The Role of Neuropilin-1–FYN Interaction in Odontoblast Differentiation of Dental Pulp Stem Cells

Abstract

Abnormal odontoblast differentiation of dental pulp stem cells (DPSCs) caused by inflammation is closely related to the development of dental caries. Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell differentiation. FYN belongs to the protein-tyrosine kinase family, which has been implicated in the control of cell growth, and the effect can be further strengthened by inflammatory factors. In our studies, we verified that NRP1 can form complexes with FYN and have the correlation changes in odontoblast differentiation of DPSCs. Therefore, we surmise that in the progress of dental caries, NRP1 interacts with FYN, by expanding inflammation and inhibition of odontoblast differentiation of DPSCs through nuclear factor kappa B (NF-κB) signaling pathway. In this subject, we first investigated the expression and interaction of NRP1 and FYN in DPSCs. And then, we researched the effect of this complex controlling downstream signal pathway in normal or inflammation stimulated DPSCs. Finally, we analyzed the relationship between this role and odontoblast differentiation of DPSCs. This research will provide the molecular mechanism of inflammation factors of dental caries through activating NF-κB signal regulating odontoblast differentiation in DPSCs for finding new potential drug targets for the clinical treatment of dental caries.

Introduction

Dental caries, a chronic infectious disease, can lead to the demineralization of enamel and dentin and subsequent pulp tissue injury (Akira et al., 2006). The polyinfection of bacterium is one of the main pathogenic factors. Numerous studies have suggested that bacteria stimulate production of pro-inflammatory cytokines (Soell et al., 1994). Cytokines are associated with the beginning and advancing of dental caries, and their levels upgrade in caries-active saliva (Evans et al., 1990; Gornowicz et al., 2012; Kim et al., 2002). Bacteria and inflammatory cytokines stimulate to produce the restorative dentin to maintain the completeness of the dental structure. However, due to the speed of the tooth decay being much faster than the speed of renovation, it eventually leads to dental caries (Larmas and Sandor, 2014). Meanwhile, dental caries is commonly treated in dental practices by removing the necrotic dental tissues, cleaning the pulp chamber, and filling the holes with dental materials, such as silver amalgams, glass ionomer cement, or composite resins. But in the tooth repair it offers nonbiological materials and has many limitations, containing bad biocompatibility, damage to surrounding normal tissues, unpleasant sensations, and unpredictable long-term curative effect (Gerritsen et al., 2010; Noble et al., 2013). Therefore, the understanding of the mechanisms of restraining inflammatory cytokine release and finding an alternative to traditional root canal therapy may provide new therapeutic strategies for dental caries.

Neuropilin-1 (NRP1) as a single-pass transmembrane glycoprotein comprises an N-terminal extracellular region that consists of two CUB domains, named a1 and a2, two blood coagulation factor V/VIII homology domains, termed b1 and b2, and a c domain that finds in the single transmembrane domain and small cytoplasmic domains and mediates interactions with other receptors (Takagi et al., 1991). NRP1 was initially identified as a coreceptor binding with the class 3 semaphorins (Sema3) and several members of growth factors, including vascular endothelial growth factor, hepatocyte growth factor, platelet derived growth factor, transforming growth factor β, and so on (Kumanogoh and Kikutani, 2013; Pellet-Many et al., 2008). For example, NRP1 is a high-appealing receptor for combining with Sema3A (also termed collapsin-1) and recognizing Plexin-A1 and its mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathway to participate in the inflammatory response (Ito et al., 2014). Several recent works reported that Sema3A unites with NRP1 and sequesters Plexin-A1 from triggering receptor expressed in myeloid cells 2/DNAX-activating protein 12, thus inhibiting RANKL-induced osteoclast differentiation. In addition, once Plexin-A1 binds to Sema3A-NRP1 to form trimer, it triggers Rac1 activity and promotes osteoblast differentiation (Hayashi et al., 2012; Liu et al., 2016). In our previous studies, we verified that NRP1 can accelerate odontoblast differentiation of dental pulp stem cells (DPSCs), but the exact mechanism is unclear.

