Tomoyuki Iwata

DEC1 Modulates the Circadian Phase of Clock Gene Expression

ABSTRACT DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E′ boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E′-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E′ box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1−/− mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.

Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase.

Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia‐induced changes was studied by using neutrophil‐like HL‐60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL‐60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL‐60 cells incubated with HG and S100B. HL‐60 cells cultured in HG and S100B exhibited a 1.8‐fold increase in fMLP‐induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE‐ERK1/2 pathway.

Diabetes-Induced Oxidative Stress Is Mediated by Ca2+-Independent Phospholipase A2 in Neutrophils

Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A2 (PLA2)-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA2 isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA2 isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA2 mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47phox phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA2 inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA2 assay, Western blotting, and PCR confirmed that there was increased iPLA2 activity and expression in neutrophils from people with diabetes. AA (10 μM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA2 in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling.

PDK1 Regulates Chemotaxis in Human Neutrophils

Phosphoinositide-dependent kinase (PDK1) plays a central role in signal transduction mediated by phosphatidylinositol 3-kinases (PI3K) and regulates cellular functions in neutrophils. Neutrophils from individuals diagnosed with localized aggressive periodontitis (LAP) present an in vivo phenotype with depressed chemotaxis. The aim of this study was to test the hypothesis that PDK1 regulates chemotaxis in neutrophils and is responsible for the abnormal neutrophil chemotaxis LAP. Neutrophil chemotaxis was significantly suppressed by the PDK1 inhibitor staurosporine. When cells were transfected with PDK1 siRNA, there was a significant reduction in chemotaxis, while superoxide generation was not significantly affected. In primary neutrophils from persons with LAP, PDK1 expression and activation levels were significantly reduced, and this reduction was associated with the reduced phosphorylation of Akt (Thr308) and chemotaxis. Analysis of these data demonstrates that PDK1 is essential for the chemotactic migration of neutrophils, and in the absence of PDK1, neutrophil chemotaxis is impaired.

Ceruloplasmin Induces Polymorphonuclear Leukocyte Priming in Localized Aggressive Periodontitis

BACKGROUND Polymorphonuclear leukocytes (PMNs) from subjects with localized aggressive periodontitis (LAgP) present multiple functional abnormalities associated with a phenotypically primed PMN phenotype. Local inflammation is characterized by hypoxia, which leads to increased production of superoxide (O(2)(-)) by PMNs. Ceruloplasmin (CP) is also induced by hypoxia and inflammation. The aim of this study was to investigate the role of CP in O(2)(-) generation in PMNs from healthy subjects and patients with LAgP. METHODS PMNs were isolated from healthy subjects and those with LAgP (N = 36). Superoxide was measured by cytochrome-C reduction at 550 nm. Intracellular CP expression was analyzed by real-time polymerase chain reaction and Western blotting. Serum levels of CP were measured by enzyme-linked immunosorbent assay. Intracellular iron ion conversion was spectrophotometrically determined by measuring the absorbance of sigma-phenanthroline at 510 nm. RESULTS O(2)(-) generation was significantly higher in LAgP PMNs before and after stimulation with formyl-methionyl-leucyl-phenylalanine (100 nM). CP expression in PMNs and CP levels in serum were significantly higher in subjects with LAgP compared to the PMNs and serum samples from matched healthy donors (P <0.05). LAgP PMNs also had significantly higher levels of Fe(3+) and lower levels of Fe(2+) compared to healthy PMNs (P <0.05), suggesting increased iron conversion. Exogenous CP treatment of healthy PMNs resulted in significant increases in O(2)(-) generation and iron ion conversion similar to LAgP PMNs. CONCLUSION LAgP PMNs are primed to express higher levels of CP, leading to hypoxia-mediated O(2)(-) generation in PMNs and increased oxidative stress and neutrophil-mediated tissue injury in LAgP.

