Biochemical and Biophysical Research Communications 518 (2019) 154e160

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

YAP balances the osteogenic and adipogenic differentiation of

hPDLSCs in vitro partly through the Wnt/b-catenin signaling pathway
Linglu Jia a, b, Yunpeng Zhang a, b, c, Yawen Ji a, b, d, Yixuan Xiong a, b, Wenjing Zhang a, b,
Yong Wen a, b, **, 1, Xin Xu a, b, *, 1
a
School of Stomatology, Shandong University, Jinan, China
b
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, China
c
Department of Oral Implantology, The Affiliated Stomatology Hospital of Kunming Medical University, Kunming, China
d
Department of Stomatology, Jinan Central Hospital, Jinan, China

a r t i c l e i n f o a b s t r a c t

Article history: Human periodontal ligament stem cells (hPDLSCs) are potential seed cells for bone tissue engineering,
Received 26 July 2019 but the molecular regulatory mechanisms of their multi-differentiation remain unclear. Here, we found
Accepted 6 August 2019 that Yes-associated protein (YAP), a transcriptional coactivator in Hippo signaling pathway, regulated the
Available online 14 August 2019
multi-differentiation ability of hPDLSCs: overexpressing YAP contributed to an enhancement of osteo-
genic differentiation and a decrease in adipogenic differentiation, while knocking down YAP inhibited
Keywords:
the osteogenic differentiation and promoted the adipogenic differentiation of hPDLSCs. In addition, YAP
YAP
promoted the stabilization and nuclear transfer of b-catenin in hPDLSCs, probably through regulating
Periodontal ligament stem cells
Differentiation
several upstream proteins of the Wnt/b-catenin signaling pathway, including LRP6 and DVL3. Treatment
Wnt with DKK1 or Wnt3a reversed the effects of overexpressing or knocking down YAP on non-phospho b-
catenin (stabilized b-catenin) and cell differentiation. Taken together, YAP promoted osteogenic and
inhibited adipogenic differentiation of hPDLSCs in vitro, which was partly via LRP6 and DVL3 mediated
Wnt/b-catenin signaling pathway. YAP could be a candidate regulatory target for facilitating the appli-
cation of hPDLSCs in bone regeneration.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction adipogenic differentiation in hPDLSCs [4], but the precise mecha-

Human periodontal ligament stem cells (hPDLSCs) are a type of
adult mesenchymal stem cells (MSCs) with the ability of self-
renewal and multi-lineage differentiation [1]. Recently, hPDLSCs
have been regarded as essential candidate seed cells for tissue
engineering, especially in alveolar bone regeneration [2,3]. Under-
standing the mechanisms underlying the fate of hPDLSCs is
conducive to their application in bone regeneration. Since osteo-
blast lineage and adipocyte lineage, which share a common origin,
are two important directions of MSCs differentiation, many
scholars devote to explain the inner regulation of osteogenic and
* Corresponding author. No. 44-1, Wenhua Xi Road, Jinan, Shandong, 250012, PR
China.
** Corresponding author. No. 44-1, Wenhua Xi Road, Jinan, Shandong, 250012, PR
China.
E-mail addresses: wenyong@sdu.edu.cn (Y. Wen), xinxu@sdu.edu.cn (X. Xu).
1
Both authors have contributed equally to the work.
nism remains unclear.
Yes-associated protein (YAP), as well as its paralogue TAZ, is a
transcriptional coactivator that is regulated by Hippo signaling
pathway [5,6]. Generally, YAP can translocate into nucleus from
cytoplasm and associate with a range of transcription factors [5].