FYN, belonging to the protein-tyrosine kinase family, is one member of Src family of protein tyrosine kinases (SrcPTKs), including Fyn, Yes, c-Src, Lyn, Lck, Hck, Blk, and Fgr. Fyn, Yes, and c-Src are widely expressed in the human body, while Hck, Blk, and Fgr are only expressed in specific tissues (Thomas and Brugge, 1997). The location of FYN is in the inner layer of the cytomembrane and it is attached by palmitic and myristic acids (Anderson et al., 1990). Similar to other family members of SrcPTKs, the activity of FYN is possessed by molecular interactions affected by tyrosine phosphorylation and dephosphorylation (Yeatman, 2004). FYN activation leads to participate in the control of cell growth, survival, adhesion, motility, cytoskeletal reconstruction, neural development, tumor metastasis, and many important physiological and pathological processes (Elias and Ditzel, 2015). On the one hand, some scholars have found that TLR4 initiates the interaction with FYN leading to an accelerated tyrosine phosphorylation in both molecules under the stimulation of lipopolysaccharide. The enhanced kinase activity activates classical inflammatory signaling cascades involving phosphatidylinositol 3-kinase/Akt/NF-κB pathways (Ko et al., 2015). On the other hand, some scholars have found that a common Cbl mediated negative feedback mechanism controlling both FYN and LYN and fibroblast growth factor receptor 2 (FGFR2) degradation in reaction to constitutive FGFR2 overactivity and indicated a role for the molecular mechanisms by which constitutive FGFR2 activation promotes osteoblast differentiation (Kaabeche et al., 2004). Thus, in the current study, we investigated whether NRP1 could control odontoblast differentiation of DPSCs associated with the interaction of FYN.

Materials and Methods

Cell cultures

All samples were acquired from generally healthy patients (20–28 years of age) after giving the informed consents, which were approved by the Ethics Committee of the Affiliated Hospital of Nantong University. Normal pulp tissues (n = 8) were isolated from complete wisdom teeth of patients without caries or pulp disease. Carious pulp tissues (n = 8) were obtained from molars diagnosed with deep caries. The diagnosis of deep caries was determined by endodontic doctors based on clinical evaluation, including the caries progressing to dental deep layer, the amount of remaining dentin thickness being <2 mm, and with no history of spontaneous and intense pain.

We isolated DPSCs from normal pulp tissues and carious pulp tissues by cleaning the tooth surface, cutting around the cementoenamel junction using sterilized dental fissure burs, and then opening to reveal the pulp chamber. The pulp was minced and digested in a solution of 3 mg/mL collagenase type I for 1 hour at 37°C. Single-cell suspensions were obtained by passing the digested tissues through a 70-μm cell strainer (BD Falcon). Cell suspensions of dental pulp were seeded into 25 cm² culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂ (Ledesma-Martinez et al., 2016). The medium was changed every 3 days. Approximately 7–10 days after seeding, the cells became nearly confluent. Cells were passaged at the ratio of 1:3 when they reached 80%–85% confluence. Stem cells from normal pulp tissues were named normal dental pulp stem cells (N-DPSCs), and those from carious pulp tissues were named inflammatory dental pulp stem cells (I-DPSCs). The cell populations were characterized by positive staining with anti-CD34, STRO-1, and c-kit and the absence of CD45 (Feng et al., 2013; Yu et al., 2010). Cells from the third to fifth passages were used in all experiments.

Odontoblast differentiation

DPSCs (2 × 10⁴ cells/dish) were cultured in 35 mm culture dishes (Costar, Cambridge, MA) in odontogenic differentiation medium containing 15% FBS (Gibco-BRL; Life Technologies, Inc., Gaithersburg, MD), a minimum essential medium (Invitrogen, Carlsbad, CA), 10 mmol/L β-glycerophosphate, 0.292 mg/mL glutamine, 50 mg/mL α-ascorbic acid, 10 nmol/L dexamethasone (Sigma-Aldrich, St Louis, MO), 100 U/mL penicillin G, and 100 mg/mL streptomycin, respectively, for 7 and 21 days, replacing the medium every 2 days. Then, the cells were collected for determination of the odontoblast differentiation by evaluating protein expression of odontogenic markers, including dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP). After induced for 7 and 21 days, cells were prepared for alizarin red S staining, alkaline phosphatase (ALP) staining, and immunofluorescence. Protein and RNA were extracted for Western blot analysis and real-time reverse transcription–polymerase chain reaction (RT-PCR).

