Sousuke Kanaya

Wnt signaling inhibits cementoblast differentiation and promotes proliferation

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells.

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca(2+)Ca(2+)(o) has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca(2+)(o) signaling in odontogenesis remains unclear. We found that elevated Ca(2+)(o) increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca(2+) increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca(2+) channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca(2+), suggesting that the Ca(2+) influx from Ca(2+) channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca(2+)-sensing receptors (CaSR) and only respond slightly to other cations such as Sr(2+) and spermine, suggesting that dental pulp cells respond to Ca(2+)(o) to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca(2+)(o) among cations.

Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype

Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL‐4 and GM‐CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real‐time quantitative RT‐PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up‐regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA‐DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS‐stimulated DC was lower than on Ec LPS‐stimulated DC. With regard to IL‐6, IL‐8, IL‐10, IL‐12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Elevated extracellular calcium increases fibroblast growth factor-2 gene and protein expression levels via a cAMP/PKA dependent pathway in cementoblasts

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Wnt3a signaling induces murine dental follicle cells to differentiate into cementoblastic/osteoblastic cells via an osterix-dependent pathway

BACKGROUND AND OBJECTIVE Dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwigs epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial-mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. MATERIAL AND METHODS The expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. RESULTS We identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix-small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. CONCLUSION These findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/β-catenin signaling pathway.

Nanohydroxyapatite increases BMP-2 expression via a p38 MAP kinase dependent pathway in periodontal ligament cells

OBJECTIVE Bone morphogenetic protein (BMP)-2 promotes the osteoblastic differentiation of human periodontal ligament (PDL) cells, which play a pivotal role in periodontal regeneration. Recently, nano-sized hydroxyapatite (nano-HA) has been highlighted due to its advantageous features over micro-sized materials. DESIGN AND RESULTS We investigated the effect of nano-HA on BMP-2 expression in human PDL cells. Real time PCR analysis revealed that the expression of BMP-2 increased upon stimulation with nano-HA in dose- and time-dependent manners. An immunofluorescence assay demonstrated the synthesis of BMP-2 proteins. Concentrations of Ca(2+) as well as phosphate (Pi) in culture supernatants were unchanged, suggesting that nano-HA functioned as a nanoparticle rather than as a possible source for releasing Ca(2+) and/or Pi extracellularly, which were shown to also enhance the expression of BMP-2. Nano-HA-induced BMP-2 expression was dependent on the p38 MAP kinase pathway because increases in BMP-2 expression were inhibited by treatment with SB203580, a p38 inhibitor, and phosphorylation of p38 was detected by Western blotting. CONCLUSIONS This novel mechanism of nano-HA will be important for the rational design of future periodontal regeneration.

Cleavage of PDGF Receptor on Periodontal Ligament Cells by Elastase

Human leukocyte elastase, a neutrophil serine protease, is considered to be a potential immunoregulatory protease. Since the PDGF receptor (PDGFR) on periodontal ligament (PDL) cells is a crucial element for various functions, such as wound healing in periodontal tissue, we investigated the effect of elastase on the expression of PDGFR on PDL cells by flow cytometry and Western blotting. We found that PDGFR-α disappeared with an increasing dose of elastase, and PDGFR-β was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation

BACKGROUND AND OBJECTIVE While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-kappaB (NF-kappaB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins. MATERIAL AND METHODS Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-kappaB were measured using enzyme-linked immunosorbent assay. RESULTS The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-kappaB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-kappaB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts. CONCLUSION These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.

Calcium-mediated increased expression of fibroblast growth factor-2 acts through NF-κB and PGE2/EP4 receptor signaling pathways in cementoblasts

We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca2+ and that elevated extracellular Ca2+ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10 mM CaCl2 induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl2-induced increase in Fgf-2 gene expression, indicating that PGE2 synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE2 receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl2-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl2 had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl2 also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl2 is indispensable for FGF-2 induction. Furthermore, CaCl2-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl2 and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration.