Yorimasa Ogata

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Effects of reactive oxygen species (ROS) on antioxidant system and osteoblastic differentiation in MC3T3-E1 cells

Oxidative stress regulates cellular functions in multiple pathological conditions, including bone formation by osteoblastic cells. However, little is known about the cellular mechanisms responsible for the effects of oxidative stress on osteoblast functions in senescence. To clarify the inhibitory effects of oxidative stress on osteoblastic mineralization, we examined the relationship between the antioxidant system and bone formation in MC3T3‐E1 cells. After a single exposure to H2O2 within range of a non‐toxic concentration for cells, the mineralization level was diminished half. Under the same conditions, gene expression of the transcription factor Nrf2, which regulates antioxidant enzymes, was up‐regulated. In addition, gene expression for the osteogenic markers Runx2, ALP, and BSP was lower than that in non‐treated cells, whereas expression of the osteocalcin gene was up‐regulated following H2O2 exposure. These results suggest that reduced mineralization by MC3T3‐E1 cells after H2O2 exposure is the result of an up‐regulated antioxidant system and altered osteogenic gene expression. IUBMB Life, 59: 27‐33, 2007

Transforming growth factor‐β1 regulation of bone sialoprotein gene transcription: Identification of a TGF‐β activation element in the rat BSP gene promoter

Transforming growth factor‐β (TGF‐β) increases steady‐state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF‐β1 regulation of bone proteins, we have analyzed the effects of the TGF‐β1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF‐β1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells ∼8‐fold; the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF‐β1, and nuclear “run‐on” transcription analyses revealed only a ∼2‐fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post‐transcriptional mechanism. Moreover, the effects of TGF‐β1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 μg/ml). To identify the site of transcriptional regulation by TGF‐β1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt −801 to −426 of the promoter sequence were found to enhance transcriptional activity ∼1.8‐fold in cells treated with TGF‐β1. Within this sequence, ∼500 nt upstream of the transcription start site, a putative TGF‐β activation element (TAE) was identified that contained the 5′‐portion of the nuclearfactor‐1 (NF‐1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein‐2 (AP‐2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF‐β1 stimulated cells in gel mobility shift assays and from the attenuation of TGF‐β1‐induced luciferase activity when cells were co‐transfected with a double‐stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF‐1 nuclear protein. These studies have therefore identified a TGF‐β activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF‐β1 on BSP gene transcription. J. Cell. Biochem. 65:501–512.

Bone sialoprotein and its transcriptional regulatory mechanism.

BACKGROUND AND OBJECTIVE Bone sialoprotein is a mineralized tissue-specific noncollagenous protein that is glycosylated, phosphorylated and sulfated. The temporo-spatial deposition of bone sialoprotein into the extracellular matrix of bone, and the ability of bone sialoprotein to nucleate hydroxyapatite crystal formation, indicates a potential role for bone sialoprotein in the initial mineralization of bone, dentin and cementum. Bone sialoprotein is also expressed in breast, lung, thyroid and prostate cancers. MATERIAL AND METHODS We used osteoblast-like cells (rat osteosarcoma cell lines ROS17/2.8 and UMR106, rat stromal bone marrow RBMC-D8 cells and human osteosarcoma Saos2 cells), and breast and prostate cancer cells to investigate the transcriptional regulation of bone sialoprotein. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene, we conducted northern hybridization, transient transfection analyses with chimeric constructs of the bone sialoprotein gene promoter linked to a luciferase reporter gene and gel mobility shift assays. RESULTS Bone sialoprotein transcription is regulated by hormones, growth factors and cytokines through tyrosine kinase, mitogen-activated protein kinase and cAMP-dependent pathways. Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. Expression of bone sialoprotein by cancer cells could play a major role in the mineral deposition and in preferred bone homing of breast cancer cells. CONCLUSION Bone sialoprotein protects cells from complement-mediated cellular lysis, activates matrix metalloproteinase 2 and has an angiogenic capacity. Therefore, regulation of the bone sialoprotein gene is potentially important in the differentiation of osteoblasts, bone matrix mineralization and tumor metastasis. This review highlights the function and transcriptional regulation of bone sialoprotein.