The antimicrobial peptide LL37 promotes bone regeneration in a rat calvarial bone defect

LL37, an antimicrobial peptide, exhibits multiple bio-functions in various types of cells, including migration, cytokine production, apoptosis, and angiogenesis. Neovascularization and the subsequent recruitment of stem cells are essential for tissue engineering therapy, including bone regeneration. We hypothesized that LL37 can facilitate successful bone regeneration. To prove this hypothesis, the present study tested the effects of LL37 on bone formation in a rat calvarial bone defect model. Synthesized LL37 markedly induced newly formed bone. Interestingly, morphologically fibroblastic cells were observed in animals treated with LL37 on day 7, the early stage of tissue regeneration, which were positive for STRO-1, a marker of mesenchymal stem cells (MSCs), and accumulated in the bone defect area where cells positive for CD34, a marker of endothelial cells, were also localized. In addition, LL37 stimulated tube formation by endothelial cells and the proliferation of MSCs in vitro. These findings demonstrated for the first time that LL37 can regulate angiogenesis and the recruitment of stem cells to promote bone regeneration.

Clumps of a mesenchymal stromal cell/extracellular matrix complex can be a novel tissue engineering therapy for bone regeneration

BACKGROUND AIMS The transplantation of mesenchymal stromal cells (MSCs) to damaged tissue has attracted attention in scientific and medical fields as an effective regenerative therapy. Nevertheless, additional studies are required to develop an MSC transplant method for bone regeneration because the use of an artificial scaffold restricts the number of transplanted cells and their function. Furthermore, regulating the degree of cell differentiation in vitro is desirable for a more effective regenerative therapy. To address these unresolved issues, with the use of a self-produced extracellular matrix (ECM), we developed clumps of an MSC/ECM complex (C-MSCs). METHODS MSCs isolated from rat femur were cultured in growth medium supplemented with 50 μg/mL of ascorbic acid for 7 days. To obtain C-MSCs, confluent cells were scratched with the use of a micropipette tip to roll up the cellular sheet, which consisted of ECM produced by the MSCs. The biological properties of C-MSCs were assessed in vitro and their bone regenerative activity was tested by use of a rat calvarial defect model. RESULTS Immunofluorescent confocal microscopic analysis revealed that type I collagen formed C-MSCs. Osteopontin messenger RNA expression and amount of calcium content were higher in C-MSCs cultured in osteo-inductive medium than those of untreated C-MSCs. The transplantation of osteogenic-differentiated C-MSCs led to rapid bone regeneration in the rat calvarial defect model. CONCLUSIONS These results suggest that the use of C-MSCs refined by self-produced ECM, which contain no artificial scaffold and can be processed in vitro, may represent a novel tissue engineering therapy.

Irsogladine maleate regulates the inflammatory related genes in human gingival epithelial cells stimulated by Aggregatibacter actinomycetemcomitans

Periodontitis is an infectious inflammatory disease. Our previous studies have revealed that irsogladine maleate (IM) regulates intercellular junctional function and chemokine secretion in gingival epithelium, resulting in the suppression of the onset of periodontal disease in a rat model. Therefore, it is plausible that IM is a promising preventive remedy for periodontal disease. In this study, to gain a better understanding of IM in gingival epithelial cells, we employed a DNA microarray analysis. More specifically, human gingival epithelial cells (HGEC) were exposed to Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in the presence or absence of IM. Then, a human genome focus array was used. A. actinomycetemcomitans facilitated the expression of several inflammatory-related genes, including these for matrix metalloproteinase (MMP)-3, interleukin (IL)-6, and intercellular adhesion molecule-1 (ICAM-1) in HGEC, while these mRNA levels were attenuated by IM treatment. Importantly, consistent with mRNA levels, immunoblotting, immunofluorescence staining and ELISA analysis indicated that IM also abrogated the A. actinomycetemcomitans-induced increase in MMP-3, IL-6, and ICAM-1 at the protein level. In addition, inhibition of the ERK or p38 MAP kinase signaling cascade, previously reported to be disturbed by IM treatment in HGEC, clearly blocked A. actinomycetemcomitans-induced MMP-3, IL-6, or ICAM-1 protein expression. Moreover, animal study revealed that IM-pretreatment inhibited the A. actinomycetemcomitans-induced increase of ICAM-1 in gingival junctional epithelium. Taken together, these results suggested that IM can regulate inflammatory responses in HGEC by inhibiting the ERK or p38 MAP kinase signaling cascade, which may result in suppression of inflammation in gingival tissue, thereby contributing to the prevention of periodontitis.

miR-584 Expressed in Human Gingival Epithelial Cells Is Induced by Porphyromonas gingivalis Stimulation and Regulates Interleukin-8 Production via Lactoferrin Receptor

BACKGROUND MicroRNAs (miRNAs) are short, non-coding RNAs that are involved in post-transcriptional regulation of gene expression. Differential miRNA expression in innate and acquired immunity has been shown to regulate immune cell development and function. miRNA expression has been demonstrated to affect pathophysiology of inflammatory diseases, such as rheumatoid arthritis and lupus. As such, this study explores the role of miRNA in the context of pathophysiology of destructive periodontitis. Specifically, this investigation profiles the differentially expressed miRNA of Porphyromonas gingivalis (Pg)-stimulated human gingival epithelial cells (HGECs). METHODS The specific miRNAs differentially expressed in Pg-stimulated OBA-9, immortalized HGECs, were analyzed using microarray. Real-time polymerase chain reaction (PCR) and Western blotting were performed to confirm the level of miRNA expression and determine target production of miRNA in OBA-9. The production of interleukin (IL)-8 was measured to determine the bioactivity of target protein regulated by miRNA. RESULTS miR-584, which targets lactoferrin receptor (LfR), was 3.39-fold upregulated by Pg stimulation. This upregulation of miR-584 was confirmed by real-time PCR. Pg stimulation resulted in the suppression of LfR at mRNA and protein levels. The transfection of the miR inhibitor for miR-584 in OBA-9 recovered Pg-induced suppression of LfR. The addition of human lactoferrin (hLf) had a suppressive effect on IL-8 production in Pg-stimulated OBA-9. However, hLf also decreased IL-8 production strongly in Pg-stimulated OBA-9 in the presence of the miR inhibitor for miR-584. CONCLUSION These findings suggest that the upregulation of miR-584 by Pg in OBA-9 inhibits the anti-inflammatory effects of hLf via the suppression of LfR.

Introduction of a Mixture of β-Tricalcium Phosphate Into a Complex of Bone Marrow Mesenchymal Stem Cells and Type I Collagen Can Augment the Volume of Alveolar Bone Without Impairing Cementum Regeneration

BACKGROUND The purpose of this study is to evaluate whether β-tricalcium phosphate (β-TCP) could be a promising modality to help augment alveolar bone in periodontal tissue regeneration by bone marrow mesenchymal stem cells (BMMSCs). METHODS Expanded BMMSCs and atelocollagen (Col) were mixed together (MSC/Col). A combination of β-TCP with MSC/Col was also prepared (MSC/Col/TCP). MSC/Col/TCP or MSC/Col was transplanted into experimental periodontal Class III furcation defects that had been exposed to inflammation in beagle dogs. Periodontal tissue regeneration was evaluated by histologic and morphometric analyses at 4 and 8 weeks after transplantation. RESULTS MSC/Col and MSC/Col/TCP enhanced periodontal tissue regeneration compared to Col and TCP/Col according to hematoxylin and eosin staining. The percentage of new cementum length in the MSC/Col/TCP group was not significantly different from that in the MSC/Col group at 4 and 8 weeks. On the other hand, the percentage of new bone area in the MSC/Col/TCP group was much higher than that in the MSC/TCP group at 4 weeks. However, at 8 weeks, no significant difference in new bone area was found between the two groups. In the MSC/Col/TCP group, β-TCP was surrounded by newly formed bone. Multinucleated cells, which were positive for osteopontin and tartrate-resistant acid phosphatase, were present in the interconnected macropores of β-TCP. CONCLUSION These findings suggest that β-TCP is applicable as a scaffold for BMMSCs transplantation and helps augment alveolar bone without impairing regeneration of cementum.