Wnt regulatory molecules coordinate Title osteoblast differentiation.

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1 Wnt regulatory molecules coordinate Title osteoblast differentiation Author(s) 月野和, 隆 Journal, (): - URL Right Posted at the Institutional Resources for Unique Colle Available from

2 Wnt regulatory molecules coordinately regulate osteoblast differentiation Takashi Tsukinowa Department of Endodontics and Clinical Cariology Tokyo Dental College

3 1 ABSTRACT Wnt signaling has attracted increasing attention from many clinical fields because Wnt pathways are commonly associated with human diseases. 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. In our study, BMP2/7 treatment increased alkaline phosphatase (ALP) activity modestly, and the combination of BMP2/7 with dexamethasone (Dex) greatly enhanced ALP activity. Sclerostin (SOST) expression increased sharply following BMP2/7 treatment; however, adding BMP2/7 and Dex drastically reversed this increase in SOST. The SOST expression induced by BMP2/7 was reversed by adding Dex and was associated with a significant increase in ALP expression and activity. Adding exogenous SOST reduced ALP activity. Dickkopfs (DKKs), and secreted frizzled-related proteins (sfrps) were first reported as Wnt inhibitors. However, several studies have indicated mutual antagonism between DKK1 and DKK2 or sfrp1 and sfrp2, and DKK1 and DKK2 or sfrp1 and sfrp2 clearly revealed opposite trends following BMP2/7 and Dex treatment. We found that adding BMP2/7 and Dex synergistically decreased DKK2 and sfrp2 expression. In contrast, BMP2/7 and Dex had a significant inductive effect on DKK1 and sfrp1 expression. Because DKK1 and sfrp1 have an inhibitory antagonistic effect and their counterparts DKK2 and

4 2 sfrp2 may have an agonistic effect on Wnt signaling, their expression trends may represent a feedback mechanism. In conclusion, well-balanced mutual antagonistic regulation among Wnt inhibitors may be crucial for bone tissue homeostasis. KEY WORDS: Wnt Signaling Pathway, Bone Morphogenetic Protein, Dexamethasone, SOST, Periodontal Ligament, Osteogenesis

5 3 INTRODUCTION Bone is a dynamic tissue that is remodeled to maintain skeletal integrity by removing old bone and replacing it with young matrix. However, bone tissue damage exceeds bone renewal capacity under some circumstances. This is often the case in periodontal disease and implant treatment. 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 diseases (Logan and Nusse 2004; Voronkov and Krauss 2013). Studies on the molecular mechanisms of Wnt signaling related to osteoblast function have been stimulated by identifying the underlying genetic cause of the pediatric syndrome osteoporosis pseudoglioma (OPPG) (Gong et al. 1996). OPPG is characterized by severe, early-onset osteoporosis. An inactivating mutation in the gene encoding low-density lipoprotein-related receptor 5 (LRP5) is responsible for this autosomal recessive disorder. Subsequent generation of mice carrying germline inactivating mutations in Lrp5 further confirmed the importance of this gene by accurately modeling phenotypes observed in OPPG syndrome (Gong et al. 2001). However, opposite phenotypes were observed in cases of LRP5 mutations, which can cause high bone mass (Boyden et al. 2002; Little et al. 2002). In contrast, the other type of LRP5 mutation that renders the co-receptor resistant to extracellular Wnt inhibitors, such as Dickopff-related 1 (DKK1) and SOST, causes high bone mass syndrome (Ai et al. 2005; Semenov and He 2006). These complex phenotypic changes have drawn increased interest to the