When the Hippo signaling pathway is activated, the cascade signals
cause the phosphorylation of YAP, then promote the sequestration
and degradation of YAP in cytoplasm [6]. Recently, numerous studies
reveal that YAP regulates the biological characteristics of various
stem cells [7e9], but its roles on MSCs multi-differentiation are
controversial: some studies reported YAP was a positive regulator for
osteogenesis [7,10] or adipogenesis [11], while others supported YAP
inhibited the osteogenic [12e14] or adipogenic differentiation [7,10]
of MSCs. Thus, the regulation of YAP on stem cell muti-
differentiation could be complicated and cell type-dependent. Our
previous studies prove that YAP have a great impact on the prolif-
eration, apoptosis and senescence of hPDLSCs [15,16], but the its
roles on multi-differentiation of hPDLSCs remain unclear.

https://doi.org/10.1016/j.bbrc.2019.08.024
0006-291X/© 2019 Elsevier Inc. All rights reserved.
 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160
Several studies reported that YAP affected the activation of the
Wnt/b-catenin signaling pathway, but their conclusions were
inconsistent and controversial [10,17e20]. In this pathway, Wnt
ligands interact with the transmembrane receptor such as Frizzled
family and LRP5/6, then recruit the Dishevelled (Dvl) protein and
lead to the disassembly of a complex consisting of Axin, APC, GSK-
3b and CK1. This complex facilitates the phosphorylation and
degradation of cytoplasmic b-catenin, so the activation of the
pathway promotes non-phospho b-catenin, the stabilized form of
b-catenin, entering the nucleus and thus affect the transcription of
downstream target genes [21,22]. Wnt/b-catenin signaling could
enhance the osteogenic lineage commitment and inhibit the adi-
pogenic differentiation of stem cells [21,22], but whether YAP af-
fects differentiation through Wnt/b-catenin pathway in hPDLSCs
has not been reported, which is worth exploring.
This study established YAP-overexpression and YAP-knockdown
hPDLSCs to investigate the regulatory roles of YAP on osteogenic
and adipogenic differentiation of hPDLSCs. The influence of YAP on
the Wnt/b-catenin signaling pathway was also detected to pre-
liminarily reveal the regulatory mechanism of YAP.
2. Materials and methods
2.1. Cell culture
The hPDLSCs were isolated according to the previous study [1]. 6
healthy premolars extracted for orthodontic treatment were
collected from 6 systemically healthy donors aged 16e20 (3 male
and 3 female). Periodontal ligament tissues were scraped from the
middle third of the root surface and cut into small pieces, then were
digested and maintained in the complete culture medium con-
taining a-MEM (Biological Industries, BI, Beit Haemek, Israel) and
10% fetal bovine serum (FBS) (BI) at 37 ! C in 5% CO2. The medium
was refreshed every 3 days, and hPDLSCs between passage 3 and 6
were used in the following experiment. All the procedures were
approved by Ethics Committee of Shandong University (Protocol
Number: 20170303).
2.2. Phenotype analysis
Cells were labeled with the CD90 FITC, CD44 PE, CD105 PerCP-
Cy, CD73 APC, PE-negative cocktail (CD34PE, CD11 b PE, CD19 PE,
CD45 PE, HLA-DR PE) and respective isotype control according to
the instructions of BD Human MSC Analysis Kit (BD Biosciences,
New Jersey, USA). Then the cells were analyzed by flow cytometry
and FlowJo software.
2.3. Lentiviruses package and cell transfection
The YAP overexpression vector (pGLV3H1-GFP-puro) and the
short hairpin RNA (shRNA) duplex oligo targeting YAP (pEF-1aF-
GFP-puro) were packaged to lentiviruses by Genechem Company
(Shanghai, China). The pGLV3H1-GFP-puro empty vector and non-
targeting shRNA pEF-1aF-GFP-puro were employed as the negative
control. The cells were incubated in lentiviral supernatant for 6 h,
then were cultured in the complete culture medium containing
puromycin for 7d. Finally, hPDLSCs with YAP overexpressed
(OEYAP), hPDLSCs with YAP knocked-down (SHYAP), and their
respective control groups (OENC, SHNC) were obtained. The over-
expression and knockdown efficiency was analyzed by quantitative
real-time PCR (qRT-PCR) and Western Blot.
