Takashi Tsukinowa et al.: Osteogenic Regulation of Wnt Inhibitors Journal of Hard Tissue Biology 24[4] (2015) 311- 318 © 2015 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649
Original Synergistic and Mutual Antagonistic Regulations of Wnt Inhibitors Play an Important Role in Osteoblast Differentiation of Human Periodontal Ligament Cells Takashi Tsukinowa1), Shoko Onodera2), Yuusei Yoshizawa1), Akiko Saito2), Takashi Muramatsu1), Masahiro Furusawa1) and Toshifumi Azuma2) Department of Endodontics and Clinical Cariology, Tokyo Dental College, Tokyo, Japan Department of Biochemistry, Tokyo Dental College, Tokyo, Japan (Accepted for publication August 26, 2015) 1) 2)
Abstract: Recent studies have suggested that a balance between Wnt and bone morphogenetic protein (BMP) output may be a key factor for regulatory networks involved in bone regeneration. Here we report that Wnt inhibitors coordinately regulate osteoblastic differentiation induced by BMPs of human periodontal ligament cell. Human periodontal ligament (HPDL) cells were stimulated with BMP2/7, Dexamethasone (Dex), or a combination of these reagents. After treatment, we analyzed the phosphorylated Smad1/5 and osteoblast differentiation markers. Specific antagonists such as Dickkopfs (Dkk1, Dkk2) , secreted Frizzled-related proteins (SFRP1,SFRP2), and sclerostin(SOST) which are believed to play important roles in osteoblast differentiations were also examined by quantitative RT-PCR. BMP2/7 treatment increased ALP activity modestly, and the combination of BMP2/7 with Dex greatly enhanced ALP activity. SOST expression increased sharply following BMP2/7 treatment; however, adding BMP2/7 and Dex drastically reversed this increase in SOST. BMP2/7 and Dex treatment synergistically decreased DKK2 and sFRP2 expression. In contrast, BMP2/7 and Dex had a significant inductive effect on DKK1 and sFRP1 expression. BMP2/7 treatment resulted in Smad1/5 phosphorylation, but adding Dex did not induce p-Smad1/5. However, BMP2/7-induced Smad1/5 phosphorylation was inhibited in the combined treatment group. These opposite trends support the previous reports showing mutual antagonism between DKK1 and DKK2 or sFRP1 and sFRP2. In conclusion, wellbalanced Wnt inhibitors may be crucial for bone tissue homeostasis. Key words: Wnt Signaling Pathway, Bone Morphogenetic Protein, SOST, Periodontal Ligament, Osteogenesis
Introduction Bone is a dynamic tissue that is remodeled to maintain skeletal integrity by removing old bone and replacing it with young matrix. Recent studies have causally linked alterations in Wnt signaling to changes in bone development and homeostasis, and alterations in this pathway are commonly associated with human diseases1,2). It was previously found that Wnt signaling plays essential roles in osteogenesis by stimulating runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) gene expression promoting osteogenesis3,4). Wnt/ -catenin signaling regulates cell proliferation and survival. Therefore, activity of this signaling is controlled closely by various inhibitors so that a course is not activated excessively. Secreted frizzled-related proteins (sFRPs), dickkopfs (DKKs), and sclerostin are secreted Wnt inhibitors. Of note, sclerostin is almost exclusively expressed in osteocytes, which have been hypothesized The first two authors contributed equally to this work. Correspondence to: Dr. Toshifumi Azuma, Department of Biochemistry, Tokyo Dental College, 2-9-18 Misaki-cho, Chiyoda-ku, Tokyo, 101-0061 Japan; Tel: +81-3-5275-9263; fax: +81-3-6380-9354; Email address:
[email protected]
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to integrate bone remodeling and sense mechanical load 5) . Transgenic overexpression of DKK1 and SOST specific to osteoblasts significantly decreased bone mass and bone formation rate6,7). These phenotypic changes have drawn increased interest to the Wnt pathway as a potential target for osteoporosis therapy and have helped link Wnt/ -catenin signaling to bone responses to mechanical loading. In vitro and in vivo studies have shown that activating the canonical Wnt/ -catenin pathway promotes osteoblastic cell proliferation or differentiation and reduces the adipogenic differentiation of mesenchymal stem cells8). In addition, Wnt signaling promotes osteoblast survival and interacts with bone morphogenetic protein 2 (BMP2) 9,10). BMP2 increases the level of -catenin in the nuclei of preosteoblasts and induces several Wnts including Wnt3a 11); however, both pathways appear to function in opposition to one another, and Wnt/BMP mutual antagonism is recognized as a common theme12). Therefore, a balance between Wnt and BMP output may be a key factor for regulatory networks involved in bone regeneration. One example explaining this theme is the result shown following the deletion
J.Hard Tissue Biology Vol. 24(4):311 -318, 2015 of BMP receptor 1A (BMPR1A) in preosteoblasts and osteoblasts. Deleting BMPR1A results in an unexpected increase in bone mass,
that they did not receive BMP2/7 or Dex.