6 4 Wnt pathway as a potential target for osteoporosis therapy and have helped link Wnt/β-catenin signaling to bone responses to mechanical loading (Robling et al. 2008). Wnt signaling comprises a family of 19 secreted cysteine-rich glycoproteins with functions related to cell specification, body plan formation, cell growth, differentiation, and apoptosis (Akiyama 2000; Katoh and Katoh 2007). Wnts activate several signaling cascades at the cellular level, including the most commonly studied canonical pathway, which results in stabilizing the β-catenin protein (MacDonald et al. 2009). This pathway is initiated when the Wnt protein binds to a receptor complex that includes a member of the Frizzled (Fz) family of seven-transmembrane receptors plus either Lrp5 or Lrp6 (Bhanot et al. 1996; Pinson et al. 2000). Formation of this receptor complex inhibits the activity of glycogen synthase kinase 3β toward β-catenin and induces nuclear translocation of β-catenin, which binds to members of the LEF/TCF family of DNA binding proteins and transactivates target-gene promoters (Krishnan et al. 2006; Kubota et al. 2009). In vitro and in vivo studies have shown that activating the canonical Wnt/β-catenin pathway promotes osteoblastic cell proliferation or differentiation and reduces the adipogenic differentiation of mesenchymal stem cells (Ling et al. 2009). In addition, Wnt signaling promotes osteoblast survival and interacts with bone morphogenetic protein 2 (BMP2) (Chan and Little 2012; Itasaki and Hoppler 2010). BMP2 increases the level of β-catenin in the nuclei of preosteoblasts and induces several Wnts including Wnt3a (Rawadi et al. 2003); however, both pathways appear to function in opposition to

7 5 one another, and Wnt/BMP mutual antagonism is recognized as a common theme (Minear et al. 2010). 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 of BMP receptor 1A (BMPR1A) in preosteoblasts and osteoblasts. Deleting BMPR1A results in an unexpected increase in bone mass, which may be caused by reduced SOST expression (Kamiya et al. 2008). Secreted Fz-related proteins (sfrps), which antagonize Wnt signaling by binding to Wnts, downregulate the Wnt signaling pathway by forming an inhibitory complex with Fz receptors (Kubota et al. 2009). sfrp1 inhibits bone formation and attenuates the anabolic action of parathyroid hormone on bone (Bodine et al. 2004). However, sfrp2 has an opposite function to that of sfrp1; it augments Wnt 3a-induced gene expression through a direct interaction with Fz receptors to synergize Wnt activity, and subsequently stabilizes β-catenin, resulting in its nuclear translocation (Cho et al. 2008; von Marschall and Fisher 2010). Thus, these Wnt inhibitors may play an important role in regulating osteogenesis. The BMP2/7 heterodimer is the most potent inducer of osteoblasts (Sun et al. 2012; Zhao et al. 2005). We treated periodontal ligament (PDL) cells with BMP2/7 with or without dexamethasone (Dex). The PDL is a connective tissue comprising a heterogeneous cell population, including undifferentiated mesenchymal cells, osteoprogenitors, fibroblasts, and cementoblasts (Tomokiyo et al. 2008). We previously reported that the phosphatidylinositide 3-kinase

8 6 (PI3K)/Akt/mTOR/p70S6K pathway plays different roles in the regulation of osteogenic differentiation by transforming growth factor-β1 (TGF-β1) since mtor plays an important role in TGF-β1 in HPDL cells.(aida et al.,2012) We also reported that an experimental model that repeated or high-dose administration of TGF-β1 inhibited osteoblast differentiation of HPDL cells by decreasing insulin-like growth factor-1 (IGF-1) expression and subsequently downregulating PI3K/Akt signaling (Ochiai et al., 2012, Ochiai et al.,2014), but Dex combined with BMP2/7 promotes osteoblast differentiation independently of IGF-1 expression unlike TGF-β1. (Yamashita et al., 2014). We found that increased alkaline phosphatase (ALP) expression occurred following a BMP2/7 and Dex combined treatment of HPDL cells. However, it is unclear how HPDL cells differentiate into osteoblasts. Thus, in this study, we elucidated how crosstalk between BMP and the Wnt pathway functions during the differentiation of HPDL cells. We found that BMP2/7 and DEX administration induced ALP expression and increased osteoblast markers. BMP2/7 and DEX administration reduced SOST expression and smad1/5 phosphorylation. DKKs and sfrps, which are Wnt inhibitors, were coordinately regulated by BMP2/7 and Dex.