2.4. Osteogenic differentiation assay
Cells were cultured in the osteogenic medium which contained
155
90% a-MEM (BI), 10% FBS (BI), 10 nM dexamethasone (Solarbio,
Beijing, China), 10 mM b-glycerophosphate (Solarbio) and 50 mg/l
ascorbic acid (Solarbio). The Wnt/b-catenin antagonist Dickkopf1
(DKK1) (200 ng/ml) (Peprotech, Rocky Hill, New Jersey, USA) or
agonist Wnt3a (200 ng/ml) (R&D Systems Inc., Minneapolis, MN,
USA) recombinant proteins were added into the medium when
needed. To evaluate the osteogenic differentiation abilities, the
alkaline phosphatase (ALP) was measured by the ALP assay kit
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China) ac-
cording to the manufacturer's instructions. ALP staining was per-
formed by the NBT/BCIP staining kit (Beyotime, Shanghai, China).
For Alizarin Red staining, cells were fixed with 4% para-
formaldehyde and incubated with the Alizarin Red solution (Sigma-
Aldrich). Western Blot and qRT-PCR were used to detect the
expression of osteoblastic marker genes including type I collagen
(COLI), runt-related transcription factor 2 (RUNX2) and osteocalcin
(OCN).
2.5. Adipogenic differentiation assay
Cells were cultured in the adipogenic medium which contained
90% a-MEM (BI), 10% FBS (BI), 1 mM dexamethasone (Solarbio),
0.2 mM indomethacin (Solarbio), 0.01 g/l insulin (Solarbio) and
0.5 mM isobutyl-methylxanthine (Solarbio). DKK1 or Wnt3a re-
combinant proteins were added into the medium when needed. To
evaluate the adipogenic differentiation abilities, Western Blot or qRT-
PCR were applied to detect the expression of adipocyte markers
including peroxisome proliferator-activated receptor g (PPARg), li-
poprotein lipase (LPL) and CCAAT/enhancer binding protein Alpha
(C/EBPa). For Oil Red O staining, cells were fixed with 4% para-
formaldehyde and incubated with the Oil Red O solution (Solarbio).
2.6. Chondrogenic differentiation assay
hPDLSCs were centrifuged to form cell precipitation and
cultured in the chondrogenic medium (Cyagen, Santa Clara, CA,
USA) in a suspension state for 4 weeks. After embedded in paraffin,
cells were stained with the Alcian Blue solution (Cyagen).
2.7. Protein extraction and Western Blot
For total protein extraction, cells were lysed with RIPA buffer
(Solarbio) containing PMSF (Solarbio) and phosphatase inhibitor
(Bosterbio, Wuhan, China). For nuclear and cytoplasmic protein
extraction, the Nuclear and Cytoplasmic Protein Extraction Kit
(Bosterbio) was used. Then the proteins were separated by SDS-
PAGE gel and transferred to PVDF membranes. After blocked with
5% nonfat milk, the membranes were incubated with primary an-
tibodies overnight at 4 ! C, followed by incubating with secondary
antibodies. The signals on the membrane were detected by the
enhanced chemiluminescence reagent (Millipore, Billerica, MA,
USA), and the gray values of protein bands were analyzed using
Image-J software. The information of primary antibodies was
shown in Supplementary Table 1.
2.8. Total RNA extraction and qRT-PCR
The total RNA was extracted using the RNAios Plus reagent
(Takara, Tokyo, Japan), and was reverse transcribed to cDNA using
the PrimeScript ™ RT reagent Kit with gDNA Eraser (Takara). The
qRT-PCR reactions were performed in a 10 ml reaction volume with
the TB Green PCR Core Kit (Takara). Changes in gene expression
were calculated by the 2-DDCT method. GAPDH was used as the
internal control. The information of primers was shown in
Supplementary Table 2.