Alizarin red S and ALP staining

DPSCs were fixed with 4% paraformaldehyde (PFA) for 1 h and washed with phosphate-buffered saline (PBS). Cells were then stained with 40 mmol/L alizarin red S (pH = 4.2) for 10 minutes under conditions of gentle agitation. Absorbance of the extracted Alizarin red S stain was measured at 570 nm. DPSCs were subjected to ALP staining using the ALP Assay Kit (JianCheng, Nanjing, China) according to the manufacturer's instructions.

Western blot analysis

Total extracts of cells were lysed in buffer consisting of 50 mM TRIS, 150 mM NaCl, 2% sodium dodecyl sulfate (SDS), and a protease inhibitor mixture and centrifuged at 12,000 rpm for 12 minutes at 4°C. The proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes at 350 mA for 2.5 hours in a blotting apparatus (Bio-RAD, CA). After blocking in Tris-Buffered Saline with Tween with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with primary antibodies. Then, the membranes were reacted with corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. GAPDH and β-tubulin were used as the internal control for the cytoplasmic and nuclear proteins. The following primary antibodies were used: GAPDH (anti-mouse; Santa Cruz), β-tubulin (anti-mouse; Sigma), NRP1 (anti-rabbit; Santa Cruz), FYN (anti-mouse; Santa Cruz), DMP1 (anti-rabbit; Santa Cruz), DSPP (anti-rabbit; Santa Cruz), β-catenin (anti-mouse; Cell Signaling), p65 (anti-mouse; Santa Cruz), interleukin (IL)-6 (anti-mouse; Santa Cruz), and IL-1β (anti-mouse; Santa Cruz).

Immunofluorescence staining

DPSCs were seeded into the 24-well plates, fixed with 4% PFA for 1 hour, rinsed with PBS containing 0.1% Triton X-100 (PBST), and blocked for 30 minutes in PBST supplemented with 10% FBS. Cells were incubated with primary antibodies against p65 (1:100, anti-mouse; Santa Cruz) or β-catenin (1:100, anti-mouse; Cell Signaling) in the same solution overnight at 4°C. Then cells were rinsed and incubated with secondary antibodies for 2 hours at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (4060-diamidino-2-phenylindole dihydrochloride) (1:1000; Santa Cruz). Finally, the cells were examined using a Leica fluorescence microscope (Germany).

Real-time RT-PCR

Total cellular RNA was extracted from the cells and reverse transcribed using conventional protocols. PCR amplification was performed using the following primer sets: GAPDH, forward: 5′-TCCATGACAACTTTGGTATCG-3′ and reverse: 5′-TGTAGCCAAATTCGTTGTCA-3′; NRP1, forward: 5′-TGAGCCCTGTGGTTTATTCC-3′ and reverse: 5′-CGTACTCCTCTGGCTTCTGG-3′; FYN, forward: 5′-CTCAGCACTA-CCCCAGCTTC-3′ and reverse: 5′-ATCTCCTTCCGAGCTGTTCA-3′; DMP1, forward: 5′-TGGGGATTATCCTGTGCTCT-3′ and reverse: 5′-GCTGTCACTGGGGTCTTCA T-3′; DSPP, forward: 5′-GGAGACAAGACCTCCAAGAGTA-3′ and reverse: 5′-TGCTGGGACCCTTGATTTCTA-3′; IL-1β, forward: 5′-ATAAGCCCACTCTACAGCT-3′ and reverse: 5′-ATTGGCCCTGAAAGGAGAGA-3′; IL-6, forward: 5′-GTACCCCCAGGAGAAGATTC-3′ and reverse: 5′-CAAACTGCATAGCCACTTTC-3′; β-catenin, forward: 5′-GCCATCTGTGCTCTTCGTCA-3′ and reverse: 5′-TGGTGGGTGCAGGAGTTA-3′; and p65, forward: 5′-GCAGCACTACTTCTTGACCAC-3′ and reverse: 5′-AACATGGCAGGCTATTGCTCA-3′. All the primer sequences were determined using established GenBank sequences. The primers were used to amplify the duplicate PCRs. Each sample was calculated from three independent experiments, and GAPDH was used as an internal control.

