Katsuhiro Takeda

Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

Brain-derived neurotrophic factor (BDNF), recognized as essential in the developing nervous system, is involved in differentiation and proliferation in non-neuronal cells, such as endothelial cells, osteoblasts, and periodontal ligament cells. We have focused on the application of BDNF to the regeneration of periodontal tissue and indicated that BDNF promotes the regeneration of experimentally created periodontal defects. Cementoblasts form cementum, mineralized tissue, which is key to establishing a functional periodontium. The application of BDNF to the regeneration of periodontal tissue requires elucidation of the mechanism by which BDNF regulates the functions of cementoblasts. In this study, we examined how BDNF regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase (ALP), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2)) in cultures of immortalized human cementoblast-like (HCEM) cells. BDNF elevated the mRNA levels of ALP, OPN, and BMP-2 in HCEM cells. Small interfering RNA (siRNA) for TRKB, a high affinity receptor of BDNF, siRNA for ELK-1, which is a downstream target of ERK1/2, and PD98059, an ERK inhibitor, obviated the increase in the mRNA levels. BDNF increased the levels of phosphorylated ERK1/2 and Elk-1, and the blocking of BDNF signaling by treatment with siRNA for TRKB and PD98059 suppressed the phosphorylation of ERK1/2 and Elk-1. Furthermore, BDNF increased the levels of phosphorylated c-Raf, which activates the ERK signaling pathway. These findings provide the first evidence that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the BDNF-induced mRNA expression of ALP, OPN, and BMP-2 in HCEM cells.

Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin αVβ3-FAK cascade.

Brain‐derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal‐regulated kinase (ERK), integrin αVβ3, and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin αVβ3 and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti‐integrin αVβ3 antibody suppressed the BDNF‐induced migration. BDNF increased the levels of integrin αVβ3 and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin αVβ3 and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin αVβ3/FAK, and this may help to enhance the regeneration of periodontal tissue. J. Cell. Physiol. 227: 2123–2129, 2012.

Macrophage inflammatory protein-3α and β-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells

Abstract Macrophage inflammatory protein (MIP)-3α and β-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3α and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3α and BD-2 can bind. Real-time PCR showed that MIP-3α and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3α. MIP-3α and BD-2 increased OPN mRNA levels very slightly. MIP-3α and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3α was lower than that of BD-2. These findings suggest the MIP-3α and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.

Brain-derived neurotrophic factor protects cementoblasts from serum starvation-induced cell death.

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNFs actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009.

The Antimicrobial Peptide LL37 Induces the Migration of Human Pulp Cells: A Possible Adjunct for Regenerative Endodontics

INTRODUCTION The antimicrobial peptide LL37 has multiple functions, such as the induction of angiogenesis and migration. Pulp cell migration is a key phenomenon in the early stage of pulp-dentin complex regeneration. In this study, we examined the effect of LL37 on the migration of human pulp (HP) cells. METHODS HP cells at the sixth passage were exposed to LL37. The migration of HP cells was assessed by a wound-healing assay. The phosphorylation of epidermal growth factor receptor (EGFR) and c-Jun N-terminal kinase (JNK) was analyzed by immunoblotting. RESULTS LL37 as well as heparin binding (HB)-EGF, which is an agonist of EGFR, induced HP cell migration. LL37 increased the level of phosphorylated EGFR. An anti-EGFR antibody, an EGFR tyrosine kinase inhibitor, and a JNK inhibitor abolished the migration induced by both LL37 and HB-EGF. Furthermore, the two peptides increased the levels of phosphorylated JNK. CONCLUSIONS LL37 activates EGFR and JNK to induce HP cell migration, and it may contribute to enhancing the regeneration of pulp-dentin complexes.

Irsogladine maleate regulates neutrophil migration and E-cadherin expression in gingival epithelium stimulated by Aggregatibacter actinomycetemcomitans

Irsogladine maleate (IM) counters Aggregatibacter actinomycetemcomitans-induced reduction of the gap junction intercellular communication and the expression of zonula occludens-1, which is a major tight junction structured protein, in cultured human gingival epithelial cells (HGEC). In addition, IM obviates the A. actinomycetemcomitans-induced increase in interleukin (IL)-8 levels in HGEC. Thus, by regulating the intercellular junctional complex and chemokine secretion in HGEC, IM may be useful to prevent periodontal disease. To clarify the effects and regulatory mechanism of IM in vivo and in vitro, we examined the expression of E-cadherin and neutrophil chemotaxis induced by A. actinomycetemcomitans under IM pretreatment. Immunohistochemical studies revealed that A. actinomycetemcomitans application to the gingival sulcus decreased the number of cells positive for E-cadherin and increased those positive for cytokine-induced neutrophil chemoattractant-2alpha (CINC-2alpha) in rat gingival epithelium. However, in IM-pretreated rats, A. actinomycetemcomitans application had little effect on CINC-2alpha and E-cadherin in gingival epithelium. In cultured HGEC, real-time PCR and Western blotting showed that IM and the ERK inhibitor PD98059 abolished the A. actinomycetemcomitans-induced increase in CXCL-1 and IL-8 in HGEC. On the other hand, IM, PD98059, and the p38 MAP kinase inhibitor SB203580 recovered the decrease in E-cadherin expression. In addition, conditioned medium from A. actinomycetemcomitans-stimulated HGEC enhanced human neutrophil chemotaxis, compared to that from un-stimulated HGEC or that from A. actinomycetemcomitans-stimulated HGEC under IM pretreatment. Furthermore, IM down-regulated the p38 MAP kinase and ERK phosphorylations induced by A. actinomycetemcomitans. In conclusion, IM may control A. actinomycetemcomitans-induced gingival inflammation by regulating neutrophil migration and E-cadherin expression in gingival epithelium.