156 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160

Fig. 1. Effects of YAP on osteogenic and adipogenic differentiation of hPDLSCs. YAP-overexpression hPDLSCs (OEYAP), YAP-knockdown hPDLSCs (SHYAP) and their corresponding
control group (OENC, SHNC) were cultured in the osteogenic or adipogenic induction medium. (A) (G) ALP activity assay after osteogenic induction for 3, 7 and 14d. (B) (H) ALP
staining after osteogenic induction for 7 and 14d. (C) (I) Alizarin Red staining after osteogenic induction for 21d. (D) (J) Western Blot analysis of COLI and RUNX2 after osteogenic
induction for 7 and 14d. The histograms represent the relative expression levels of proteins normalized by GAPDH. (E) (F) (K) (L) qRT-PCR analysis of COLI, RUNX2 and OCN after
 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160
2.9. Statistical analysis
All the experiments were repeated at least three times. Stu-
dent's t-test or one-way ANOVA was used to determine statistical
differences. The statistical analyses were conducted by GraphPad
software and the values of P < 0.05 were considered statistically
significant.
3. Results
3.1. Culture and identification of hPDLSCs
The hPDLSCs presented spindle-shaped morphology
(Supplementary Fig. 1A). Cells were positive to MSCs specific
markers (CD90, CD105, CD73, CD44), and negative to hematopoi-
etic and endothelial cell-specific markers (CD34, CD11b, CD19,
CD45, HLA-DR) (Supplementary Fig. 1E). After osteogenic, adipo-
genic and chondrogenic induction, Alizarin Red-positive calcium
deposits (Supplementary Fig. 1B), Oil Red O-positive lipid droplets
(Supplementary Fig. 1C) and Alcian Blue-positive proteoglycans
(Supplementary Fig. 1D) were observed, which verified the multi-
differentiation ability of hPDLSCs.
3.2. YAP overexpression and knockdown efficiency in hPDLSCs
After lentivirus transfection, observation with fluorescence mi-
croscope indicated that the transfection efficiency was over 90%
(Supplementary Fig. 1F). The mRNA or protein level of YAP and its
downstream targets, such as CTGF and CYR61, was much higher in
OEYAP group than that in OENC group (Supplementary Figs. 1G and
H), while was significantly lower in SHYAP group than in SHNC
group (Supplementary Figs. 1I and J).
3.3. Effects of YAP on osteogenic differentiation of hPDLSCs
When YAP was overexpressed, the ALP activities of OEYAP group
were much higher than that in OENC group after osteogenic in-
duction for 3, 7 and 14 days (Fig. 1A). The ALP staining was stronger
in OEYAP group than in OENC group (Fig. 1B). After 21 days of in-
duction, more mineralized matrix was formed in OEYAP group than
in OENC group (Fig. 1C). In addition, the proteins and mRNAs of
COLI, RUNX2 or OCN were upregulated in OEYAP group on day 7
and 14 as compared with OENC group (Fig. 1D, E, F). Therefore,
OEYAP hPDLSCs showed stronger osteogenic capacities than OENC
hPDLSCs.
When YAP was knocked down, SHYAP group showed lower ALP
activities (Fig. 1G) and weaker ALP staining (Fig. 1H), and formed
less mineralized matrix (Fig. 1I) than SHNC group. The expression of
COLI, RUNX2 and OCN in SHYAP group decreased as compared with
SHNC group (Fig. 1J, K, L). Above results proved knocking down YAP
inhibited the osteogenic differentiation of hPDLSCs.
3.4. Effects of YAP on adipogenic differentiation of hPDLSCs
When YAP was overexpressed, the Oil Red O staining on 28d
showed that less lipid droplets were formed in OEYAP group than in
OENC group (Fig. 1M). The protein and mRNA levels of PPARg and C/
EBPa in OEYAP group were lower than that in OENC group (Fig. 1O,
P, Q). Overall, OEYAP hPDLSCs owned weaker adipogenic differen-
tiation abilities than the control group.
osteogenic induction for 7 and 14d. (M) (N) Oil Red O staining after adipogenic induction for 28d. (O) (R) Western Blot analysis of PPARg and C/EBPa after adipogenic induction for 7
and 14d. The histograms represent the relative expression levels of proteins normalized by GAPDH. (P) (Q) (S) (T) qRT-PCR analysis of PPARg, LPL and C/EBPa after adipogenic
induction for 7 and 14d. *P < 0.05, yP<0.01, z P>0.05.