this may be caused by reduced SOST expression13). sFRP1 inhibits bone formation and attenuates the anabolic action of parathyroid
ALP activity assay The cells were washed twice with phosphate-buffered saline
hormone on bone14). However, sFRP2 has an opposite function to that of sFRP1; it augments Wnt 3a-induced gene expression
(Life Technologies, Carlsbad, CA, USA) at 3 days after stimulation, fixed in 4 % paraformaldehyde for 5 min at room
through a direct interaction with Fz receptors to synergize Wnt activity, and subsequently stabilizes -catenin, resulting in its
temperature, and washed three times with distilled water. An ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was
nuclear translocation15,16). Thus, these Wnt inhibitors may play an important role in regulating osteogenesis.
added to the fixed cells for staining and incubated for 60 min at room temperature. After staining, the cells were washed three times
The BMP2/7 heterodimer is the most potent inducer of osteoblasts17,18). We treated periodontal ligament (PDL) cells with
with distilled water, and the images were analyzed.
BMP2/7 with or without dexamethasone (Dex). The PDL is a connective tissue comprising a heterogeneous cell population,
Quantitative real-time polymerase chain reaction (qRT-PCR) After 72 h stimulation, total cellular RNA was extracted using
including undifferentiated mesenchymal cells, osteoprogenitors, fibroblasts, and cementoblasts.19) We previously reported that the
QIAzol reagent (Qiagen Inc., Valencia, CA, USA), according to the manufacturer’s instructions in order to examine the early
phosphatidylinositide 3-kinase (PI3K)/Akt/mTOR/p70S6K pathway plays pivotal roles in the regulation of osteogenic
osteoblast differentiations. cDNA was synthesized using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems,
differentiation by transforming growth factor- 1 (TGF- 1)20). We also reported that an experimental model that repeated or high-
Foster City, CA, USA). qRT-PCR was performed using the Premix Ex Taq reagent (Takara Bio Inc., Shiga, Japan), according to the
dose administration of TGF- 1 inhibited osteoblast differentiation of HPDL cells by decreasing insulin-like growth factor-1 (IGF-1)
manufacturer’s instructions. The target genes were ALP, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2),
expression and subsequently downregulating PI3K/Akt signaling21,22), but Dex combined with BMP2/7 promotes osteoblast
collagen type 1 alpha 1 (COL1A1), osterix (OSX), distal-less homeobox 5 (DLX5), SOST, sFRP1, sFRP2, DKK1, and DKK2;
differentiation independently of IGF-1 expression unlike TGF1 23). We found that increased alkaline phosphatase (ALP)
18S rRNA was used as the internal control. All primers and probes are presented in Table 1. Relative expression of the genes of interest
expression occurred following a BMP2/7 and Dex combined treatment of HPDL cells. However, it is unclear how HPDL cells
was estimated using the △△ Ct method.