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells.

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue‐related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)‐3 and NT‐4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein‐2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard‐tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard‐tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.

BDNF protects human vascular endothelial cells from TNFα-induced apoptosis

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation that directs the quality of tissue repair. In this study, we investigated the anti-apoptotic effect of BDNF against the toxicity of tumor necrosis factor α (TNFα), which is known for its pro-apoptotic action in human microvascular endothelial cells (HMVECs). We demonstrate that BDNF attenuates TNFα-increased Annexin V-positive cells, lactic dehydrogenase (LDH) release, and intercellular adhesion molecule 1 (ICAM-1) mRNA and cleaved caspase-3 expression. In addition, biochemical analyses indicate that TNFα increases phosphatase and tensin homolog (PTEN) expression; however, it decreases phosphorylated PTEN. BDNF did not affect PTEN expression, but it did increase the phosphorylation of PTEN. BDNF-induced Akt phosphorylation was inhibited by TNFα. Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay showed that the PTEN inhibitor bpV(pic) rescues HMVECs from TNFα-induced apoptosis. In conclusion, BDNF protects HMVECs from toxicity of TNFα through the regulation of the PTEN/Akt pathway.

The expressions of claudin-1 and E-cadherin in junctional epithelium

BACKGROUND AND OBJECTIVE The epithelium provides an important barrier against microbial invasion. Tight junction structural proteins called claudins are known to contribute to the epithelial cell barrier. Junctional epithelium is located at a strategically important interface between gingival sulcus and is interconnected by desmosomes and gap junctions, but not by tight junctions. Although claudins are tight junction-associated proteins, they are also expressed in the epithelium despite its lack of tight junctions in invertebrates. Therefore, claudins may play an important role in junctional epithelium without tight junctions. E-cadherin is a key molecule in the formation of adherence junctions and desmosomes. In the present study, we aimed to investigate the expressions of claudin-1,claudin-3, claudin-7 and E-cadherin in the junctional epithelium of Fischer 344 rats. MATERIAL AND METHODS Gingival tissues from Fischer 344 rats were analyzed by immunohistochemical staining for claudin-1, claudin-3, claudin-7, and E-cadherin. RESULTS Intense staining for claudin-1 and E-cadherin were observed in the junctional epithelium. In contrast to claudin-1, claudin-3 was mainly expressed in oral gingival epithelium and claudin-7 could not be detected on immunohistochemical analysis of the rat gingiva. CONCLUSION These data suggest that claudin-1 and E-cadherin exist in the junctional epithelium and may play an important role in epithelial barrier function.

Antimicrobial peptide LL37 promotes vascular endothelial growth factor-A expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE LL37, originally found in the innate immune system, is a robust antimicrobial peptide. LL37 exhibits multiple bio-functions in various cell types, such as migration, cytokine production, apoptosis, and angiogenesis besides its antimicrobial activity Periodontal ligament (PL) cells play a pivotal role in periodontal tissue regeneration. Based on these findings, we hypothesized that LL37 can regulate PL cell function to promote regeneration of periodontal tissue. To prove this hypothesis, we investigated the effect of LL37 on the potent angiogenic inducer vascular endothelial growth factor (VEGF) expression in cultures of human PL (HPL) cells because neovascularization is indispensable for the progress of tissue regeneration. Moreover, we investigated the signaling cascade associated with LL37-induced VEGF expression. MATERIAL AND METHOD HPL cells were treated with synthesized LL37 in the presence or absence of PD98059, a MEK-ERK inhibitor, or PDTC, an NF-κB inhibitor. VEGF expression levels were assessed by real-time polymerase chain reaction analysis and an enzyme-linked immunoassay. Phosphorylation levels of ERK1/2 or NF-κB p65 were determined by Western blotting. RESULTS LL37 upregulated VEGF-A expression at the mRNA and protein levels in HPL cells, while VEGF-B mRNA expression was not affected. Both ERK and NF-κB inhibitors clearly abrogated the increase in VEGF-A levels induced by LL37 in HPL cells. Importantly, LL37 increased phosphorylated levels of ERK1/2 and NF-κB p65 in HPL cells. CONCLUSION LL37 induces VEGF-A production in HPL cells via ERK and NF-κB signaling cascades, which may result in angiogenesis, thereby contributing to periodontal regeneration.