9 7 MATERIALS & METHODS Cell culture and osteogenic differentiation Normal HPDL cells were purchased from Lonza (Basel, Switzerland) and cultured in BulletKit stromal cell growth medium (SCGM, Lonza). Cells at passage numbers 5 8 were seeded at a density of cells/cm 2 in SCGM at 37 C in 5% CO 2 for each assay. The following day, osteoblast differentiation was induced by replacing the medium with α-mem (Invitrogen, Carlsbad, CA, USA) supplemented with or without 100 ng/ml BMP2/7 (R&D Systems, Minneapolis, MN, USA), 100 nm Dex (Sigma-Aldrich, St Louis, MO, USA), or a combination of these reagents. Recombinant human SOST/sclerostin was purchased from R&D Systems. Control cells were treated identically except that they did not receive BMP2/7 or Dex. ALP activity assay The cells were washed twice with phosphate-buffered saline (Life Technologies, Carlsbad, CA, USA) 3 days after stimulation, fixed in 4% paraformaldehyde for 5 min at room temperature, and washed three times with distilled water. An ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was added to the fixed cells for staining and incubated for 60 min at room temperature. After staining, the cells were washed three times with distilled water, and the images were analyzed.

10 8 Quantitative real-time polymerase chain reaction (qrt-pcr) Total cellular RNA was extracted using QIAzol reagent (Qiagen Inc., Valencia, CA, USA), according to the manufacturer s instructions. cdna was synthesized using the High-Capacity cdna Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qrt-pcr was performed using the Premix Ex Taq reagent (Takara Bio Inc., Shiga, Japan), according to the manufacturer s instructions. The target genes were ALP, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 1 (COL1A1), osterix (OSX), distal-less homeobox 5 (DLX5), SOST, sfrp1, sfrp2, DKK1, and DKK2; 18S rrna was used as the internal control. All primers and probes are presented in Table 1. Relative expression of the genes of interest was estimated using the Ct method. Protein extraction and immunoblotting Cells were lysed in the lysis buffer [10 mm Tris-HCL (ph 7.5), 150 nm NaCl, complete protease inhibitor mixture, 1 mm sodium orthovanadate, and 1% Nonidet P-40], and protein concentrations were measured using a DC protein assay kit (Bio-Rad, Marnes-la-Coquette, France). Equivalent protein concentrations were resolved by electrophoresis on NuPAGE 4% 12% Bis Tris gels (Life Technologies) and transferred to a PVDF membrane. The membrane was probed with anti-smad1 and anti-phosphorylated Smad1/5 antibodies (1:1000; Cell Signaling Technology,

11 9 Danvers, MA, USA), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG. Bound antibodies were visualized using a chemiluminescent substrate (ECL TM Primer Western Blotting Detection Reagent; GE Healthcare Ltd., Buckinghamshire, UK) and the ImageQuant LAS 4000 mini instrument (GE Healthcare). Statistical analysis Data are expressed as mean ± standard deviation. Multiple comparisons of each experimental group were performed using the Bonferroni test when analysis of variance indicated differences among the groups. All data are representative of at least three independent experiments. A p < 0.05 was considered significant.

12 10 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). BMP2/7 and Dex treatment induces the expression of osteoblast differentiation markers in HPDL cells We examined the expression of the osteoblast differentiation-related 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, 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 phosphorylated Smad1/5

13 11 (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. 3). However, BMP2/7-induced Smad1/5 phosphorylation was inhibited in the combined treatment group. Total Smad1/5 was observed in the 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. 4A). The combination of BMP2/7 and Dex strongly induced DKK1 expression compared with other treatments (Fig. 4B). DKK2 and sfrp2 expression decreased significantly in the combined BMP2/7 and Dex treatment group (Fig. 4C, E). However, the BMP2/7 and Dex combination modesty increased sfrp1 expression compared with BMP2/7 alone (Fig. 4D); however, no significant difference was observed between these groups. sfrp1 expression decreased slightly in BMP2/7-treated cells, but no significant difference was 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

14 12 BMP2/7 and Dex combination. As shown in Fig. 4F, SOST-treated cells revealed suppressed ALP activity induced by BMP2/7 and Dex treatment.

