Shinya Shirasu

Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation

Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases. Here the cytotoxic effects of a purified preparation of a P. gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro. The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen. Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases.

TNF-α enhances MMP-2 production in deciduous dental pulp fibroblasts

Abstract Inflammation in dental pulp tissue is associated with tissue degradation, and matrix metalloproteinases (MMPs) are believed to participate in this destruction. Elevated levels of some MMPs have been reported in inflamed pulp and periapical lesions. Moreover, in inflamed pulp, many kinds of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, are released from inflammatory cells like macrophages, lymphocytes and neutrophils. In the present study, we examined whether TNF-α affected the production of MMP-2 in deciduous dental pulp fibroblasts and its signaling pathways utilizing gelatin zymography and western blotting analysis. TNF-α increased the expression of MMP-2 in a dose-dependent manner in deciduous dental pulp fibroblasts. LY294002 and Wortmannin, which are PI3-K (phosphoinositide 3-kinase) inhibitors, inhibited the MMP-2 production induced by TNF-α in deciduous dental pulp fibroblasts. Moreover, in deciduous dental pulp fibroblasts cultured with TNF-α, AKT (protein kinase B) was phosphorylated in a time-dependent manner with the maximum phosphorylation at 30 min, and LY294002 and Wortmannin abolished this phosphorylation of AKT in TNF-α-stimulated deciduous dental pulp fibroblasts. These results suggest that TNF-α may enhance pulp tissue destruction during pulp inflammation in part by regulating MMP-2, and that the AKT pathway is involved in MMP-2 production in deciduous dental pulp fibroblasts.

DNA microarray analysis of dental pulp fibroblasts exfoliated from deciduous teeth

Abstract Dental pulp plays an important role in tooth vitality. Previous studies have indicated that stem cells can be isolated from dental pulp, and dental pulp exfoliated from deciduous teeth has become a useful alternative for dental tissue engineering because of its higher proliferation rate. In the present study, we analyzed the differences in gene expressions between human dental pulps exfoliated from deciduous and permanent teeth by DNA microarray assays. A scatter plot of mRNA levels based on fluorescent signals in human dental pulps from deciduous and permanent teeth indicated a dispersed distribution pattern. In a scatter plot of the genes, 2,573 genes were expressed at >2-fold higher levels in dental pulp from deciduous teeth, compared with permanent teeth. To confirm the microarray results, insulin-like growth factorbinding protein 5 (IGFBP5) and vascular endothelial growth factor A (VEGFA) showing higher mRNA expression levels were selected and analyzed for their mRNA levels by RT-PCR. Both the IGFBP5 and VEGFA mRNA levels were upregulated by about 3-fold in dental pulp from deciduous teeth compared with permanent teeth. Thus, the RT-PCR results for these genes were consistent with the microarray data. The dental pulps in deciduous and permanent teeth differ significantly with regard to their developmental processes, tissue structures and functions. The present findings using DNA microarray analyses to detect differences in the gene expressions of deciduous and permanent teeth may be useful for dental pulp tissue engineering.

Comparative Studies of the Effects of Adenosine and ATP on All-trans Retinoic Acid, 1,25-dihydroxy-vitamin D3 and Phorbol 12-myristate 13-acetate-induced Differentiation in U937 Human Leukemia Cells

Abstract Adenosine and ATP have been implicated in the regulation of inflammatory responses. Furthermore, adenosine and ATP affect cell growth, cell differentiation, and apoptosis. The aim of this study was to evaluate the differentiation-inducing effects of adenosine and ATP on human monocytic leukemia U937 cells. Adenosine at 10 -3 M markedly induced differentiation in leukemia U937 cells as assessed by evaluating the expression of CD11b. Similarly, ATP at 10 -4 and 10 -3 M significantly induced differentiation in U937 cells. Simultaneously, adenosine at 10 -3 M and ATP at 10 -4 and 10 -3 M significantly inhibited the cell number in U937 cells. Next, we evaluated the effects of adenosine and ATP on U937 cells differentiated by differentiation inducers all-trans retinoic acid (ATRA), 1,25-dihydroxy-vitamin D 3 , (VD 3 ) and phorbol. 12-myristate 13-acetate (PMA). Interestingly, adenosine at 10 -4 and 10 -3 M significantly potentiated ATRAinduced CD11b expression. However, ATRA failed to affect adenosine at 10 -3 M-induced CD11b expression. Similarly, ATP at 10 -4 and 10 -3 M significantly potentiated ATRA-induced CD11b expression. In addition, ATRA potentiated ATP-induced CD11b expression. In contrast, VD 3 and PMA further increased the expression of CD11b in the presence of adenosine or ATP, respectively. These results suggest that adenosine and ATP may induce differentiation in human leukemia U937 cells, and may further increase ATRAinduced differentiation in U937 cells.

15-Deoxy-Δ12,14-prostaglandin J2 and Its Precursors Target Phosphoinositide 3-kinase and p38 MAPK to Accelerate Proliferation in the Human T Cell Leukemia Cell Line MOLT-4F

Abstract 15-Deoxy-Δ 12,14 -prostaglandin J 2 (15dPGJ 2 ), which is a ligand for peroxisome proliferatoractivated receptor γ (PPARγ), induced apoptosis of several human tumors including gastric, lung, colon, prostate and breast. However, the role of PPARγ signals in other types of cancer cells such as leukemia, except solid cancer cells, is still unclear. The aim of this study was to evaluate the ability of 15dPGJ 2 on the proliferation of the human T cell leukemia cell line MOLT-4F. 15dPGJ 2 at 5 μM stimulated proliferation in MOLT-4F at 1 to 3 days after incubation. In contrast, 15dPGJ 2 at concentrations of above 10 μM inhibited proliferation. PGD 2 , PGJ 2 and Δ 12 -PGJ 2 (APGJ 2 ), which are precursors of 15dPGJ 2 , had similarly proliferative effects, whereas they showed anti-proliferatioe effects at high concentrations. Both SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, and LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, prevented proliferation accelerated by 15dPGJ 2 and its three precursors in MOLT-4F. In contrast, PD98059, an extracellular signal-related kinase 1/2 inhibitor, did not affect proliferation accelerated by 15dPGJ 2 and its three precursors. Immunoblotting analysis revealed that PGD 2 at 5 μM, PGJ 2 at 5 μM, APGJ 2 at 1 μM and 15dPGJ 2 at 5 μM potentiated the expression of cyclin A, without affecting the expression of Cdk inhibitors including p18, p21, and p27 in MOLT-4F. These results suggest that PGD 2 , PGJ 2 , ΔPGJ2 and 15dPGJ 2 may, through the activation of p38 MAPK and/or PI3K, potentiate the expression of cyclin A, leading to acceleration of proliferation in MOLT-4F.