differentiate into osteoblasts. Thus, in this study, we elucidated how crosstalk between BMP and the Wnt pathway functions during
Protein extraction and immunoblotting After 3 h stimulation, cells were lysed in the lysis buffer [10
the differentiation of HPDL cells. We found that BMP2/7 and DEX administration induced ALP
mM Tris-HCL (pH 7.5), 150 nM NaCl, complete protease inhibitor mixture, 1 mM sodium orthovanadate, and 1 % Nonidet P-40],
expression and increased osteoblast markers. BMP2/7 and DEX administration reduced SOST expression and smad1/5
and protein concentrations were measured using a DC protein assay kit (Bio-Rad, Marnes-la-Coquette, France). Equivalent
phosphorylation. DKKs and sFRPs, which are Wnt inhibitors, were coordinately regulated by BMP2/7 and Dex.
protein concentrations were resolved by electrophoresis on NuPAGE 4-12 % Bis Tris gels (Life Technologies) and transferred
Materials and Methods
to a PVDF membrane. The membrane was probed with anti-Smad1 and anti-phosphorylated Smad1/5 antibodies (1:1000; Cell
Cell culture and osteogenic differentiation Normal HPDL cells were purchased from Lonza (Basel,
Signaling Technology, Danvers, MA, USA), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG. Bound
Switzerland) and cultured in BulletKit® stromal cell growth medium (SCGM, Lonza). Cells at passage numbers 5-8 were
antibodies were visualized using a chemiluminescent substrate (ECLTM Primer Western Blotting Detection Reagent; GE
seeded at a density of 1 × 105 cells/cm2 in SCGM at 37 °C in 5 % CO2 for each assay. The following day, osteoblast differentiation
Healthcare Ltd., Buckinghamshire, UK) and the ImageQuant LAS 4000 mini instrument (GE Healthcare).
was induced by replacing the medium with -MEM (Invitrogen, Carlsbad, CA, USA) supplemented with or without 100 ng/ml
Statistical analysis
BMP2/7 (R&D Systems, Minneapolis, MN, USA), 100 nM Dex (Sigma-Aldrich, St Louis, MO, USA), or a combination of these
Data are expressed as mean ± standard deviation. Multiple comparisons of each experimental group were performed using
reagents. Recombinant human SOST/sclerostin was purchased from R&D Systems. Control cells were treated identically except
the Bonferroni test when analysis of variance indicated differences among the groups. All data are representative of at least three 312
Takashi Tsukinowa et al.: Osteogenic Regulation of Wnt Inhibitors Table 1. Primers Used for Quantitative Real-Time Polymerase Chain Reaction Analysis Gene symbol
GenBankTM accession no.
ALP
NM_000478.3
DLX5
NM_005221.5
OSX
NM_152860.1
RUNX2
NM_001024630.2
BSP
NM_004967.3
COL1A1
NM_000088.3
SOST
NM_025237.2
DKK1
NM_012242.2
DKK2
NM_014421.2
sFRP1
NM_003012.4
sFRP2
NM_003013.2
18SrRNA
M11188.1
Primsequence:sense/antisense 5'-caaccctggggaggagac-3' 5'-gcattggtgttgtacgtcttg-3' 5'-ctacaaccgcgtcccaag-3' 5'-gccattcaccattctcacct-3' 5'-catctgcctggctccttg-3' 5'-caggggactggagccata-3' 5'-gtgcctaggcgcatttca-3' 5'-gctcttcttactgagagtggaagg-3' 5'-caatctgtgccactcactgc-3' 5'-tcattttggtgattgcttcct-3' 5'-gggattccctggacctaaag-3' 5'-ggaaacctcgctctcca-3' 5'-agctggagaacaacaagacca-3' 5'-gctgtactcggacacgtcttt-3' 5'-caggcgtgcaaatctgtct-3' 5'-aatgattttgatcagaagacacacata-3' 5'-ggcagtaagaagggcaaaaa-3' 5'-cctcccaacttcacactcct-3' 5'-gctggagcacgagaccat-3' 5'-tggcagttcttgttgagca-3' 5'-gctagcagcgaccacctc-3' 5'-tttttgcaggcttcacatacc-3' 5'-cggacaggattgacagatttg-3'
Probe no.