157
When YAP was knocked down, more lipid droplets were
observed after induction for 28d (Fig. 1N). The expression of PPARg
and C/EBPa was higher in SHYAP group than that in SHNC group
(Fig. 1R, S, T). Thus, knocking down YAP promoted adipogenesis of
hPDLSCs.
3.5. YAP affected Wnt/b-catenin signaling pathway in hPDLSCs
First, since significant differences were observed on differenti-
ation ability of hPDLSCs with YAP overexpressed or knocked down
after induction for 7d, we detected b-catenin at this time point. As
shown in Fig. 2A, the expression of total b-catenin between
different groups changed slightly (even though there was statistical
significance), but the protein level of non-phospho b-catenin (the
active state of b-catenin) was much higher in OEYAP hPDLSCs than
in OENC hPDLSCs, and it was lower in SHYAP hPDLSCs than in SHNC
hPDLSCs. Next, the nuclear and cytoplasmic proteins of hPDLSCs
were analyzed, and more nuclear b-catenin was detected in OEYAP
group than in OENC group, while less b-catenin was located in
nucleus of SHYAP cells than in SHNC cells (Fig. 2B). Then, over-
expressing or knocking down YAP did not alter the mRNA of gene
CTNNB, which encodes b-catenin protein (Fig. 2C). Finally, the
protein levels of LRP6 and DVL3, which are upstream proteins of
Wnt/b-catenin signaling pathway, was higher in OEYAP group than
in OENC group, while was lower in SHYAP group than in SHNC
group (Fig. 2D).
3.6. DKK1 and Wnt3a reversed the effects of YAP on hPDLSCs
differentiation
After DKK1 or Wnt3a was added to the culture medium, the
expression of non-phospho b-catenin in OEYAP plus DKK1 group
was lower than in OEYAP group, and that in SHYAP plus Wnt3a
group was higher in SHYAP group, indicating DKK1 or wnt3a
reduced the effects of YAP on non-phospho b-catenin (Fig. 2E).
The subsequent functional experiments further studied
whether YAP regulated hPDLSCs differentiation through the Wnt/
b-catenin signaling pathway. As shown in Fig. 3, lower ALP activity
(Fig. 3 A), lighter ALP staining (Fig. 3 C), less mineralization of
extracellular matrix (Fig. 3 B) and lower expression level of osteo-
genic markers (Fig. 3 D, E) were observed in OEYAP plus DKK1
group than in OEYAP group. In addition, stronger ALP activity (Fig. 3
H), deeper ALP staining (Fig. 3 F), more mineralization of extra-
cellular matrix (Fig. 3 G), and higher expression level of osteogenic
markers (Fig. 3 I, J) were observed in SHYAP plus Wnt3a group as
compared with SHYAP group. In adipogenesis assay, more lipid
droplets (Fig. 3 K) and higher expression level of PPARg and C/EBPa
(Fig. 3 L, M) were observed in OEYAP plus DKK1 group as compared
with OEYAP group. While cells in SHYAP plus Wnt3a group pro-
duced less fat droplets (Fig. 3 N) and expressed less PPARg and C/
EBPa (Fig. 3 O, P) than SHYAP group. Taken together, the effects of
overexpressing or knocking down YAP on hPDLSCs osteogenesis or
adipogenesis could be partly reversed by inhibiting or activating
the Wnt/b-catenin signaling pathway.
