Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells
- Shan Yang†1,
- Lijia Guo†2,
- Yingying Su3,
- Jing Wen2,
- Juan Du1,
- Xiaoyan Li1,
- Yitong Liu1,
- Jie Feng1,
- Yongmei Xie1,
- Yuxing Bai2,
- Hao Wang3 and
- Yi Liu1Email author
© The Author(s). 2018
Received: 21 December 2017
Accepted: 12 April 2018
Published: 2 May 2018
Critical tissues that undergo regeneration in periodontal tissue are of mesenchymal origin; thus, investigating the regulatory mechanisms underlying the fate of periodontal ligament stem cells could be beneficial for application in periodontal tissue regeneration. Nitric oxide (NO) regulates many biological processes in developing embryos and adult stem cells. The present study was designed to investigate the effects of NO on the function of human periodontal ligament stem cells (PDLSCs) as well as to elucidate the underlying molecular mechanisms.
Immunofluorescent staining and flow cytometry were used for stem cell identification. Western blot, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescent staining, and flow cytometry were used to examine the expression of NO-synthesizing enzymes. The proliferative capacity of PDLSCs was determined by EdU assays. The osteogenic potential of PDLSCs was tested using alkaline phosphatase (ALP) staining, Alizarin Red staining, and calcium concentration detection. Oil Red O staining was used to analyze the adipogenic ability. Western blot, RT-PCR, and staining were used to examine the signaling pathway.
Human PDLSCs expressed both inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) and produced NO. Blocking the generation of NO with the NOS inhibitor l-NG-monomethyl arginine (l-NMMA) had no influence on PDLSC proliferation and apoptosis but significantly attenuated the osteogenic differentiation capacity and stimulated the adipogenic differentiation capacity of PDLSCs. Increasing the physiological level of NO with NO donor sodium nitroprusside (SNP) significantly promoted the osteogenic differentiation capacity but reduced the adipogenic differentiation capacity of PDLSCs. NO balances the osteoblast and adipocyte lineage differentiation in periodontal ligament stem cells via the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway.
NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/MAPK signaling pathway.
Periodontitis is one of the most widespread infectious diseases and is characterized by chronic bacterial infection of the supporting structures of the teeth, leading to tooth loss in adults . Stem cell therapy has been shown to be a promising strategy for the treatment of periodontitis . Although many signaling pathways and molecules have been identified that regulate the differentiation of mesenchymal stem cells (MSCs), the precise mechanisms determining the fate of stem cells are unclear . This results in limited clinical translation of stem cell therapy. Understanding the mechanisms underlying the fate of stem cells would be helpful for application in regenerative medicine.
Nitric oxide (NO) is a gaseous radical that is recognized as one of the smallest known bioactive products of mammalian cells. Endogenous NO is primarily generated by NO synthase (NOS) enzymes. Three distinct isoforms of NOS have been identified: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) . Accumulating evidence suggests that many biological processes are regulated by NO in developing embryos and adult stem cells [5, 6]. The modulation of cell function depends on specific local concentrations of NO, and opposite effects can be observed when low and high levels of NO are compared. NO at physiological concentrations has been shown to promote MSC survival, homing, and differentiation, and NO also maintains the self-renewal potential of neuronal stem cells and their efficacy against ischemic conditions . Moderate levels (100 μM) of NO donor can enhance the survival rate of MSCs via protection from renal ischemic injury after kidney damage . These results highlight the importance of NO as a potential modulator of stem cell therapies.
Periodontal ligament stem cells (PDLSCs) have been shown to possess great potential in periodontal regenerative therapies. It has been suggested that NO mediates the differentiation of PDLSCs into osteoblasts, as demonstrated by an increase in NO production during osteogenic differentiation of PDLSCs . Periodontal ligament (PDL) cells treated with exogenous NO exhibit enhanced osteogenic potential [9, 10]. However, the role of endogenous NO in regulating the fate of PDLSCs, as well as the underlying molecular mechanisms, remain poorly understood. The present study was designed to investigate the effects of NO on the functions of human PDLSCs and the possible signaling pathway underlying the process. Our study found that blocking the production of NO with a NOS inhibitor decreased the osteogenic capacity of PDLSCs but promoted their adipogenic capacity, whereas adding additional physiological levels of NO with sodium nitroprusside (SNP) decreased the adipogenic capacity of PDLSCs but promoted their osteogenic capacity, indicating the importance of NO in balancing the osteogenic and adipogenic potential of human PDLSCs. In addition, we show that NO regulates the differentiation function of PDLSCs through the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway.
The study was performed according to an informed protocol for handling human tissue approved by the Research Ethical Committee of Capital Medical University, China (2012-x-53). The method for culturing periodontal ligament stem cells has been described previously . Experiments were performed using the third generation of human PDLSCs.