Immunoprecipitation assay

DPSCs were lysed with 1 mL of lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, 5 mM ethylenediaminetetraaceticacid, 2 mM Na₃VO₄, 2.5 mM Na₄PO₇, 100 mM NaF, 200 nM microcystin lysine–arginine, and protease inhibitors). Then, cells were centrifuged at 13,000 rpm for 5 minutes, and supernatants were transferred to new cups and incubated on ice. For co-immunoprecipitation, cell lysate was immunoprecipitated with indicated antibodies and protein G/A agarose (Roche Diagnostics). The immunoprecipitates were subjected to immunoblotting. After immunoprecipitation, the beads were washed thrice, and the bound proteins were released from the beads by boiling in SDS-PAGE sample buffer for 5 minutes. Samples were analyzed by western blotting with indicated antibodies.

Small interfering RNAs and transfection

Small interfering RNA (siRNA) transfection was carried out using a commercially available kit (Genpharm). For siRNA inhibition studies, DPSCs were washed with the siRNA transfection medium and then incubated (at 37°C and 5% CO₂) for 12 hours with transfection medium containing the transfection reagent and either siRNA targeting NRP1 (50 nmol/L) or control siRNA (50 nmol/L), according to the manufacturer's instructions. After transfection, the cells were harvested at 72 hours for RNA or protein extraction. Oligo sequences used were as follows: siNRP1-RNA, 5′-AACGATAAATGTGGCGATA-3′; Negative control siRNA, 5′-TTCTCCGAACGTGTCACGT-3′; siFYN-RNA, 5′-UAAAGCGCCACAAACAGUGUCACUC-3′; Negative control siRNA, 5′-TTCTCCGAACGTGTCACGT-3′.

Plasmid constructs and plasmid transfection

The full-length NRP1 (GenBank Accession Number NM_003873) was extracted from the human cDNA library and connected to P-CMV-Flag. The primers used for NRP1 were as follows: 5′-TGAGCCCTGTGGTTTATTCC-3′ (sense), 5′-CGTACTCCTCTGGCTTCTGG-3′ (anti-sense). Transfection of P-CMV-Flag and P-CMV-Flag-NRP1 in DPSCs was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Statistical analysis

All experiments were repeated at least in triplicate, and values are presented as the mean ± standard deviation. The significance of differences between the experimental groups and controls was analyzed using analysis of variance. Statistical significance was evaluated by the independent samples t-test using SPSS v17.0 software. Differences in which p was <0.05 were considered to be statistically significant.

Results

Effect of NRP1 and FYN in DPSCs in noninflammatory and inflammatory microenvironment

To demonstrate whether NRP1 and FYN expressions in carious pulp were different from that in normal pulp, we isolated and characterized DPSCs from pulp tissues of teeth that had been clinically diagnosed as deep caries and compared them with those from normal teeth. We obtained that NRP1 was highly expressed in N-DPSCs than in I-DPSCs, and the levels of odontogenic markers, including DMP1 and DSPP, significantly declined in I-DPSCs. In contrast, FYN was lowly expressed in N-DPSCs than in I-DPSCs (Fig. 1A). As demonstrated in Figure 1B, the mRNA levels of NRP1, DMP1, DSPP, and FYN were consistent with the results of protein analysis. Alizarin red S staining for mineralized matrix results showed that mineralized matrix was strongly decreased in I-DPSCs. Likewise, the ALP activity was also decreased in comparison with N-DPSCs and I-DPSCs (Fig. 1C). It is well known that classical Wnt/β-catenin pathway plays an important role in promoting odontoblastic differentiation of DPSCs (Lian et al., 2016). Meanwhile, NF-κB is a noted transcription factor concerned in the process of inflammation. As expected, our data showed that nuclear β-catenin expression was higher in the N-DPSC group compared to the I-DPSC group, while nuclear p65 presented the exact opposite result (Fig. 1D). Immunofluorescence research also confirmed the same results (Fig. 1E, F). These data indicate that NRP1 may promote odontoblast differentiation of DPSCs and that FYN is related to inflammation.

FIG. 1.

