway by decreasing the degradation of b-catenin. Hence, GSK3b inhibitors may provide a new therapeutic strategy for DPSC-based bone regeneration. In the past decades, a number of small molecule GSK3b inhibitors have been identified; 19e21 however, more than half of the published inhibitors not only target GSK3b, but also affect cyclindependent kinase 2 (cdk2) or cdk5. 22 In 2003, Bhat et al. 23 identified an amino thiazole in a high-throughput biochemical screening using purified recombinant GSK3b, which was named as AR-A014418 (AR). AR can inhibit GSK3b activity in an ATP-competitive way, with high specificity for GSK3b as it does not affect the activity of other 26 protein kinases, especially cdk2 and cdk5 (IC50 > 100 mM) that are inhibited by the majority of GSK3 inhibitors. 23 Several studies reported that AR had potent activities against diverse types of cancer with no adverse effects in rodents. 24,25 However, to our knowledge, the role of AR in migration and osteogenic differentiation of DPSCs has been rarely studied before.
The aim of this study is to investigate the effects of GSK3b inhibitors, AR, on migration and differentiation of rat DPSCs (rDPSCs) into osteoblasts in vitro, and preliminarily explore the underlying molecular mechanisms.

Materials and methods rDPSCs isolation and characterization
SpragueeDawley rats (3 months, SPF grade) were purchased from Guangdong Medical Laboratory Center (Guangzhou, China), and rDPSCs were isolated from the dental pulp of incisors of rats, as Alksne et al. 26 previously described. The isolated rDPSCs were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 C in a 5% CO 2 incubator. The third rDPSC passage was subjected to characterization, which included identification of the cell surface markers and multidirectional differentiation of rDPSCs. Every procedure was approved by the Animal Care and Use Committee of Fujian Provincial Hospital.

Osteogenic and adipogenic differentiation
For osteogenic differentiation, 5 Â 10 4 rDPSCs were maintained in cultured media containing 10 mM b-glycerol phosphate, 50 mg/mL L-ascorbic acid, 10 nM dexamethasone, and 2 mM glutamine for 7 or 14 days. During the cultivation, the media were renewed three times every week.

Alizarin red S (ARS) staining
ARS staining was conducted to confirm osteogenic differentiation and assess calcium-rich deposits in rDPSCs. In brief, after incubated with osteogenic media for 14 days, rDPSCs were fixed with 4% paraformaldehyde (PFA) for 15 min and subsequently washed with PBS. 2% ARS solution was added and incubated for 15 min. Thereafter, the stained rDPSCs were observed under an optical microscope. For quantitative evaluation, ARS was dissolved in 5% perchloric acid by measuring absorbance at 562 nm under a microplate spectrophotometer.

Oil red O staining
As a fat-soluble diazo dye that can stain lipid droplets in cells, Oil Red O is usually utilized to confirm the adipogenic Journal of Dental Sciences 17 (2022) 802e810 differentiation of MSCs. Briefly, rDPSCs were fixed with 4% PFA, followed by staining with Oil Red O solution in isopropanol for 15 min. Finally, differentiated oil red O-positive rDPSCs were observed and imaged under a microscope.

Western blot
rDPSCs were lysed in RIPA buffer (Abcam, Cambridge, MA, USA) on ice to extract total protein. The total protein was separated by SDS-PAGE gels and electrophoretically transferred onto PVDF membranes, which were subsequently blocked in skimmed milk (12%) for 2 h. The membranes were incubated overnight at 4 C with primary antibodies, and subsequently rinsed three times before incubation with the secondary antibody. Finally, protein bands were visualized using ECL Substrate Kit (Abcam), and quantified using Image J software (Bethesda, MD, USA). The information of antibodies used in this study was described in Supplementary Table S1.

Transwell assay
Briefly, 700 mL cultured media were added to the bottom chamber. Then, 200 mL rDPSCs suspension (serum-free) with different treatments was added to the top chamber with a filter membrane (8 mm pores), followed by 24-h incubation. The cells which migrated to the bottom chamber were fixed and stained. They were then photographed under a microscope.

Alkaline phosphatase (ALP) assay
After incubated with osteogenic media for 7 days, the ALP activity of rDPSCs was determined using colorimetric Alkaline Phosphatase Assay Kit (Abcam). In short, rDPSCs were incubated with ALP substrate for 15 min in the dark. Then, the reaction was stopped by 2 M NaOH. The absorbance of the product was detected at 405 nm under a microplate reader.

Statistical analysis
All data were expressed as mean AE standard deviation (SD) of three independent experiments. The Student's t-test was used for comparison between two groups. For multiplegroup condition, one-way ANOVA was performed with Bonferroni's method. P < 0.05 indicated significant difference.

Results rDPSC characterization
rDPSCs were isolated from incisors of rats, of which surface antigens were characterized using Western blot. Compared with non-DPSCs, CD29, CD44, CD90, and CD105 were obviously up-regulated, while CD34 and CD45 were downregulated in the isolated rDPSCs (Fig. 1A). ARS staining and Oil red O staining respectively confirmed the role of rDPSCs in osteogenic differentiation and adipogenic differentiation (Fig. 1B). All these data suggested successful isolation of rDPSCs.