Amplicon
#19
78bp
#20
76bp
#69
91bp
#29
78bp
#38
74bp
#67
63bp
#77
96bp
#4
89bp
#43
80bp
#40
100bp
#83
98bp
#77
78bp
independent experiments. A p < 0.05 was considered significant. Results BMP2/7 and Dex treatment increases ALP activity in HPDL cells The combination of BMP2/7 and Dex induced greater expression of ALP, an early mineralization marker indicating commitment to osteoblast differentiation, compared with that induced by other treatments (Fig. 1A). Similarly, adding Dex to BMP2/7 upregulated ALP mRNA expression more than that upregulated by Dex or BMP2/7 alone (Fig. 1B). BMP 2/7 and Dex treatment induces the expression of osteoblast differentiation markers in HPDL cells We examined the expression of the osteoblast differentiationrelated genes DLX5, OSX, RUNX2, BSP, and COL1A1. The combination of BMP2/7 and Dex increased DLX5 and OSX expression significantly compared with other treatments (Fig. 2A,
Figure 1. Bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) treatment increases alkaline phosphatase (ALP) activity in human periodontal ligament (HPDL) cells. (A) Confluent HPDL cells were cultured in -MEM (1), -MEM with BMP2/7 (2), -MEM with Dex (3), and -MEM with both BMP2/7 and Dex (4) for 72 h. ALP activity was visualized by ALP cell staining.(B) The mRNA expression level of the osteogenic differentiation gene ALP in HPDL cells cultured for 72 h under the same conditions indicated in (A) was examined by quantitative real-time polymerase chain reaction. Data represent means ± standard deviations (n = 7).
B). RUNX2 expression increased slightly in cells treated with BMP2/7 with or without Dex (Fig. 2C). No differences were observed in BSP or COL1A1 expression (Fig. 2D, E). Combination of BMP2/7 and Dex treatment inhibits Smad1/5 phosphorylation in HPDL cells We conducted a western blot analysis to measure the level of 313
J.Hard Tissue Biology Vol. 24(4):311 -318, 2015
Figure 2.Bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) treatment induces expression of osteoblast differentiation markers and inhibits Smad1/5 phosphorylation in human periodontal ligament (HPDL) cells (A–E) mRNA expression levels of the osteogenic differentiation genes distal-less homeobox 5 (DLX5) (A), osterix (OSX) (B), runtrelated transcription factor 2 (RUNX2) (C), bone sialoprotein (BSP) (D), and collagen type 1 alpha 1 (COL1A1) (E) in HPDL cells cultured in -MEM (1), -MEM with BMP2/7 (2), -MEM with Dex (3), and -MEM with both BMP2/7 and Dex (4) for 72 h. Data represent means ± standard deviations (n = 5). (F) The combination bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) treatment Confluent HPDL cells were cultured in -MEM and treated with BMP2/7, Dex, or both BMP2/7 and Dex for 3 h. The level of phosphorylated Smad1/5 was determined by western blot analysis. Smad1/5 was detected as a control.
phosphorylated Smad1/5 (p-Smad1/5), a downstream effecter of BMP2/7. BMP2/7 treatment resulted in Smad1/5 phosphorylation, but adding Dex did not induce p-Smad1/5 (Fig. 2F). However, BMP2/7-induced Smad1/5 phosphorylation was inhibited in the combined treatment group. Total Smad1/5 was observed in the
observed between the BMP2/7 and the combined BMP2/7 and Dex treatments (Fig. 2D). We investigated whether SOST is a negative regulator in cells treated with the BMP2/7 and Dex combination. As shown in Fig. 3F, SOST-treated cells revealed suppressed ALP activity induced by BMP2/7 and Dex treatment.