Effect of NRP1 and FYN in DPSCs in N-DPSCs and I-DPSCs. N-DPSCs and I-DPSCs were cultured in odontogenic differentiation medium for 21 days. (A) Western blot analysis showed that the levels of NRP1, DMP1, and DSPP significantly increased in N-DPSCs compared with I-DPSCs. In contrast, FYN was lowly expressed in N-DPSCs than in I-DPSCs. (B) The mRNA levels of NRP1, DMP1, DSPP, and FYN were detected by RT-PCR (*p < 0.05). (C) Mineralized matrix was strongly decreased in I-DPSCs by ALP staining and Alizarin red S staining. (D) Western blot analysis showed that nuclear β-catenin expression was higher in the N-DPSC group, while nuclear p65 expression was higher in the I-DPSC group. (E) The expression of β-catenin in the cytoplasm and nucleus of DPSCs was examined by immunofluorescent staining. Nuclei are stained with DAPI. Scale bar 100 μm. All experiments were performed thrice independently. (F) The expression of p65 in the cytoplasm and nucleus of DPSCs was examined by immunofluorescent staining. Nuclei are stained with DAPI. Scale bar 100 μm. All experiments were performed thrice independently. DPSC, dental pulp stem cell; NRP1, neuropilin-1; DMP1, dentin matrix protein-1; DSPP, dentin sialophosphoprotein; I-DPSC, inflammatory dental pulp stem cell; N-DPSC, normal dental pulp stem cell; DAPI, 4′,6-diamidino-2-phenylindole; RT-PCR, reverse transcription–polymerase chain reaction; ALP, alkaline phosphatase.

Effect of NRP1 and FYN on tumor necrosis factor-α-induced inflammatory microenvironment

To further extend the relevance of these observations, tumor necrosis factor-α (TNF-α) was used to stimulate DPSCs to imitate the inflammatory microenvironment of deep caries to examine the effect of NRP1 and FYN on odontoblast differentiation in DPSCs. After stimulation with TNF-α, NRP1 expression dramatically increased along with DMP1 and DSPP. These expressions had their maximums at the dose of 10 ng/mL and then significantly decreased at the dose of 100 ng/mL. The results showed that the FYN protein expression level was influenced by TNF-α in a dose-dependent manner (Fig. 2A). Consistent with the protein results, the mRNA levels, alizarin red S staining for mineralized matrix, and the ALP activity also displayed a similar change in DPSCs (Fig. 2B, C). At the same time, in the group treated with TNF-α of 10 ng/mL nuclear β-catenin expression increased and after treated with 100 ng/mL TNF-α suppressed β-catenin nuclear translocation. But nuclear p65 progressively rose stimulated by TNF-α in a dose-dependent manner (Fig. 2D). Immunofluorescence analysis also sustained them (Fig. 2E, F). These results suggest that NRP1 accelerates odontoblast differentiation through classical Wnt/β-catenin signaling, and FYN regulates inflammation by activating NF-κB pathway.

FIG. 2.

Effect of NRP1 and FYN in TNF-α-induced DPSCs. DPSCs were cultured in odontogenic differentiation medium and added TNF-α (10 or 100 ng/mL) for 21 days. (A) Western blot analysis showed that NRP1 expression dramatically increased along with DMP1 and DSPP with the stimulation of 10 ng/mL TNF-α, then significantly decreased at the dose of 100 ng/mL, and the FYN protein expression level was influenced by TNF-α in a dose-dependent manner. (B) The mRNA levels of NRP1, DMP1, DSPP, and FYN were detected by RT-PCR (*p < 0.05). (C) The mineralized nodule formation was detected by ALP staining and Alizarin red S staining. (D) Western blot analysis showed that in the TNF-α of 10 ng/mL group nuclear β-catenin expression increased, DPSCs treated with 100 ng/mL TNF-α suppressed β-catenin nuclear translocation, and nuclear p65 progressively rose stimulated by TNF-α in a dose-dependent manner. (E) The expression of β-catenin in the cytoplasm and nucleus of DPSCs was examined by immunofluorescent staining. Nuclei are stained with DAPI. Scale bar 100 μm. All experiments were performed thrice independently. (F) The expression of p65 in the cytoplasm and nucleus of DPSCs was examined by immunofluorescent staining. Nuclei are stained with DAPI. Scale bar 100 μm. All experiments were performed thrice independently. TNF-α, tumor necrosis factor-α.

The interaction of NRP1 and FYN

To reveal the molecular mechanism underlying NRP1-mediated control of odontoblast differentiation, we searched for NRP1-interacting proteins through which NRP1 may play its encouraging role in odontoblast differentiation indirectly. Co-immunoprecipitation experiments were conducted to examine the physical interaction between NRP1 and FYN induced by TNF-α with regard to the dose interval. Co-immunoprecipitated NRP1 and FYN were verified by means of Western blot analysis (Fig. 3A, B). Stimulation with TNF-α significantly and persistently increased interaction of NRP1 and FYN (Fig. 3C–F).

