Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells
Yang et al. Stem Cell Research & Therapy
Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells
Shan Yang 0 1 4
Lijia Guo 0 3
Yingying Su 2
Jing Wen 3
Juan Du 1 4
Xiaoyan Li 1 4
Yitong Liu 1 4
Jie Feng 1 4
Yongmei Xie 1 4
Yuxing Bai 3
Hao Wang 2
Yi Liu 1 4
0 Equal contributors
1 Laboratory of Tissue Regeneration and Immunology and Department of Periodontics, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University , Tian Tan Xi Li No.4, Beijing 100050 , People's Republic of China
2 Department of Stomatology, Beijing Tiantan Hospital, Capital Medical University , Beijing , People's Republic of China
3 Department of Orthodontics, Capital Medical University School of Stomatology , Beijing , People's Republic of China
4 Laboratory of Tissue Regeneration and Immunology and Department of Periodontics, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University , Tian Tan Xi Li No.4, Beijing 100050 , People's Republic of China
Background: 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. Methods: 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. Results: 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. Conclusions: NO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/ MAPK signaling pathway.
Periodontal ligament stem cells; Nitric Oxide; Osteogenesis; Adipogenesis; 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
]. 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
]. 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 [
]. 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
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 [
ligament (PDL) cells treated with exogenous NO exhibit
enhanced osteogenic potential [
]. 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
cJun 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 [
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
antieNOS 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
fluoresceinactivated 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
βglycerophosphate, 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;
SigmaAldrich). 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
We first identified whether all three isoforms of NOS
were expressed in PDLSCs. Since both iNOS and eNOS
have been reported to be expressed in human umbilical
vein endothelial cells (HUVECs) [
] and nNOS is
expressed in glioma cell (U251) [
], we used HUVECs
and U251 as a positive control. We found that PDLSCs
highly expressed iNOS and eNOS but very little nNOS,
as shown by Western blot, immunofluorescent staining,
and flow cytometry (Fig. 1c–e). Moreover, the NOS
inhibitor L-NMMA significantly downregulated the
expression of iNOS/eNOS (Fig. 1f ). Considering that a
high dose of inhibitor may be cytotoxic, we used
1 mM L-NMMA in subsequent experiments. We next
showed that PDLSCs produced 6–8 μM NO in culture
supernatants, and the levels of NO were significantly
downregulated and upregulated by L-NMMA and NO
donor SNP, respectively (Fig. 1g).
Blocking the production of NO has no effect on PDLSC proliferation and apoptosis
To analyze the effects of NO on PDLSC proliferation
and apoptosis, we added L-NMMA and SNP to PDLSCs
and then performed cell proliferation and apoptosis
assays. EdU and apoptosis assays showed that reduction of
NO or increasing the physiological concentration of NO
had no influence on PDLSC proliferation and apoptosis
(Fig. 2a, b).
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-NMMAtreated cells than in osteogenic-inducing
mediumtreated cells, and SNP partially rescued the impaired
osteogenic potential (Fig. 2c). Consistently, real-time
RTPCR 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
We next investigated the effect of NO on the adipogenesis
of PDLSCs. After 4 weeks of adipogenic induction, cells
were treated with L-NMMA resulting in adipogenic
conversion as indicated by increased cellular lipid
accumulation revealed by Oil Red O staining, and this phenotype
was reversed by the NO donor SNP (Fig. 3a, b). In parallel,
we evaluated the expression of adipogenesis-induced
genes, including those encoding the late marker
lipoprotein lipase (LPL) and the transcription factors peroxisome
proliferator-activated receptor (PPAR)γ2 and
CCAATenhancer binding protein (C/EBP)α. Consistent with the
Oil Red O staining, L-NMMA stimulated the expression
of adipogenic markers, while NO donor reduced the
upregulation of these genes (Fig. 3c).
NO balances the osteogenic and adipogenic potential of
PDLSCs through the JNK/MAPK signaling pathway
We next investigated the possible signaling pathway
involved in the differentiation of PDLSCs induced by NO.
The MAPK family includes extracellular signal-regulated
kinase (ERK), P38 kinase, and JNK, which are involved
in regulating numerous cell functions, such as
differentiation, proliferation, and apoptosis [
]. There is
substantial evidence that MAPK signaling plays an important
role in regulating cell differentiation [
]. We found that
NO enhanced the phosphorylation of JNK during
osteogenesis and adipogenesis, while blocking NO production
led to the opposite result (Fig. 4a, b). Based on this
result, further experiments were performed for verification
purposes. ALP staining was applied on the fourth day
after osteogenic induction, showing that SNP effectively
promoted osteogenic conversion, and treatment with the
JNK signal inhibitor SP600125 slowed this conversion
(Figs. 4c and 5a). Furthermore, L-NMMA remarkably
inhibited the osteogenic function of PDLSCs, while
activating JNK signaling with anisomycin rescued the
repression of osteogenesis (Fig. 4c and Fig. 5a). After 3
weeks of osteogenic induction, Alizarin Red staining
showed that SNP treatment promoted osteogenic
conversion as shown by the increase in cellular calcium
nodules, as well as elevated calcium concentrations.