Western blot analysis
The protocol has been described previously . Proteins of interest were detected using anti-iNOS (1:1000, Novus), anti-eNOS (1:1000, Abcam), anti-alkaline phosphatase (ALP; 1:1000, Abcam), anti-runt-related transcription factor 2 (Runx2; 1:1000, Abcam), anti-peroxisome proliferator-activated receptor (PPAR)γ (1:1000, Abcam), anti-p-JNK (1:1000, Abcam), or anti-JNK (1:1000, Abcam) antibodies, while β-actin was detected with anti-β-actin antibody (1:2000, Abcam) as a control.
Flow cytometric analysis
For flow cytometric analysis of iNOS and eNOS expression, PDLSCs were harvested and fixed with 80% methanol and then permeabilized with 0.1% PBS-Tween for 20 min. Cells were then incubated in phosphate-buffered saline (PBS)/10% normal goat serum/0.3 M glycine to block nonspecific protein-protein interactions by the anti-iNOS and anti-eNOS antibodies (Abcam, 1 μg/1 × 106 cells). Fluorescein isothiocyanate (FITC) goat anti-mouse immunoglobulin (Ig)G was used as a secondary antibody (BioLegend, 1 μg/1 × 106 cells). Cells were analyzed with a fluorescein-activated cell sorter (FACS) Calibur flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA).
For flow cytometric analysis of stem cell identification, PDLSCs were harvested and fixed with 80% methanol for 20 min. Fixed cells were incubated in sealing buffer for 30 min and then incubated in 3% bovine serum albumin (BSA)/PBS with anti-CD44, anti-CD45, and anti-CD146 antibodies (Abcam, 1 μg/1 × 106 cells). FITC goat anti-rabbit IgG was used as a secondary antibody (BioLegend, 1 μg/1 × 106 cell). Cells were analyzed with a FACS Calibur flow cytometer (BD Immunocytometry Systems).
EdU assay for cell proliferation
PDLSCs were seeded in six-well plates (Nunc) and cultured for 2–3 days. The cultures were incubated with EdU solution (1:1000, Invitrogen) for 24 h and stained with Click-iT EdU Flow Cytometry Assay Kits (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed with a flow cytometer (BD Immunocytometry Systems). The number of EdU-positive cells was indicated as a percentage of the total cell number.
Determination of apoptotic cell percentage
To detect apoptotic cells, we utilized the Annexin V Apoptosis Detection Kit FITC (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions.
In vitro osteogenic differentiation assay
PDLSCs were grown in mineralization-inducing media containing 100 μM/ml ascorbic acid, 2 mM β-glycerophosphate, and 10 nM dexamethasone. For detecting mineralization, cells were induced for 3 weeks, fixed with 70% ethanol, and stained with 2% Alizarin Red (Sigma-Aldrich). Calcium nodule areas were quantified using NIH ImageJ. After Alizarin Red staining, the relative concentration of calcium was measured after extracting dye with 10% cetylpyridinium chloride (CPC; Sigma-Aldrich) for 1 h. Absorbance values were measured in a microplate reader (Bio-Rad Labs) at 562 nm. The relative concentration of calcium was calculated against a standard curve.
In vitro adipogenic differentiation assay
For adipogenic induction, a StemPro® Adipogenesis Differentiation Kit (Invitrogen, USA) was used. Four weeks after induction, cultured cells were stained with Oil Red O, and positive cells were quantified using NIH ImageJ.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from PDLSCs using Trizol reagent (Invitrogen, USA). For real-time RT-PCR, cDNA was synthesized from 2 μg RNA using random hexamers or oligo dT and reverse transcriptase according to the manufacturer’s protocol (Invitrogen, USA). Real-time PCR reactions were performed using the QuantiTect SYBR Green PCR kit (Qiagen, Germany) and iCycler iQ Multi-color Real-time PCR Detection System. The specific primers used for RT-PCR are listed in Additional file 1: (Table S1).
Measurement of NO
Cell culture supernatants were collected for measurement of NO levels. NO levels were measured using a Griess Reagent kit (Beyotime) according to the manufacturer’s instructions. Absorbance values were measured in a microplate reader (Bio-Rad Laboratories) at 540 nm. SNP (75 μM, Sigma-Aldrich) was used as an NO donor, and l-NG-monomethyl arginine (l-NMMA; 1 mM, Sigma-Aldrich) was used as an NO inhibitor. The NO concentration was calculated with a standard curve.
Cells were grown on glass coverslips, fixed in 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked in 10% normal goat serum, and incubated with primary antibodies (1:200) overnight at 4 °C. The samples were then treated with rhodamine/FITC-conjugated secondary antibodies (1:400, Sigma-Aldrich, St. Louis, MO, USA) and mounted with Vectashield mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images were captured with a confocal microscope (AX10, Carl Zeiss, Gottingen, Germany).