FIG. 3.

FYN is identified as a NRP1-interacting protein. DPSCs were cultured in odontogenic differentiation medium and added TNF-α (10 or 100 ng/mL) for 7 days. (A, B) Immunoprecipitation and western blot analysis for the interaction between endogenous NRP1 and FYN. (C, D) Immunoprecipitation and western blot analysis for the interaction between endogenous NRP1 and FYN cultured with TNF-α of 10 ng/mL. (E, F) Immunoprecipitation and western blot analysis for the interaction between endogenous NRP1 and FYN cultured with TNF-α of 100 ng/mL. IgG, immunoglobulin G.

FYN weakens the NRP1-mediated signaling in odontoblast differentiation

Our previous study demonstrated that NRP1 promotes odontoblast differentiation and that FYN is related to inflammation, so we next examined whether NRP1–FYN plays roles in odontoblast differentiation. DPSCs infected with FYN siRNA downregulated FYN protein level (Fig. 4A) and FYN mRNA level (Fig. 4B). First, knockdown of FYN using siRNA increased NRP1 and DMP1 levels and decreased IL-6 and IL-1β levels (Fig. 4A, C). Furthermore, knockdown of FYN in DPSCs significantly enhanced nuclear β-catenin and reduced nuclear p65 (Fig. 4B). In addition, TNF-α (100 ng/mL) statistically attenuated DPM1 expression (Fig. 4D).

FIG. 4.

FYN inhibits the NRP1-mediated signaling in odontoblast differentiation. DPSCs infected with FYN siRNA were cultured in odontogenic differentiation medium for 21 days. (A, B) Western blot analysis showed that knockdown of FYN increased NRP1, DMP1, and nuclear β-catenin levels and decreased IL-6, IL-1β, and nuclear p65 levels. (C) The mRNA levels of FYN, NRP1, DMP1, IL-6, IL-1β, β-catenin, and p65 were detected by RT-PCR (*p < 0.05). (D) Western blot analysis showed that TNF-α (100 ng/mL) statistically attenuated DMP1 expression. IL, interleukin; siRNA, small interfering RNA.

NRP1 does not interfere with the FYN-mediated signaling in inflammatory reaction

To further identify the function of NRP1–FYN in odontoblast differentiation, NRP1 was stably knocked down or overexpressed in DPSCs. On the one hand, the protein and mRNA levels of NRP1 and DMP1 changed obviously following siNRP1 infected or NRP1 full-length plasmid transfection (Fig. 5). These findings demonstrate that NRP1 acts as a key factor in regulating odontoblast differentiation of DPSCs. But the expression level of FYN showed no statistically significant differences by Western blot and RT-PCR (Fig. 5). On the other hand, upon stimulation with TNF-α (100 ng/mL) and siNRP1, compared with the control group, the expressions of FYN and IL-6 by siNRP1 were further strengthened by TNF-α pretreatment (Fig. 5E). Meanwhile, TNF-α (100 ng/mL) resulted in significant increase in releasing inflammation factors compared to the control group, but overexpression of NRP1 did not obviously inhibit the effect of TNF-α (Fig. 5F). Together, these results demonstrated that FYN cooperates with NRP1 to control odontoblast differentiation and FYN might be upstream of NRP1.

FIG. 5.

NRP1 does not influence the FYN-mediated signaling in inflammatory reaction. DPSC infected NRP1 siRNA or transfected NRP1 full-length plasmid was cultured in odontogenic differentiation medium or added TNF-α (100 ng/mL) for 21 days. (A) Western blot analysis showed that knockdown of NRP1 suppressed DMP1 expression, but it did not change the protein of FYN. (B) The mRNA levels of NRP1, FYN, and DMP1 were detected by RT-PCR. (C) Western blot analysis showed that overexpression of NRP1 promoted DMP1 expression, but it did not change the protein of FYN (*p < 0.05). (D) The mRNA levels of NRP1, FYN, and DMP1 were detected by RT-PCR (*p < 0.05). (E) Western blot analysis showed that the levels of FYN and IL-6 were significantly increased in the siNRP1-challenged cells compared to the normal cells, and TNF-α further strengthened this effect for both FYN and IL-6. (F) Western blot analysis showed that TNF-α resulted in significant increase in FYN and IL-6 expressions compared to the normal group, but overexpression of NRP1 did not obviously inhibit the effect of TNF-α.