4. Discussion
Recently, the regulation of Hippo-YAP/TAZ pathway on stem cell
biology has been paid much attention to, but the effects of YAP on
stem cell multi-differentiation is not as fully defined. This study
158 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160

Fig. 2. YAP overexpression and knockdown affected the Wnt/b-catenin signaling pathway. (A) Western Blot analysis of total b-catenin and non-phospho b-catenin in YAP-
overexpression hPDLSCs (OEYAP), YAP-knockdown hPDLSCs (SHYAP) and their corresponding control group (OENC, SHNC) after osteogenic and adipogenic induction for 7d. (B)
Western Blot analysis of b-catenin and YAP proteins in nucleus and cytoplasm. (C) qRT-PCR analysis of CTNNB mRNA in hPDLSCs. (D) Western Blot analysis of LRP6, DVL3 and non-
phospho b-catenin. (E) Western Blot analysis of non-phospho b-catenin in YAP-overexpression hPDLSCs treated with DKK1 (OEYAP þ DKK1) and YAP-knockdown hPDLSCs treated
with Wnt3a (SHYAP þ WNT3A). All the histograms represent the relative expression levels of proteins normalized by GAPDH or Histone H3. *P < 0.05, yP<0.01, z P>0.05, xP < 0.001.

showed that YAP balances the osteogenic and adipogenic differ-
entiation of hPDLSCs in vitro: overexpressing YAP contributed to an
enhancement of osteogenesis and a decrease in adipogenesis, while
knocking down YAP inhibited osteogenesis and promoted adipo-
genesis of hPDLSCs. In fact, several studies support there is a
reciprocal relationship between the osteoblastic and adipocytic
lineages differentiation of stem cells [23,24], and our findings
indicated that YAP participated in the osteogenic-adipogenic-
lineage fate choice of hPDLSCs. Combined with our previous
studies that overexpressing YAP promotes proliferation, inhibits
apoptosis, and delays senescence of hPDLSCs [15,16], we believe
that YAP is a potential regulatory target of hPDLSCs, and over-
expressing YAP could facilitate the application of hPDLSCs in bone
tissue engineering. Of course, more in vivo studies are needed to
verify the regulation of YAP in hPDLSCs, and the biosafety of
overexpressing YAP requires further detection.
Similar to our findings, Pan proved that bone marrow stem cells
(BMSCs) of YAP knockout mice owned lower osteogenic and
stronger adipogenic differentiation ability [10,25]. Another study
reported YAP promoted osteogenesis and inhibited adipogenesis in
rat BMSCs stimulated by fluid flow [26]. However, some scholars
hold different opinions. Seo found that knocking down YAP in mice
BMSCs enhanced their ability to undergo osteogenic differentiation,
while both knocking down and overexpressing YAP led to a sig-
nificant reduction of adipocyte formation [18]. Another study
proved YAP inactivation promoted osteogenesis and inhibited
adipogenesis in rat adipose-derived stem cells [11]. These incon-
sistent conclusions may be related with different sources of stem
cells, different culture environment, different YAP intervention
models and so on. It also reflects that the regulation of YAP on stem
cell differentiation is complicated, and requires further research
and confirmation.
