Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli

Abstract The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.


| INTRODUCTION
Dental pulp stem cells (DPSCs) derived from dental pulp are capable of differentiating into odontoblasts, chondroblasts, osteoblasts, angiogenic and active neurons under the appropriate environmental conditions. [1][2][3][4][5][6] Adult human DPSCs are considered as a valuable source for pulpo-dentinal complex regeneration in tooth loss 7 and also a new tool in bone tissue engineering which can fabricate vascularized woven bone tissue. 8 Although the mineralizationrelated molecules involved in DPSC differentiation into odontoblasts/osteoblasts (such as BMP, SHH, TRPM7, etc.) are known, [9][10][11] knowledge of their mechanism remains limited. Therefore, important issue for efficient tooth regeneration is to target hDPSCs to promote their osteogenic/odontoblastic differentiation potential.
Stathmin (also called OP18), a key endogenous regulator of microtubule dynamic, [12][13][14] is a ubiquitously expressed, small cytosolic phosphoprotein that was originally identified as an important factor involved in regulating cell proliferation. 15,16 Microtubules, which are involved in bone metabolism and microtubule assembly modulation, are major components of the cytoskeleton and can regulate changes in bone mass. Microtubule assembly affects bone mass by preventing the proteolytic degradation of GLI2, a major mediator of hedgehog signalling, and in turn stimulates bone morphogenetic protein 2 expression in osteoblast cells. 17 Stathmin plays an essential role in the maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. Although Stathmin seems to be a crucial regulator responsible for the initiation and mineralization of bone, its role in dental pulp is poorly documented.
In our previous study, we showed higher expression of Stathmin in DPSCs than in DPSCs derived from caries 18 [20][21][22] We have been suggested that Stathmin, as an important microtubule regulator, plays an important role in hDPSC osteogenic/odontoblastic differentiation via the Shh signalling pathway.

| Human dental pulp stem cells culture
Human dental pulp tissues were collected from clinically extracted, periodontally healthy and noncarious human mandibular third molars from Nanfang Hospital affiliated Southern Medical University, Guangzhou, China. Human subjects who participated in the study all provided written informed consent, which was approved by the Ethics Committee of Nanfang Hospital. All patients were performed under local anaesthesia by nerve-block of the inferior alveolar nerve, lingual nerve and buccal nerve with two 1.8 mL cartridges of 4% articaine with epinephrine 1:200 000. 23 Before the tooth extracted, the dental crown is handled with 0.3% chlorexidin gel (Forhans, NY, USA) for 2 minutes. Dental pulp was gently removed from the crown and root and dissected into 1 mm 3 pieces with ophthalmic scissors. to remove the tissue pieces not digested. Then, cells were rinsed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin (HyClone, NY, USA). After centrifugation, the supernatant was removed, and the tissue blocks were added to DMEM low glucose medium containing 10% foetal bovine serum and then transferred to 25 cm 2 culture flasks (Corning Inc., NY, USA), which were inverted and incubated in a 5% CO 2 atmosphere at 37°C. The culture flasks were turned over 24 hours later. The individual hDPSCs were released from the pulp after 3-5 days. 24 DPSCs were enriched by collecting multiple colonies. DPSCs at passage 3 were used in each experiment.

| Immunocytochemistry
Human dental pulp stem cells cultured in a confocal dish were fixed in 4% paraformaldehyde for 40 minutes and washed three times with PBS. Then, 0.25% (v/v) Triton X-100 in PBS was used to permeabilize the cell membranes for 15 minutes at room temperature.
The cells were rinsed in 2% BSA for 30 minutes and then incubated overnight with rabbit anti-human Stathmin monoclonal antibody (Abcam, Cambridge, UK) at a dilution of 1:100 in 2% BSA in PBS at 4°C.
After they were washed in PBS, the hDPSCs were covered with FITC-labelled goat anti-rabbit IgG antibody (1:100) and DAPI.

| CCK8 assay
hDPCSs transduced with shRNA-Stathmin lentiviruses, shRNA-Stathmin lentiviruses + purmorphamine and shRNA-Ctrl lentiviruses were seeded at 2 9 10 3 cells in 96-well culture plates for the CCK8 assay. Detection was performed on days 1, 3, 5 and 7. Then, 10 lL of CCK8 reagent was added to each culture well and the cells were incubated avoiding light for 2 hours at 37°C. The absorbance was measured using a SpectraMax M5 multi-functional microplate reader (BD Falcon, San Jose, USA) at a wavelength of 490 nm. The CCK8 assay was repeated in triplicate.  Table 1.

| Alizarin red S staining
Human dental pulp stem cells infected with shRNA-Stathmin lentiviruses and shRNA-Ctrl lentiviruses were cultured in 6-well plates with 2 mL of complete culture medium. When the cell density reached 70%, complete medium supplemented with 0.1 lmol/L dexamethasone, 50 lmol/L ascorbic acid and 10 mmol/L b-glycerol phosphate was used to induce the osteogenic differentiation of the cells for 21 days. After cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature, the induced cells were stained with 0.1% alizarin red S. The assay was performed 3 times.

| Statistical analysis
Outcome measurements were analysed and displayed as the mean AE standard deviation. Statistical significance was performed by the two-sample t test using SPSS v.20.0 software (SPSS Inc.,   Figure 1C-G, **P < .01, ***P < .001), which showed effective silencing of Stathmin with specific shRNA.

CONFLI CT OF INTERESTS
The authors confirm that there are no conflict of interests.