15 13 DISCUSSION Wnt signaling plays central roles in bone development and homeostasis, and alterations in this pathway are commonly associated with human diseases. Wnts are antagonized by several extracellular inhibitors. These inhibitors have attracted attention because of important advances from studies on sclerosteosis and van Buchem disease, which are two disorders associated with increased bone mass (Brunkow et al. 2001). Both disorders are caused by loss of SOST gene expression, which encodes the sclerostin protein. BMP2/7 treatment modestly increases ALP activity, but the BMP2/7 and Dex combined treatment greatly enhances ALP activity (Mikami et al. 2010). We observed a sharp increase in SOST expression in HPDL cells following BMP2/7 treatment; however, the BMP2/7 and Dex combined treatment induced ALP activity in HPDL cells, significantly decreased SOST expression. Thus, SOST expression induced by BMP2/7 was reversed by adding Dex. This decreased SOST expression induced by BMP2/7 and Dex was accompanied by decreased Smad1 phosphorylation. This result suggests 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 DKK1 and sfrp1 (Monroe et al. 2012), and both play roles in regulating bone mass. DKK1 inhibits osteoblast differentiation and

16 14 bone formation by binding to Lrp5/6, and sfrp1 inhibits Wnt signaling by binding to Wnts and preventing their binding to the Frizzled complex. Mice with deleted sfrp1 have increased trabecular bone mineral density, and their osteoblasts show increased proliferation and differentiation into osteocytes in vitro (Bodine et al. 2004). Deletion of a single Dkk1 gene allele leads to increased bone formation and bone mass (Morvan et al. 2006). However, several reports have indicated that even though DKK1 and DKK2 or sfrp1 and sfrp2 have similar molecular structures, they may have opposite functions. Mutual antagonism between Dkk1- and Dkk2-regulated Wnt/β-catenin signaling has been reported (Olivares-Navarrete et al. 2010). DKK1 is a master regulator of osteogenesis (Diarra et al. 2007), whereas DKK2 acts as a fine-tuning regulator of terminal differentiation and mineralization. Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation (Li et al. 2005). This mutual antagonism is also observed with sfrp1 and sfrp2. Some studies have reported that myeloma cells treated with sfrp2 or sfrp4 have significantly higher Wnt3a-induced ALP activity (Cho et al. 2008). This result indicates that DKK1 and sfrp1 negatively regulate Wnt signaling or ALP activity in some circumstances, whereas DKK2 and sfrp2 positively regulate Wnt signaling or ALP activity. We found that DKKs and sfrps showed similar trends. The combination of BMP2/7 and Dex had a synergistic stimulatory effect on the expression of the Wnt antagonists DKK1 and sfrp1 but reduced expression of the Wnt agonists DKK2 and sfrp2. These similar trends support the idea of mutual antagonism between DKK1 and DKK2 or

17 15 sfrp1 and sfrp2, which could function as a feedback mechanism during BMP2/7- and Dex-induced osteoblastic differentiation. Further investigation is necessary to elucidate whether this mutual antagonism between DKK1 and DKK2 or sfrp1 and sfrp2 shown here is ubiquitously present. However, the altered expression of these Wnt inhibitors is notable, and drugs to modify these inhibitors are now available or under development. Clarification of Wnt inhibitor expression is essential for clinical use of the newly developed Wnt inhibitor drugs.

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19 17 negatively regulates bone mass through sclerostin by inhibiting the canonical Wnt pathway. Development 135: Katoh M, Katoh M (2007). WNT signaling pathway and stem cell signaling network. Clin Cancer Res 13: Krishnan V, Bryant HU, Macdougald OA (2006). Regulation of bone mass by Wnt signaling. J Clin Invest 116: Kubota T, Michigami T, Ozono K (2009). Wnt signaling in bone metabolism. J Bone Miner Metab 27: Li X, Liu P, Liu W, Maye P, Zhang J, Zhang Y, et al. (2005). Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation. Nat Genet 37: Ling L, Nurcombe V, Cool SM (2009). Wnt signaling controls the fate of mesenchymal stem cells. Gene 433:1-7. Little RD, Carulli JP, Del Mastro RG, Dupuis J, Osborne M, Folz C, et al. (2002). A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 70: Logan CY, Nusse R (2004). The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: MacDonald BT, Tamai K, He X (2009). Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9-26. Mikami Y, Asano M, Honda MJ, Takagi M (2010). Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells. J Cell Physiol 223: Minear S, Leucht P, Miller S, Helms JA (2010). rbmp represses Wnt signaling and influences skeletal progenitor cell fate specification during bone repair. J Bone Miner Res 25: Monroe DG, McGee-Lawrence ME, Oursler MJ, Westendorf JJ (2012). Update on Wnt signaling in bone cell biology and bone disease. Gene 492:1-18. Morvan F, Boulukos K, Clement-Lacroix P, Roman Roman S, Suc-Royer I, Vayssiere B, et al. (2006). Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass. J Bone Miner Res 21: Ochiai H, Okada S, Saito A, Hoshi K, Yamashita H, Takato T, et al. (2012). Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-beta1 (TGF-beta1) administration suppresses osteoblast differentiation. J Biol Chem 287 (27): Ochiai-Shino H, Kato H, Sawada T, Onodera S, Saito A, Takato T, et al. (2014). A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination. PLoS One 9(6):e99534.