AR enhances migration and osteogenic differentiation of rDPSCs
Using the CCK-8 kit, the effect of AR (0, 1, 2.5, 5, 10, and 20 mM) on rDPSCs proliferation was observed ( Fig. 2A). On day 1 and day 3, there was no significant discrepancy in rDPSCs proliferation between groups. After 5-day and 7-day incubation, both 10 and 20 mM of AR suppressed proliferation of rDPSCs. Notably, after 5-day and 7-day incubation, 15 mM, 2.55 mM and 5 mM of AR enhanced proliferation of rDPSCs. Hence, 15 mM, 2.55 mM and 5 mM of AR were selected to investigate the effect of AR on rDPSCs migration and osteogenic differentiation. As shown in Fig. 2B, the migratory capacity of rDPSCs was promoted by AR in a concentration-dependent way.
The biological role of AR in osteogenic differentiation of rDPSCs was also explored using ALP assay, and ARS staining, and the expression of osteogenic markers (RUNX2, OPN, OCN, and OSX) was determined. The results showed that the ALP activity of rDPSCs in 2.5 mM and 5 mM groups was significantly higher than that in the control group after osteogenic induction for 7 days (Fig. 2C). Similar to the result of the ALP assay, the ARS staining showed there was no significant difference between the control and 1 mM groups (Fig. 2D). Besides, the deposition of calcium induced by osteogenic media in rDPSCs was significantly increased by 2.5 mM and 5 mM of AR (Fig. 2D). Moreover, both 2.5 and 5 mM of AR significantly elevated the expression of RUNX2, OPN, OCN, and OSX in rDPSCs at mRNA and protein levels ( Fig. 2E and F). Taken together, our results indicated that AR plays a positive role in migration and osteogenic differentiation of rDPSCs.

AR promoted migration and osteogenic differentiation of rDPSCs via the Wnt/b-catenin/ PI3K/Akt signaling
To preliminarily explore the potential mechanisms of AR acting in migration and osteogenic differentiation, the effect of AR on the activity of Wnt/b-catenin, PI3K/Akt, ERK1/2, and JNK signaling pathways in rDPSCs was investigated. The results were depicted in Fig. 3. Compared with the control group, AR treatment decreased the phosphorylation of GSK3b at Tyr216 along with an increase in the expression of b-catenin. Besides, the phosphorylation of GSK3b at Ser9 was increased by the treatment of 5 mM AR. These data suggested that AR could reduce the activation of GSK3b, thereby increasing the expression of b-catenin to activate Wnt/b-catenin signaling. In addition, the treatment of AR also increased the phosphorylation of both PI3K and Akt in a concentration-dependent way. The phosphorylation of ERK1/2 was obviously increased when the concentration of AR reached 5 mM. However, there was no obvious difference in the phosphorylation of JNK among all groups. Collectively, our results suggested that the Wnt/ b-catenin/PI3K/Akt signaling was involved in the effect of AR on migration and osteogenic differentiation of rDPSCs.

Inhibiting PI3K reversed the effect of AR on migration and osteogenic differentiation of rDPSCs
To confirm the role of PI3K/Akt signaling in the function of AR in rDPSCs, rDPSCs were pretreated with LY294002 (15 mM), a specific PI3K inhibitor, for 1 h before AR treatment (5 mM). LY294002 significantly weakened the migration ability of AR-treated rDPSCs (Fig. 4A). ALP assay and ARS staining indicated that LY294002 effectively reversed the increased osteogenic differentiation induced by AR ( Fig. 4B and C). Additionally, LY294002 also significantly decreased the expressions of osteogenic markers in ARtreated rDPSCs at both mRNA and protein levels ( Fig. 4D and E). We further investigated the effect of LY294002 interference on Wnt/b-catenin/PI3K/Akt signaling in ARtreated rDPSCs. As expected, the treatment of LY294002 noticeably decreased expression of p-PI3K and p-Akt in ARtreated rDPSCs. In the meantime, LY294002 also effectively suppressed the decrease of p-GSK3b (Tyr216) and increase of p-GSK3b (Ser9) and b-catenin induced by AR treatment. In short, these data suggested that PI3K/Akt signaling pathway was involved in the role of AR in migration and osteogenic differentiation of rDPSCs.