Activation of NFκB is necessary for IL-1β-induced cyclooxygenase-2 (COX-2) expression in human gingival fibroblasts

The immediate-early cyclooxygenase-2 (COX-2) gene encodes an inducible prostaglandin synthase enzyme which is implicated in inflammatory and proliferative diseases. COX-2 is highly induced during cell activation by various factors, including mitogens, hormones and cytokines. Since pro-inflammatory cytokine IL-1β has been shown to induce prostaglandin E2 (PGE2) release in human gingival fibroblasts (HGF), here we analyzed the effect of IL-1β on the expression of COX-2 and the activation of NFκB in HGF. Northern hybridization analysis revealed that IL-1β (200 pg/ml) increased the expression of COX-2 mRNA in HGF. The effect of IL-1β was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1β-induced PGE2 release was blocked by the tyrosine kinase inhibitor and increased by the tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human COX-2 promoter (nt -1432 ~ +59) ligated to a luciferase reporter gene indicated that IL-1β stimulated the transcriptional activity ~ 1.5-fold. Gel mobility shift assays with a radiolabelled COX-2-NFκB oligonucleotide (nts -223 to -214) revealed an increase in the binding of nuclear proteins from IL-1β-stimulated HGF. This increase of DNA-protein complex formation induced by IL-1β was blocked by herbimycin A and another tyrosine kinase inhibitor, genistein. These results suggest that NFκB is an important transcription factor for IL-1β-induced COX-2 gene expression, and is involved in inducing COX-2 gene transcription through tyrosine phosphorylation in HGF.

AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter.

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.

Molecular Cloning of Bovine Amelogenin cDna

Molecular cloning of a bovine amelogenin cDNA was accomplished by construction of a cDNA expression library (λgt11 cDNA library) from the bovine ameloblast mRNA and then screening of the library with antibodies to bovine amelogenins. The complete primary structure of an amelogenin was deduced from cloned cDNA. One of the cDNA clones isolated from a bovine ameloblast phage λgt11 library had an 864-base-pair-long insert that encoded a protein with 216 amino acid residues. This cDNA clone appears to represent the complete coding region of amelogenin mRNA, including a putative AUG initiation codon and a signal peptide sequence. The predicted bovine amelogenin sequence has 87% amino acid homology with murine amelogenin.

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

Insulin‐like growth factor‐I (IGF‐I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF‐I regulation of osteogenesis, we analyzed the effects of IGF‐I on the expression of BSP in osteoblast‐like Saos2 and in rat stromal bone marrow (RBMC‐D8) cells. IGF‐I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC‐D8 cells, IGF‐I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF‐I was observed at 12 h in pLUC3 construct that included the promoter sequence from −116 to +60. Effect of IGF‐I was abrogated by 2‐bp mutations in either the FGF2 response element (FRE) or homeodomain protein‐binding site (HOX). Gel shift analyses showed that IGF‐I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX‐protein complex was supershifted by Smad1 antibody, while the FRE‐protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE‐ and HOX‐protein complexes. The IGF‐I effects on the formation of FRE‐protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3‐kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF‐I effects on HOX‐protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF‐I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene. J. Cell. Physiol. 208: 326–335, 2006.

A Single Nucleotide Polymorphism in 3′-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation

Background: Genetic variation of SNP rs11536889 in 3′-UTR of TLR4 is implicated in certain diseases, including periodontitis, gastric atrophy, and prostate cancer. Results: The G allele of rs11536889 inhibited translation, but not transcription, of TLR4. Conclusion: Genetic variation of rs11536889 regulates TLR4 expression. Significance: Polymorphism in rs11536889 could be an excellent genetic marker for the diseases caused by TLR4-ligands. We have previously shown that a single nucleotide polymorphism rs11536889 in the 3′-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK4 (Pam3CSK4), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3′-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.

Purification, characterization, and biosynthesis of bovine enamelins

SummaryEnamelins were extracted from developing bovine enamel with 0.5 M EDTA, 4 M guanidine HCl, and purified by DEAE-Sephacel, Sephacryl S-200, and high-performance gel filtration chromatography. Four distinct enamelins having molecular weights of 70, 45, 30, and 28 K daltons were isolated. Their amino acid compositions were found to be rich in Pro, Glu, Gly, and Asp. Low molecular weight enamelins (45, 30, and 28 K) were more abundant in Pro, Gly, and Phen. Two-dimensional electrophoretic pattern of enamelins revealed several spots that immunoreacted to monoclonal anti-enamelin antibody raised in mice. Enamelins were found to be comparised of heterogeneous proteins as well as amelogenins. Biosynthesis of enamelins was investigated by incubating the bovine ameloblast cell layer, and several radioactive enamelins were identified by the use of two-dimensional electrophoresis. The data in this study suggest that enamelins were synthesized by the ameloblasts.