20 18 Olivares-Navarrete R, Hyzy S, Wieland M, Boyan BD, Schwartz Z (2010). The roles of Wnt signaling modulators Dickkopf-1 (Dkk1) and Dickkopf-2 (Dkk2) and cell maturation state in osteogenesis on microstructured titanium surfaces. Biomaterials 31: Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407: Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S (2003). BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res 18: Robling AG, Niziolek PJ, Baldridge LA, Condon KW, Allen MR, Alam I, Mantila SM, et al. (2008). Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem 283: Semenov MV, He X (2006). LRP5 mutations linked to high bone mass diseases cause reduced LRP5 binding and inhibition by SOST. J Biol Chem 281: Sun P, Wang J, Zheng Y, Fan Y, Gu Z (2012). BMP2/7 heterodimer is a stronger inducer of bone regeneration in peri-implant bone defects model than BMP2 or BMP7 homodimer. Dent Mater J 31: Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Akamine A (2008). Development of a multipotent clonal human periodontal ligament cell line. Differentiation 76: von Marschall Z, Fisher LW (2010). Secreted Frizzled-related protein-2 (sfrp2) augments canonical Wnt3a-induced signaling. Biochem Biophys Res Commun 400: Voronkov A, Krauss S (2013). Wnt/beta-catenin signaling and small molecule inhibitors. Curr Pharm Des 19: Yamashita H, Ochiai H, Saito A, Shintani S, Azuma T. (2014). Phosphoinositide 3-Kinase (PI3K) Activation is Differentially Regulated during Osteogenesis induced by TGF-β1 and BMP-2/BMP-7. Journal of Hard Tissue Biology 23(1): Zhao M, Zhao Z, Koh JT, Jin T, Franceschi RT (2005). Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7. J Cell Biochem 95:1-16.

21 19 TABLE 1 Primers used for quantitative real-time polymerase chain reaction analysis

22 20 FIGURE LEGENDS 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 α-mem (1), α-mem with 100 ng/ml BMP2/7 (2), α-mem with 100 nm Dex (3), and α-mem with both 100 ng/ml BMP2/7 and 100 nm Dex (4) for 72 h. ALP activity was visualized by ALP cell staining. The combination of BMP2/7 and Dex induced ALP activity. (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. The ratio of the expression of each gene to that of 18S rrna was calculated. Each experiment was performed in triplicate, and data represent means ± standard deviations (n = 7). BMP2/7 and Dex combination induced ALP expression significantly compared with other treatments.. The Boneferroni correction for multiple comparisons was applied. *p < 0.05.

23 21 Figure 2 Bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) treatment induces expression of osteoblast differentiation markers 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), runt-related transcription factor 2 (RUNX2) (C), bone sialoprotein (BSP) (D), and collagen type 1 alpha 1 (COL1A1) (E) in HPDL cells cultured in α-mem (1), α-mem with 100 ng/ml BMP2/7 (2), α-mem with 100 nm Dex (3), and α-mem with both 100 ng/ml BMP2/7 and 100 nm Dex (4) for 72 h. The ratio of the expression of each gene to that of 18S rrna was calculated. The combination of BMP2/7 and Dex increased DLX5 and OSX expression significantly compared with other treatments. Each experiment was performed in triplicate, and data represent means ± standard deviations (n = 5). The Boneferroni correction for multiple comparisons was applied. *p < Figure 3 The combination bone morphogenetic protein (BMP) 2/7 and dexamethasone (Dex) treatment inhibits Smad1/5 phosphorylation in human periodontal ligament (HPDL) cells Confluent HPDL cells were cultured in α-mem and treated with 100 ng/ml BMP2/7, 100 nm Dex,

