Tatsuji Nishihara

Differential Inhibition of Smad6 and Smad7 on Bone Morphogenetic Protein- and Activin-mediated Growth Arrest and Apoptosis in B Cells

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-β family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.

Antimicrobial Effect of Ozonated Water on Bacteria Invading Dentinal Tubules

Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. In the present study, we examined the effect of ozonated water against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with ozonated water, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). We also compared the cytotoxicity against L-929 mouse fibroblasts between ozonated water and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with ozonated water, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl. These results suggest that ozonated water application may be useful for endodontic therapy.

Mechanical stress-mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts.

The sequence of biochemical events involved in mechanical stress‐induced signaling in osteoblastic cells remains unclear. Runx2, a transcription factor involved in the control of osteoblast differentiation, has been identified as a target of mechanical stress‐induced signaling in osteoblastic cells. In this study, uniaxial sinusoidal stretching (15% strain, 115% peak‐to‐peak, at 1/12 Hz) stimulated the differentiation of osteoblast‐like MC3T3‐E1 cells and rat primary osteoblastic cells by activating Runx2. We examined the involvement of diverse mitogen‐activated protein kinase (MAPK) pathways in the activation of Runx2 during mechanical stress. Mechanical stress increased alkaline phosphatase activity, a marker of osteoblast differentiation, increased the expression of the osteoblast‐specific extracellular matrix (ECM) protein osteocalcin, and induced Runx2 activation, along with increased osterix expression. Furthermore, activation of ERK1/2 and p38 MAPKs increased significantly. U0126, a selective inhibitor of ERK1/2, completely blocked Runx2 activation during periods of mechanical stress, but the p38 MAPK‐selective inhibitor SB203580 did not alter nuclear phosphorylation of Runx2. Small interfering RNA (siRNA) targeting Rous sarcoma kinase (RAS), an upstream regulator of both ERK1/2 and p38 MAPKs, inhibited stretch‐induced ERK1/2 activation, but not mechanically induced p38 MAPK activity. Furthermore, mechanically induced Runx2 activation was inhibited by Ras depletion, using siRNA. These findings indicate that mechanical stress regulates Runx2 activation and favors osteoblast differentiation through the activation of MAPK signal transduction pathways and Ras/Raf‐dependent ERK1/2 activation, independent of p38 MAPK signaling. J. Cell. Biochem. 101:1266–1277, 2007.

Smad7 Is an Activin-inducible Inhibitor of Activin-induced Growth Arrest and Apoptosis in Mouse B Cells

Members of the transforming growth factor-β (TGF-β) family, which includes the activins, relay signals from serine/threonine kinase receptors in membrane to nucleus via intracellular Sma- and Mad-related (Smad) proteins. Inhibitory Smad proteins were found to prevent the interaction between the serine/threonine kinase receptors and pathway-restricted Smad proteins. Smad7 was identified as a TGF-β-inducible antagonist of TGF-β signaling, and it may participate in a negative feedback loop to control TGF-β signaling. Here we demonstrate that the mRNA expression of Smad7 is induced by activin A in mouse B cell hybridoma HS-72 cells, which undergo growth arrest and apoptosis upon exposure to activin A. The ectopic expression of mouse Smad7 in HS-72 cells suppressed the activin A-induced cell cycle arrest in the G1 phase by abolishing the activin A-induced expression of p21CIP1/WAF1 and hypophosphorylation of retinoblastoma protein. Furthermore, Smad7 expression suppressed activin A-induced apoptosis in HS-72 cells. Thus, our data indicate that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis of B lineage cells.

Lipopolysaccharide Promotes the Survival of Osteoclasts Via Toll-Like Receptor 4, but Cytokine Production of Osteoclasts in Response to Lipopolysaccharide Is Different from That of Macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-α, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-κB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1β, TNF-α, and receptor activator of NF-κB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.

Local delivery system of cytotoxic agents to tumors by focused sonoporation

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as ‘sonoporation’ has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechsts staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.

Formation of Dentinal Bridge on Surface of Regenerated Dental Pulp in Dentin Defects by Controlled Release of Fibroblast Growth Factor–2 From Gelatin Hydrogels

INTRODUCTION Pulp regeneration therapy is important to overcome the limitations of conventional therapy to induce reparative dentinogenesis. In the present study, we examined the effects of controlled release of different dosages of fibroblast growth factor-2 (FGF-2) from gelatin hydrogels to regenerate the dentin-pulp complex. METHODS After the amputation of dental pulp of rat molars, gelatin hydrogels incorporating various dosages of FGF-2 were individually implanted into dentin defects above the sites of the amputated pulps. Histologic changes as well as the expression of dentin matrix protein-1 (DMP-1) and nestin in the dentin defect area above the amputated pulp were analyzed. RESULTS We found that controlled release of high doses of FGF-2 from gelatin hydrogels induced DMP-1-positive calcified particles in the proliferating pulp, whereas a moderate dose of FGF-2 induced DMP-1-positive dentinal bridge on the surface of the proliferating pulp. These findings indicate that the dosage of released FGF-2 has an influence on the structure of calcified tissue regenerated in dentin defects. In addition, pulp cells near calcified tissues regenerated in dentin defects were nestin-negative, suggesting that the calcified tissues might be osteodentin. CONCLUSIONS Our results showed that the dentin regeneration on amputated pulp, not reparative dentin formation toward amputated pulp, can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16−cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21WAF1/CIP1, as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

Specific Inhibitors of Vacuolar H+‐ATPase Trigger Apoptotic Cell Death of Osteoclasts

Osteoclasts are multinucleated bone‐resorbing cells that play a critical role in bone remodeling. Specific inhibitors of vacuolar H+‐ATPase (V‐ATPase), concanamycin A and bafilomycin A1, abolish bone resorption by osteoclasts. In this study, we examined whether these V‐ATPase inhibitors trigger apoptotic cell death in osteoclasts, using murine osteoclast‐like multinucleated cells (OCLs) formed in vitro. Acridine orange staining revealed that the treatment of OCLs with concanamycin A resulted in chromatin condensation and alterations in nuclear morphology within a few hours. The TdT‐mediated dUTP‐nick‐end labeling (TUNEL) reaction confirmed the apoptotic features of OCLs treated with concanamycin A. The accelerated apoptotic cell death induced by concanamycin A occurred in OCLs treated with interleukin‐1α or macrophage colony‐stimulating factor as well, which are known to elongate the survival time of osteoclasts. In contrast, these inhibitors did not induce cell death of osteoblastic cells isolated from mouse calvaria. These results suggest that functional impairment of V‐ATPase triggers apoptotic cell death in osteoclasts.

Role of TGF-β family in osteoclastogenesis induced by RANKL

Abstract Recent studies have revealed that both transforming growth factor-β (TGF-β) and activin A play pivotal roles in osteoclastogenesis. In this report, we show that the effect of TGF-β family members, TGF-β1 and activin A, but not BMP-2, enhance multinucleated osteoclast-like cell (OCL) formation induced by receptor activator of NF-κB ligand (RANKL) in isolated bone marrow macrophages and monocytic cell line, RAW264.7. TGF-β1 and activin A caused the growth suppression and concomitant expression of tartrate-resistant acid phosphatase (TRAP) and c-Src, without inducing syncytium formation or increasing the survival rate in RAW264.7 cells. Although TGF-β1 and activin A had no effect on NF-κB and JNK activities, these factors enhanced the expression of JunB, a component of the AP-1 transcriptional complex. These results suggest that TGF-β1 and activin A may function as commitment factors in osteoclastic differentiation, not as a crucial component for terminal differentiation to form multinucleated OCLs nor in OCL survival.