To investigate the regulatory mechanism of YAP in hPDLSCs, we
focused on the connection between YAP and Wnt/b-catenin
signaling pathway. The present results showed that YAP over-
expression or knockdown did not significantly affect the protein
and mRNA of total b-catenin, but increased or decreased the protein
level of non-phospho b-catenin, which is the stabilized form of b-
catenin. Moreover, more b-catenin translocated to nucleus when
YAP was overexpressed, while nuclear b-catenin decreased when
YAP was knocked down. Above results suggested that YAP was a
positive regulator for the stabilization and nuclear transfer of b-
catenin in hPDLSCs. Our findings were in line with some previous
studies, such as YAP were reported to facilitate the stabilization of
b-catenin by promoting the phosphorylation of GSK-3b or reducing
the endogenous DKK1 [10,19]. However, some scholars supported
YAP was a negative regulator of Wnt/b-catenin signaling: YAP
induced the expression of endogenous DKK1 to block the Wnt/b-
catenin signaling [18]; phosphorylated YAP could bind with b-
catenin in the cytoplasm and suppress the translocation of b-cat-
enin to the nucleus [17]. These previous studies were based on
different cell models, and their inconsistent conclusions suggest
 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160 159

Fig. 3. DKK1 and Wnt3a reversed the effects of YAP on osteogenic and adipogenic differentiation of hPDLSCs. YAP-overexpression hPDLSCs, YAP-knockdown hPDLSCs and their
control groups were cultured in the induction medium (OENC, OEYAP; SHNC, SHYAP) or induction medium containing DKK1 or Wnt3a (OENC þ DKK1, OEYAP þ DKK1;
SHNC þ WNT3A, SHYAP þ WNT3A). (A) (F) ALP staining after osteogenic induction for 7 d. (B) (G) Alizarin Red staining after osteogenic induction for 21 d. (C) (H) ALP activity assay
after osteogenic induction for 7 d. (D) (I) Western blot analysis of COLI after osteogenic induction for 7 d. The histograms represent the relative expression levels of proteins
normalized by GAPDH. (E) (J) qRT-PCR analysis of COLI and RUNX2 after osteogenic induction for 7 d. (K) (H) Oil Red O staining after adipogenic induction for 28 d. (L) (O) Western
blot analysis of PPARg and C/EBPa after adipogenic induction for 7 d. The histograms represent the relative expression levels of proteins normalized by GAPDH. (M) (P) qRT-PCR
analysis of PPARg and C/EBPa after adipogenic induction for 7 d *P < 0.05, yP < 0.01, xP < 0.001. . (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article).

that YAP may regulate b-catenin through multiple mechanisms.
The present study showed LRP6 and DVL3, which are upstream
proteins of Wnt/b-catenin pathway, were upregulated or down-
regulated when YAP was overexpressed or knocked down, so we
speculate that YAP influences the Wnt/b-catenin signaling partly
through affecting LRP6 and DVL3 in hPDLSCs (Fig. 4). Several re-
ports suggest that YAP and DVL affected the activity of each other
[8,27], which provides clues for our further study on the regulatory
mechanism of YAP. This speculation is not fully studied in the
present study, and it will be our future research direction.
Based on the influence of YAP on Wnt/b-catenin signaling, we
further verified the regulation of YAP on hPDLSCs differentiation
was partly through Wnt/b-catenin signaling. As shown above, the
antagonist DKK1 [28] or agonist Wnt3a [29] of Wnt/b-catenin
reversed the effects of YAP overexpression or knockdown on the
expression of non-phospho b-catenin and hPDLSCs osteogenesis
and adipogenesis, which supported our hypothesis. It is noteworthy
that some other mechanisms of YAP affecting differentiation have
been reported, including YAP interfered the function of RUNX2
[5,12,13], YAP regulated the activation of the TGF-b pathway, which
is another signaling cascade that affected stem cell differentiation
[30,31] and so on. Whether these reported mechanisms of YAP
participate in regulating the differentiation of hPDLSCs requires
further investigation.
Taken together, YAP promoted osteogenic differentiation while
inhibited adipogenic differentiation of hPDLSCs, which was partly
through activating the Wnt/b-catenin signaling pathway via LRP6
and DVL3. YAP is a candidate regulatory target for improving
osteogenic differentiation of PDLSCs and promoting the application
of hPDLSCs in bone regeneration.
160 L. Jia et al. / Biochemical and Biophysical Research Communications 518 (2019) 154e160
Fig. 4. Putative mechanism for the regulation of hPDLSCs differentiation by YAP. YAP
promotes the expression of LRP6 and DVL, and further promotes the stabilization and
nuclear translocation of b-Catenin, which leads to the enhancement of osteogenic
differentiation and inhibition of adipogenic differentiation in hPDLSCs.
Acknowledgement
This work was supported by grants from Construction Engi-
neering Special Fund of Taishan Scholars (ts201511106), Shandong
Provincial Natural Science Foundation (ZR2017MH031,
ZR2018MH018) and Young Scholars Program of Shandong Univer-
sity (2015WLJH53).
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2019.08.024.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.08.024.
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