24 22 or both 100 ng/ml BMP2/7 and 100 nm Dex for 3 h. BMP2/7-induced Smad1/5 phosphorylation was inhibited by Dex. The level of phosphorylated Smad1/5 was determined by western blot analysis. Smad1/5 was detected as a control. Figure 4 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 frizzled-related protein 1 (sfrp1) (D), and sfrp2 (E) in HPDL cells cultured in α-mem (1), α-mem with 100 ng/ml BMP2/7 (2), and α-mem with both 100 ng/ml BMP2/7 and 100 nm Dex (3) for 72 h. The ratio of the expression of each gene to that of 18S rrna was calculated. SOST expression was suppressed to the control level in BMP2/7- and Dex-treated cells. The combination of BMP2/7 and Dex strongly induced DKK1 expression compared with other treatments. DKK2 and sfrp2 expression decreased significantly in the combined BMP2/7 and Dex treatment group. Each experiment was performed in triplicate, and data represent means ± standard deviations (n = 5). The Boneferroni correction for multiple comparisons was applied. *p < (F) Confluent HPDL cells were cultured in α-mem with 100 ng/ml BMP2/7, 100 ng/ml BMP2/7

25 23 and 100 nm Dex, or combined administration of 100 ng/ml BMP2/7, 100 nm Dex, and 1000 ng/ml 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.

26 24 Figure 1 (A) (B)

27 25 Figure 2 (A) (B) (C) (D) (E)

28 Figure 3 26

29 27 Figure 4 (A) (B) (C) (D) (E) (F)

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31 学位論文内容の要旨 1. 研究目的歯周病の治療やインプラント治療において 顎骨欠損部位の新生骨形成を効果的に促進する治療法の開発は重要な課題である 我々は BMP と Dexamethasone ( 以下 Dex ) を複合添加して培養すると 間葉系幹細胞を多く含むヒト歯根膜由来 ( 以下 HPDL) 細胞から骨芽細胞への分化が促進されることを見いだした Wnt 経路は骨分化調節において大きな役割を果たしていることが明らかとなり 近年特に注目されている しかし その機能は多岐に亘るため未解明な点が多い 特に他の骨分化誘導物質との関係については重要性が示唆されているが 詳細なメカニズムは不明である 本研究では HPDL 細胞が BMP 2/7 と Dex により骨芽細胞へ分化する際の骨分化誘導 特に Wnt 抑制因子について詳細に検討することにより BMP と Wnt シグナル間の相互関係を明らかにする 2. 研究方法 HPDL 細胞は Lonza より購入し SCGM (BulletKit Stromal cell growth medium, Lonza) を用いて培養した 12well プレートに cells/cm 2 で播種し 翌日 培地交換を行った 培地には α-mem (Life Technologies) を使用し 添加物として 100ng/ml BMP2/7 (R&D Systems) 100nM Dex (Sigma) をそれぞれ単独添加した群と BMP2/7 と Dex を複合添加した群に分け 72h 培養した 培養後 BCIP/TCP(Roche Diagnostics) にて ALP 活性染色を行い 骨芽細胞への分化を確認した 各群より抽出した RNA から Wnt 経路抑制因子及び骨分化マーカーについてリアルタイム PCR (Applied Biosystems 7500 Fast) で確認した さらに 各群よりタンパクを抽出し Western blot 法を用い Smad 経路の活性を確認した 3. 研究成績および結論 ALP 活性染色では BMP2/7+Dex 複合添加群は他の群と比較して濃く染色された 定量的 RT-PCR 法による各群の ALP 発現量は BMP2/7+Dex 複合添加群はコントロールに比較し約 10 倍増加していた この時 Wnt 経路抑制因子である SOST は BMP2/7 刺激で発現が増加するが Dex との複合添加により著明に ( 約 1/4 倍 ) 発現が低下していた また 外部より SOST を添加することにより ALP 活性が抑制された Wnt 経路阻害因子 SFRP2 DKK2 では BMP2/7 及び BMP2/7+Dex 複合添加群で段階的に発現が低下していた また DKK1 SFRP1 は Dex により発現量が上昇し BMP と複合添加することにより 特に DKK1 では他の群と比較して有意に増加した DKK1,2 と SFRP1,2 は互いに拮抗する作用を持つ可能性が示唆された Western blot 法より Smad の活性化の指標であるリン酸化 Smad1/5 を検討すると BMP2/7 単独群では Smad1/5 の活性が認められるが BMP2/7+Dex 複合添加群では活性が認められず BMP2/7 による Smad1/5 の活性化を Dex が著明に抑制していた 以上の結果より SOST は Wnt 経路を抑制し 骨分化を抑制する重要な物質である また 多種類の Wnt 抑制因子発現の変化は Wnt シグナル間のフィードバックメカニズムによる可能性が示唆された 結論として Wnt 阻害因子間のバランスのとれた相互の拮抗作用が骨再生の調整に重要である