Discussion
DPSCs are an attractive source of MSCs for regenerative medicine due to their relative accessibility and lack of ethical controversy. 28,29 It has been demonstrated that DPSCs display greater proliferative potential than bone MSCs. 30 During the application of DPSCs for bone regeneration, it is required for DPSCs to expand and migrate to the bone damaged/defect area and differentiate into osteoblasts. Hence, it is important to explore a strategy for promoting DPSCs migration and osteogenic differentiation in DPSC-based therapy. In this study, it was demonstrated that AR, an inhibitor specific to GSK3b, may promote cell migration and osteogenic differentiation via specifically activating Wnt/b-catenin/PI3K/Akt signaling pathways in rDPSCs.
In an attempt to determine the ideal concentration range of AR on the cell proliferation of rDPSCs, a CCK-8 assay was carried out on rDPSCs after treated with diverse concentrations of AR. In our study, after 7 days of incubation, AR suppressed cell viability of rDPSCs at a concentration of 10 up to 20 mM, while no more than 5 mM promoted the cell viability. Based on the results, a concentration range of 1e5 mM was chosen for the subsequent experiments. Thereafter, our data demonstrated that the migratory ability of hDPSCs was enhanced by 2.5 and 5 mM of AR. Previous studies indicated that GSK3 inhibitors potentially promoted osteogenic differentiation of several MSCs. 31e33 Similar to the literature, our study showed that the indicated concentrations of AR enhanced ALP activity and calcium deposition. Moreover, both the mRNA and protein expressions of RUNX2, OPN, OCN, and OSX were upregulated in a concentration-dependent way. RUNX2 is a crucial transcription factor involved in osteoblast differentiation, which regulates expression of many osteogenesis-related genes. 34 Takahashi 35 indicated that the overexpression of RUNX2 induced differentiation of preadipocytes into functional osteoblasts. RUNX2 enhances the expression of OSX, 36 thereby inducing the expression of OCN and OPN. 37 These data collectively revealed that AR promoted migration and osteogenic differentiation of rDPSCs.
It is well known that Wnt/b-catenin signaling is involved in tissue generation, regeneration, as well as self- renewal. 38 Given that GSK3b is one of the key components of Wnt/b-catenin signaling, we assessed the effect of AR on the activity of the Wnt/b-catenin pathway. The activity of GSK3b is down-regulated by phosphorylation at the Ser9 site, while up-regulated by phosphorylation at the Tyr216 site. 16 The inactivation of GSK3b might lead to a decrease in the degradation of b-catenin, thereby activating Wnt/bcatenin signaling. Previous studies demonstrated that the treatment of AR leads to a significant reduction in GSK-3b phosphorylation at Tyr216 and regulates diverse biological processes, such as cell growth, 39 apoptosis, 40 and survival. 41 Besides, AR increases GSK-3b phosphorylation at Ser9 when promoting osteogenic differentiation in human adipose-derived MSCs. 31 A similar result was obtained in the present study. Once the concentration of AR reached 5 mM, the Ser9 phosphorylation of GSK3b was significantly enhanced, but 1 mM and 2.5 mM of AR did not affect Ser9 phosphorylation of GSK3b. Meanwhile, the administration of AR resulted in an obvious decrease in the Tyr216 phosphorylation of GSK3b in a dose-dependent manner. Meanwhile, as expected, the protein level of b-catenin was elevated in rDPSCs after treated with AR. These data revealed that the activation of Wnt/b-catenin signaling might be involved in the role of AR in rDPSCs migration and osteogenic differentiation.
Moreover, our data also indicated that AR could increase the activity of the PI3K/Akt pathway in a dose-dependent manner, but displayed no obvious effect on JNK signaling in rDPSCs. Our study also revealed that the activity of the ERK1/2 in rDPSCs was obviously elevated only when the concentration of AR reached 5 mM. This suggested that a high dose of AR might further induce the activation of ERK1/2 in rDPSCs. Since our results demonstrated that AR promoted migration and osteogenic differentiation of rDPSCs once its concentration reached 2.5 mM, the subsequent experiments merely focused on the role of the PI3K/ Akt pathway in the function of AR. Akt is an important member of the protein kinase family, and it is at the center of the signaling pathway, which is a biological signal transduction pathway initiated by PI3K and can be activated by many stresses. Multiple studies implicated the PI3K/Akt signaling pathway in cell proliferation and differentiation. 42,43 Accumulating evidence has demonstrated that this pathway is involved in osteogenic differentiation. 44,45 Besides, a recent study has revealed that the migration of DPSCs is associated with the PI3K/Akt signaling. 46 In our study, LY294002 was used to inhibit PI3K/ Akt signaling, and it was found that the treatment of LY294002 reversed the enhanced migration and osteogenic differentiation induced by AR. GSK3b is an important  substrate of PI3K/Akt signaling. 47 PI3K/Akt signaling inhibits GSK3b by enhancing its phosphorylation at the Ser9 site. Our data suggested that inhibition of GSK3b activated PI3K/Akt signaling, which was related to the role of AR in promoting migration and osteogenic differentiation of rDPSCs. Our data hinted a novel regulatory loop between GSK3b and PI3K/Akt in rDPSC migration and osteogenic differentiation.
In conclusion, the present study indicated that AR, a GSK3b inhibitor, promoted migration and osteogenic differentiation of rDPSCs in vitro. Activation of theb-catenin/ PI3K/Akt signaling pathway was involved in the effect of AR on migration and osteogenic differentiation of rDPSCs.

Declaration of competing interest
The authors have no conflicts of interest relevant to this article.