All statistical calculations were performed using SPSS 18.0 statistical software. Student’s t test or one-way analysis of variance (ANOVA) were performed to determine statistical significance (P < 0.05).
iNOS and eNOS expression and NO production in PDLSCs
Blocking the production of NO has no effect on PDLSC proliferation and apoptosis
A physiological level of NO is necessary for the mineralization ability of PDLSCs
Next, to examine whether NO affected the osteogenic potential of PDLSCs, cells were treated with l-NMMA or without l-NMMA . Three weeks after osteogenic induction, Alizarin Red staining revealed that mineralization was significantly lower in l-NMMA-treated cells than in osteogenic-inducing medium-treated cells, and SNP partially rescued the impaired osteogenic potential (Fig. 2c). Consistently, real-time RT-PCR results showed that the expression levels of the osteogenic markers osteopontin (OPN), runt-related transcription factor 2 (Runx2), and osterix (OSX) were significantly reduced in l-NMMA-treated cells (Fig. 2d). SNP treatment significantly rescued the expression of these osteogenic markers (Fig. 2d).
The adipogenesis capacity of PDLSCs is enhanced when blocking endogenous NO production
NO balances the osteogenic and adipogenic potential of PDLSCs through the JNK/MAPK signaling pathway
Moreover, consistent with previous results (Fig. 4a, b), Western blots showed that p-JNK levels were increased by SNP and decreased by l-NMMA during the adipogenic process (Fig. 4g, Fig. 5e, Fig. 6d, and Fig. 7d). This result further confirmed the molecular mechanism of NO-induced PDLSC differentiation.
PDLSCs are a type of mesenchymal stem cell (MSC) with strong potential for proliferation and multipotent differentiation. MSC lineage differentiation can be regulated at different molecular levels [17, 18]. The shift between osteoblastic and adipocyte lineages is a result of crosstalk between various factors that drive MSCs toward the adipocyte lineage, inhibiting osteoblast differentiation [19, 20]. Since osteoblasts and adipocytes share a common origin, a switching mechanism in MSCs is important for regenerative medicine. In the present study, we show that decreasing endogenous NO production with a NOS inhibitor increases PDLSC-mediated adipocyte differentiation while reducing the number of osteoblasts. In contrast, the NO donor SNP reversed the effect of NOS inhibition, suggesting that endogenous NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs.
It has been suggested that NO mediates the effects of physical activity on bones, including bone development, bone healing, and bone resorption [21–24]. Mice lacking eNOS exhibit profound abnormalities in bone formation, and osteoblasts isolated from eNOS-null mice show significant delays in differentiation and a reduction in Runx2 levels, suggesting that NO regulates Runx2 expression [25, 26]. Treatment of eNOS-null osteoblastic cells with NO donors significantly rescued the levels of Runx2 and was correlated with an enhancement of cell differentiation [26, 27]. The contribution of NO in MSC osteogenesis has also been reported. Increased NO production was previously observed in human PDLSCs during their osteogenic differentiation ; however, in that case, a causal relationship was not demonstrated. The results of the present study show that, when treated with the NOS inhibitor l-NMMA to reduce NO levels, PDLSCs showed a reduced capacity for forming mineralized nodules in vitro, with downregulation of Runx2, OSX, and OPN. These observations suggest that NO is required to maintain PDLSC osteogenic differentiation.
Observations concerning the role of NO in adipocyte differentiation have remained controversial. Several studies have reported that the production of NO promotes adipocyte differentiation [28–30]. Conversely, results from other studies have suggested that NO may have the opposite effect on adipogenesis [31–33]. Among these studies, one focused on the role of NO in the adipogenesis of mesenchymal tissue-derived progenitors . NO has been shown to inhibit the adipogenesis of mesenchymal fibro-adipogenic progenitors by inducing expression of miR-27b and downregulating PPARγ. In the present study, we found that the NOS inhibitor l-NMMA increased the number of Oil Red O-positive cells and enhanced expression levels of LPL, PPARγ, and CEBP/α in PDLSCs, while NO donor treatment resulted in a significant reduction in PDLSC adipogenesis. Determining whether NO acts as a pro-adipogenic or anti-adipogenic factor requires further research.