Discussion

The aim of this project was to extend our previous review and to include the potential character of NRP1–FYN in the progress of odontoblast differentiation. The present work of this study was to demonstrate the main findings as per the following: (1) NRP1 can accelerate odontoblast differentiation of DPSCs through the classical Wnt/β-catenin pathway; (2) FYN can promote inflammatory reaction of DPSCs through NF-κB signaling; and (3) NRP1 interacts with FYN, by expanding inflammation and inhibition of odontoblast differentiation of DPSCs through NF-κB signaling pathway (Fig. 6).

FIG. 6.

Schematic showing the signaling pathway of NRP1–FYN interaction by TNF-α in endothelial cells. NRP1 activates its downstream of Wnt/β-catenin signaling pathway, increases the expression of DMP1 and DSPP, and promotes the odontoblast differentiation of DPSCs. In inflammatory microenvironment NRP1 directly interacts with FYN, forms NRP1–FYN compounds, and co-excites NF-κB signaling pathway to restrain the odontoblast differentiation of DPSCs and amplification of the inflammatory cascade effect. NF-κB, nuclear factor kappa B.

Protein interaction is crucial for the maintenance of organism function. Previous studies have shown that NRP1 is closely related to FYN in regulating biological functions of the organism (Kim et al., 2010; Sijaona et al., 2012; Zhang et al., 2015). Fyn is a nonreceptor tyrosine kinase expressed in DPSCs. FYN has been shown to mediate pro-inflammatory mediator production in macrophages, mast cells, basophils, natural killer cells, and so on (Rajasekaran et al., 2013). Many studies revealed that Fyn serves as a major upstream regulator of pro-inflammatory signaling involving PKCδ, MAPK, and the NF-κB pathways (Panicker et al., 2015). Meanwhile, NRP1, as a multifunctional protein, is expressed in neuroinflammatory disorders associated with mental disorders and also can participate in inflammatory response to promote disease progression (Ito et al., 2014). We confirmed that NRP1 interacted with FYN in the odontoblast differentiation of DPSCs, and this function can be enhanced under the stimulation of TNF-α.

The inflammatory response caused by various oral bacterial infections is an important pathological process in the development of dental caries, and the inflammation can reduce the differentiation ability of DPSCs. Our results showed that the odontoblast differentiation was significantly decreased with the comparison of I-DPSCs and N-DPSCs, and the odontoblast differentiation also decreased in the stimulation of inflammation. First, NRP1 plays an important role in this process. NRP1 would promote the odontoblast differentiation of DPSCs, but in inflammatory conditions the expression of NRP1 was decreased with the decline of the odontoblast differentiation of DPSCs. Overexpression of NRP1 upregulated DMP1, while knockdown of NRP1 induced the opposite effects. Second, FYN was highly expressed in I-DPSCs than in N-DPSCs, and the FYN protein expression level was growing by TNF-α in a dose-dependent manner. Knockdown of FYN using siRNA increased NRP1, DMP1, DSPP, and nuclear β-catenin levels and decreased IL-6, IL-1β, and nuclear p65 levels. Finally, immunoprecipitation confirmed the interaction of NRP1 and FYN, and the interaction significantly and persistently increased by stimulation with TNF-α.

According to previous reports and our findings, we hypothesize that pathogens invade and release various inflammatory factors in the process of dental caries. In the early stage of infection, the release of inflammatory factors is limited, and NRP1 activates its downstream of Wnt/β-catenin signaling pathway, increases the expression of DMP1 and DSPP, and promotes the formation of reparative dentinogenesis through the odontoblast differentiation of DPSCs. However, in the further development of infection, by the increasing release of inflammatory factors and constantly expanding the inflammatory response, NRP1 directly interacts with FYN, forms NRP1-FYN compounds, co-excites NF-κB signaling pathway to restrain the odontoblast differentiation of DPSCs, and exacerbates the dental caries process through further amplification of the inflammatory cascade effect. Therefore, inhibiting the interaction between NRP1 and FYN is of great significance to the development of dental caries.

Footnotes

Acknowledgments

The study was partly supported by Graduate Student Innovation of Science and Technology Projects funded by Jiangsu Province (No. SJLX16_0567) and Nantong University (No. YKC16092) and Nantong Science and Technology Project (MS22015091).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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