controls for each group. Combination of BMP2/7 and Dex modulates Wnt inhibitors, particularly SOST expression, in HPDL cells We examined the expression of the Wnt inhibitors SOST, DKK1, DKK2, sFRP1, and sFRP2 to investigate the effect of BMP2/7 and Dex on Wnt signaling in HPDL cells. Significantly increased SOST expression was observed in cells treated only with BMP-2/7, whereas SOST expression was suppressed to the control level in BMP2/7- and Dex-treated cells (Fig. 3A). The combination of BMP2/7 and Dex strongly induced DKK1 expression compared with other treatments (Fig. 3B). DKK2 and sFRP2 expression decreased significantly in the combined BMP2/7 and Dex treatment group (Fig. 3C, E). However, the BMP2/7 and Dex combination modesty increased sFRP1 expression compared with BMP2/7 alone (Fig. 3D); however, no significant difference was observed between these groups. sFRP1 expression decreased slightly in BMP2/7-treated cells, but no significant difference was
Discussion In bone reproduction therapy, developments of the cure using bone morphogenetic proteins (BMPs) are pushed forward than before. BMP and Wnt signaling are regulated in many biological processes by each original pathway, but BMP and Wnt are often regulating similar cellular events9). Specifically in bone formation, crosstalk between BMP and Wnt/ -catenin signaling has complement or antagonistic effects depending on various factors, including cell type and developmental stage10). BMP2/7 treatment modestly increases ALP activity, but the BMP2/7 and Dex combined treatment greatly enhances ALP activity24). In HPDL cells, we found that the BMP2/7 and Dex combined treatment had a significant increased ALP expression and ALP activity in comparison to other treatments (Fig.1A, B). In addition, combination of BMP2/7 and Dex treatment had a significant increased DLX5 expression compared with other treatments (Fig.2A), and OSX expression increased three hundredfold 314
Takashi Tsukinowa et al.: Osteogenic Regulation of Wnt Inhibitors
Figure 3. The combination of bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) modulates Wnt inhibitor genes, particularly sclerostin (SOST) expression, in human periodontal ligament (HPDL) cells. (A–E) mRNA expression levels of the Wnt inhibitor genes SOST (A), Dickopff-related 1 (DKK1) (B), DKK2 (C), secreted frizzledrelated protein 1 (sFRP1) (D), and sFRP2 (E) in HPDL cells cultured in á-MEM (1), -MEM with BMP2/7 (2), and -MEM with both BMP2/7 and Dex (3) for72 h. The ratio of the expression of each gene to that of 18S rRNA was calculated. Data represent means ± standard deviations (n = 5). (F) Confluent HPDL cells were cultured in -MEM withBMP2/7, BMP2/7 and Dex, or combined administration of BMP2/7, Dex, and SOST for 72 h.Alkaline phosphatase (ALP) activity was visualized by ALP cell staining. SOST - treated cells revealed suppressed ALP activity induced by BMP2/7 and Dex treatment.
compared with control. BMP2 and Dex regulate ALP transcription by regulating osteogenic transcription factors, such as Runx2, Osx,
extracellular inhibitors. These inhibitors have attracted attention because of important advances from studies on sclerosteosis and
and the homeobox genes Dlx525,26). It is reported that Dlx5 was interacted with Osx promoter containing Dlx5 binding elements27).
van Buchem disease, which are two disorders associated with increased bone mass28). Both disorders are caused by loss of SOST
These reports suggested that upregulation of OSX expression for combination of BMP2/7 and Dex treatment was induced by
gene expression, which encodes the sclerostin protein. We observed a sharp increase in SOST expression in HPDL cells
promoted expression of DLX5. Wnt signaling plays central roles in bone development and
following BMP2/7 treatment; however, the BMP2/7 and Dex combined treatment induced ALP activity in HPDL cells,
homeostasis, and alterations in this pathway are commonly associated with human diseases. Wnts are antagonized by several
significantly decreased SOST expression. Thus, SOST expression induced by BMP2/7 was reversed by adding Dex. This decreased 315
J.Hard Tissue Biology Vol. 24(4):311 -318, 2015 (B) Grant Numbers 25861761, 26861686 and 26861560. SOST expression induced by BMP2/7 and Dex was accompanied by decreased Smad1 phosphorylation. These evidences were supported by the previous report showing Smad dependent BMP signaling through the type 1A receptor enhanced SOST
1.
development and disease. Annu Rev Cell Dev Biol 20: 781810, 2004
expression 13). Thus, it is suggested that the BMP2/7 canonical pathway can enhance SOST expression blocked by Dex. We ectopically added human recombinant SOST and found a significant decrease in ALP activity, indicating that SOST may play a key role in BMP-Wnt induced osteogenesis. In addition to SOST, osteoblasts express the Wnt inhibitors
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