These phenotypes were reversed by treatment with the
JNK inhibitor SP600125 (Fig. 4d, e and Fig. 5b, c).
Blocking NO production with L-NMMA led to a
significant decrease in PDLSC mineralization, while the JNK
activator anisomycin partially rescued the impairment of
osteogenesis (Fig. 4d, e and Fig. 5b, c). Meanwhile,
realtime RT-PCR and Western blot results showed that
the expression levels of the osteogenic markers OPN,
OSX, Runx2, and ALP were significantly increased in
NO-treated cells, while the JNK inhibitor reduced
their induction (Fig. 4f, g and Fig. 5d, e). Anisomycin
reversed the repression of osteogenic markers caused
by L-NMMA (Fig. 4f, g and Fig. 5d, e). Furthermore,
we discovered that SNP and anisomycin significantly
reduced the expression of the adipogenesis-related
transcription factor PPARγ2 during the PDLSC
osteogenesis process, while L-NMMA and SP600125 led to
the opposite result (Fig. 5f ). Consistent with previous
results (Fig. 4a, b), p-JNK expression, as measured by
Western blot, was increased by SNP and decreased by
l-NMMA (Figs. 4g and 5e).
To investigate adipogenesis, cells were cultured with
adipogenesis-inducing medium for 4 weeks. We found that
SNP-treated PDLSCs significantly resisted differentiation
into adipocytes, which was confirmed by Oil Red O
staining. This decreased adipogenic conversion was partially
rescued by blocking the JNK signaling pathway (Fig. 6a, b
and Fig. 7a, b). In contrast, L-NMMA caused an elevated
level of adipogenic conversion compared with the control
group, while activating the JNK pathway partially reduced
the effect (Fig. 6a, b and Fig. 7a, b). These phenomena were
further confirmed with real-time RT-PCR and Western
blots for adipogenesis-induced markers including LPL,
PPARγ, and C/EBPα, and the results were consistent with
Oil Red O staining (Fig. 6c, d and Fig. 7c, d). During
induction of adipogenesis, the expression of the
osteogenic marker Runx2 was significantly reduced by
blocking NO production but increased by SNP
treatment (Fig. 7e). While the effect of SNP was reversed
by inhibiting the JNK signaling pathway, the effect of
L-NMMA was reversed by activating the JNK
signaling pathway (Fig. 7f ).
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.
We also studied the effects of NO and JNK on
PDLSCs in regular (standard) medium to verify the
above conclusions. From the results of real time
RTPCR and Western blot, we found that NO and JNK had
the same effect in regular medium compared with
induced medium. NO could induce upregulation of the
expression of the osteogenic markers ALP, Runx2, OPN,
and OSX, and reduced the expression of the adipogenic
markers LPL, PPARγ and C/EBPα. When blocking the
JNK signaling pathway, the NO-induced increase in
osteogenic markers was inhibited and adipogenic
markers were upregulated; activating the JNK signaling
pathway produced opposite effect (Fig. 8a–c). Thus, we
infer that JNK plays a major role in balancing the
osteogenic and adipogenic differentiation of PDLSCs under
the effects of NO.
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 [
]. The shift
between osteoblastic and adipocyte lineages is a result of
crosstalk between various factors that drive MSCs
toward the adipocyte lineage, inhibiting osteoblast
]. 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 [
]. 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
]. Treatment of eNOS-null osteoblastic
cells with NO donors significantly rescued the levels of
Runx2 and was correlated with an enhancement of cell
]. 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 [
]. Conversely, results from other
studies have suggested that NO may have the opposite
effect on adipogenesis [
]. 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
fibroadipogenic 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
]. Conversely, results from other studies have
suggested that JNK activity may have the opposite effect on
]. 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 [
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
]. 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 [
]. In our study, we focused on the
switch effect of JNK in NO-induced PDLSC
differentiation, and the deeper molecular mechanism of
NOinduced adipogenesis differentiation requires further
In previous studies, Runx2 and PPARγ have been
shown to be vital osteogenic and adipogenic
transcription factors, respectively, playing major roles in stem cell
]. 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.
Additional file 1: Table S1. List of the specific primers used for RT-PCR.
(JPG 58 kb)
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
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.
Consent for publication
All of the authors consent to submit the article for publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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