The MAPK signaling pathway plays vital roles in maintaining cell physiological function, and the present study confirmed that this signaling pathway can induce cell differentiation. Research concerning the role of the JNK signaling pathway in adipocyte differentiation has remained controversial. Several studies have reported that JNK activity is specifically required for the initial stage of differentiation events of adipocytes and may act with a positive impact in adipogenesis differentiation [34, 35]. Conversely, results from other studies have suggested that JNK activity may have the opposite effect on adipogenesis [36–38]. In our study, we found that, in both adipogenic and osteogenic differentiation processes, NO increased the phosphorylation of JNK/MAPK, and then p-JNK transportation to the nucleus induced the expression of osteogenic transcription factors and repressed the expression of adipogenic transcription factors, thus increasing osteogenesis and reducing adipogenesis. Conversely, blocking NO production in PDLSCs led to a decrease in phosphorylation of JNK/MAPK with the opposite differentiation result. These results strongly suggest that JNK/MAPK acts as a switch in NO-induced cellular differentiation of PDLSCs. It is hard to explain the inconsistent effect of JNK in the adipogenesis process between our results and other studies [34, 35], and we suggest the consideration that the influence of NO and cell type may regulate the function of adipogenesis. So far, the effects of NO and JNK on stem cell adipogenesis remains controversial. In addition, the components upstream of JNK/MAPK in NO-induced PDLSC differentiation are still unclear. Several reports showed that PPARγ may act upstream of JNK activation or inhibit the JNK downstream target, AP-1, to regulate cell functions. AP-1 is a heterodimer consisting of c-fos (Fra-1, Fra-2, c-Fos, FosB) and c-jun (c-Jun, JunB, JunD) and acts as a major transcription activator in cells, controlling many cellular processes. AP-1 is recognized as a JNK downstream target, activated by the JNK signaling pathway and promoting downstream gene expression to regular cell functions; it is reported to be involved in cell inflammation, apoptosis, and osteogenesis differentiation [39, 40]. In the process of PDLSC differentiation, PPARγ may block AP-1 which, combined with targeted DNA or competitive binding with CBP to inhibit AP-1 activation, leads to downregulation of osteogenesis with increased adipogenesis. Blocking the JNK signaling pathway increased the expression of PPARγ and further decreased AP-1 activity, which induced adipogenesis differentiation of PDLSCs [39, 40]. In our study, we focused on the switch effect of JNK in NO-induced PDLSC differentiation, and the deeper molecular mechanism of NO-induced adipogenesis differentiation requires further research.
In previous studies, Runx2 and PPARγ have been shown to be vital osteogenic and adipogenic transcription factors, respectively, playing major roles in stem cell differentiation . In our study, NO activated the JNK/MAPK signaling pathway during the differentiation process, thus increasing the phosphorylation level of JNK. Subsequently, p-JNK was transported to the nucleus, where it promoted Runx2 transcription activity through phosphorylation, inducing higher expression of the transcription factor Runx2 and ultimately accelerating the osteogenesis of PDLSCs. On the other hand, NO reduced the transcriptional activity of PPARγ through the JNK signaling pathway, downregulating PPARγ expression and thus suppressing the adipogenic conversion of PDLSCs. When NO generation was blocked during osteogenic differentiation, p-JNK was downregulated leading to a lower expression of Runx2 but elevated levels of PPARγ, inhibiting osteogenic differentiation while promoting adipogenic conversion. Our results further confirm that NO balances the osteogenic and adipogenic differentiation of PDLSCs by regulating the expression of Runx2 and PPARγ transcription factors through the JNK/MAPK pathway.
In conclusion, the results of this study demonstrate that blocking the production of NO in PDLSCs downregulated JNK/MAPK, thus inhibiting osteogenesis while increasing adipogenesis. In contrast, the addition of NO promotes osteogenesis by upregulating JNK/MAPK and reducing adipogenesis. NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs through JNK/MAPK signaling. These findings may be important for our understanding and clinical application of stem cell therapy.
This work was supported by grants from the National Nature Science Foundation of China (81470751 to Yi Liu, 81600891 to LG, and 81600829 to YS), the Beijing Natural Science Foundation (7172087 to Yi Liu), the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201703 to YB), and the Beijing Baiqianwan Talents Project (2017A17 to Yi Liu).
This work was supported by grants from the National Nature Science Foundation of China 81470751 to Yi Liu (supporting sample collection), 81600891 to LG (supporting experiments process), and 81600829 to YS (supporting the manuscript preparation), the Beijing Natural Science Foundation (7172087 to Yi Liu, supporting data analysis), the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201703 to YB, supporting interpretation of data), and the Beijing Baiqianwan Talents Project (2017A17 to Yi Liu, supporting the design of the study).
Availability of data and materials
Please contact the corresponding author for data requests.
YS carried out the research and experiments. LG participated in drafting the manuscript. SY carried out the signaling pathway study. JW participated in collecting periodontal tissue. JD performed the experimental facility and coordination. XL participated in the statistical analysis. YitL participated in the statistical analysis. JF helped to culture cells. YX helped to analyze the preliminary data. YB participated in the design of the study. HW participated in the design of the study and helped draft the manuscript. YiL conceived the study and participated in its design. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was performed according to an informed protocol for handling human tissue approved by the Research Ethical Committee of Capital Medical University, China (2012-x-53). All participants gave informed consent to participate in the study.
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The authors declare that they have no competing interests.
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