32 最終試験の結果の要旨および担当者 報告番号甲第号氏名月野和隆 主査片倉朗教授 副査 1 齋藤淳教授 最終試験担当者 2 古澤成博教授 3 東俊文教授 4 山本仁教授 最終試験施行日試験科目試験方法試験問題 平成 26 年 10 月 2 日 歯科保存学口頭試問主題ならびに関連問題 結果の要旨 本審査委員会は 主題ならびに関連問題について最終試験を行った結果 十分な学識を有することを認め 合格と判定した

33 学位論文審査の要旨 骨の形成と吸収は様々な細胞内シグナルと そのクロストークによって成り立っている Wnt 経路は骨分化調節において大きな役割を果たしていることが明らかとなり 近年特に注目されている 特に他の骨分化誘導物質との関係については重要性が示唆されているが 詳細なメカニズムは不明である 本研究では ヒト歯根膜由来 (HPDL) 細胞が BMP 2/7 と Dex により骨芽細胞へ分化する際の Wnt 抑制因子について詳細に検討することにより BMP と Wnt シグナル間の相互関係を検討した これらの内容により HPDL 細胞における BMP-Wnt シグナル間の骨分化促進機序には SOST 遺伝子が重要な役割を担っていることが示唆された また Wnt 抑制因子群は 相互に発現を拮抗することにより Wnt シグナルを調節している可能性が示唆された 本審査委員会では (1) なぜ HPDL 細胞を実験材料として選択したのか (2)Wnt signal が関与している Human disease には具体的にはどのようなものが含まれるのか (3) 今回の研究は HPDL 細胞以外の他の細胞においても確認できるのか (4) これらの結果の臨床的な意味は何か について討論を行い 質疑がなされた (1) については 生化学講座では 以前より HPDL 細胞を用いた情報伝達経路について研究を進めており HPDL 細胞は未分化な間葉系幹細胞を多く含むことから 再生医療において利用価値があると考えられており 分化メカニズムの解明を進めているため (2) については Wnt signal は生物の初期発生 形態形成の制御や細胞の増殖 分化 アポトーシスを制御している Wnt signal は骨形成にも関与しており そのため骨代謝異常症の原因であると考えられている また細胞の増殖や生存も管理していることから 癌などの腫瘍形成にも関与している (3) については MC3T3 にて同じ条件で ALP 活性の確認を行ったところ HPDL と同じような結果は得られなかった MC3T3 では BMP や Dex の濃度が足りなかったことと 今回の現象が細胞特異的であった可能性が考えられる (4) については 歯周炎や根尖性歯周炎による歯槽骨の破壊や 難治性の症例の骨形成の治癒遅延などにはこれらのシグナルが関わり合い 骨形成にストップが生じることで骨の再生がなされないのではないかと考えられる その分子レベルでのメカニズムの解明が病態や治療薬の開発の一助になるのではないかと考えられる との回答を得た また 英文表記 図表の修正等についての指摘が行われた 論文内容及びその質疑により概ね妥当な回答が得られたことにより 本研究は今後の歯学の進歩 発展に寄与するところ大であり 学位授与に値すると判定した

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