High Extracellular Calcium Increased Expression of Ank, PC-1 andOsteopontin in Mouse Calvarial Cells

In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium (Ca 2+ e) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and β-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High Ca 2+ e (10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high Ca 2+ e-induced expression of Ank, PC-1 and osteopontin. When high Ca 2+ e (5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high Ca 2+ e. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high Ca 2+ e-treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high Ca 2+ e stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high Ca 2+ e- induced expression of Ank, PC-1 and osteopontin

Identification of genes modulated by high extracellular calcium in coculuture of mouse osteoblasts and bone marrow cells by oligo chip assay

Calcium concentration in the bone resorption lacunae is high and is in the mM concentration range. Both osteoblast and osteoclast have calcium sensing receptor in the cell surface, suggesting the regulatory role of high extracellular calcium in bone metabolism. In vitro, high extracellular calcium stimulated osteoclastogenesis in coculture of mouse osteoblasts and bone marrow cells. Therefore we examined the genes that were commonly regulated by both high extracellular calcium and 1,25(OH)2vitaminD3 (VD3) by using mouse oligo 11 K gene chip. In the presence of 10 mM [Ca2+]e or 10 nM VD3, mouse calvarial osteoblasts and bone marrow cells were co-cultured for 4 days when tartrate resistant acid phosphatase-positive multinucleated cells start to appear. Of 11,000 genes examined, the genes commonly regulated both by high extracellular calcium and by VD3 were as follows; 1) the expression of genes which were osteoclast differentiation markers or were associated with osteoclastogenesis were up-regulated both by high extracellular calcium and by VD3; trap, mmp9, car2, ctsk, ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and chemokine receptor genes such as sdf1, scya2, scyb5, scya6, scya8, scya9, and ccr1 were up-regulated both by high extracellular calcium and by VD3, 3) the genes such as mmp1b, mmp3 and c3 which possibly stimulate bone resorption by osteoclast, were commonly up-regulated, 4) the gene such as c1q and msr2 which were related with macrophage function, were commonly down-regulated, 5) the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were commonly down-regulated; slc8a1, admr, plod2, lox, fosb, 6) the genes which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were commonly up-regulated; s100a4, npr3, mme, 7) the genes such as calponin 1 and tgfbi which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were up-regulated by high extracellular calcium but were down-regulated by VD3. These results suggest that in coculture condition, both high extracellular calcium and VD3 commonly induce osteoclastogenesis but suppress osteoblast differentiation/mineralization by regulating the expression of related genes.본 연구는 보건복지부 보건의료기술진흥사업의 지원에 의하여 이루어진 것임(03-PJ1-PG3-20500-0013)

The Expression of Matrix Metalloprotease 20 is Stimulated by Wild Type but not by 4 bp- or 2 bp- Deletion Mutant DLX3

Mutations in DLX3 are associated with both autosomal dominant hypoplastic hypomaturation amelogenesis imperfecta (ADHHAI) and tricho-dento-osseous (TDO) syndrome. ADHHAI is caused by a c.561_562delCT (2bpdel DLX3) mutation whereas TDO syndrome is associated with a c.571_574delGGGG (4bp-del DLX3) mutation. However, although the causal relationships between DLX3 and an enamel phenotype have been established, the pathophysiological role of DLX3 mutations in enamel development has not yet been clarified. In our current study, we prepared expression vectors for wild type and deletion mutant DLX3 products (4bp-del DLX3, 2bp-del DLX3) and examined the effects of their overexpression on the expression of the enamel matrix proteins and proteases. Wild type DLX3 enhanced the expression of matrix metalloprotease 20 (MMP20) mRNA and protein in murine ameloblast-like cells. However, neither a 4bp-del nor 2bpdel DLX3 increased MMP20 expression. Wild type DLX3, but not the above DLX3 mutants, also increased the activity of reporters containing 1.5 kb or 0.5 kb of the MMP20 promoter. An examination of protein stability showed that the half-life of wild type DLX3 protein was less than 12 h whilst that of both deletion mutants was longer than 24 h. Endogenous Dlx3 was also found to be continuously expressed during ameloblast differentiation. Since inactivating mutations in the gene encoding MMP20 are associated with amelogenesis imperfecta, the inability of 4bp-del or 2bp-del DLX3 to induce MMP20 expression suggests a possible involvement of such mutations in the enamel phenotype associated with TDO syndrome or ADHHAI

Tricho-dento-osseous Syndrome Mutant Dlx3 Shows Lower Transactivation Potential but Has Longer Half-life than Wild-type Dlx3

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3 (wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation

Occult bacteremia in children with simple febrile seizure in the post-pneumococcal conjugate vaccine era

Purpose The authors aimed to investigate the utility of blood culture (BC) for children with simple febrile seizure (SFS) in the emergency department (ED) in the post-10/13-valent pneumococcal conjugate vaccine (PCV) era. Methods This study was performed at the ED of a tertiary care university-affiliated women and children’s hospital, and involved 3,237 previously healthy children aged 6-60 months who visited the ED with SFS from January 2013 through December 2017. The SFS was defined according to the International Classification of Diseases, 11th Revision codes related to seizure. The children were divided into 2 groups according to the vaccination rates of the period of their visit: the 70-PCV (70%, 2013-2014) and 97-PCV (97%, 2015-2017) groups. The primary outcome was the yield, defined as a true positivity of BC. In addition, we collected information on baseline characteristics, ED length of stay, inflammatory biomarkers, and ED outcomes. Results Of the 1,578 children with SFS who underwent BC, 1,357 belonged to the 97-PCV group. The median age of the study population was 22 months (interquartile range, 16.0-30.0), and 935 children (59.3%) were boys. Of the 41 children (2.6%) with positive BC results, 3 had the yield (0.2%): Staphylococcus aureus in 2 children and Streptococcus pneumoniae in the other. All 3 children belonged to the 97-PCV group. There were 38 contaminated BCs (2.4%; 95% confidence interval, 1.6%-3.2%). The 97-PCV group showed a shorter median ED length of stay (166.0 minutes [108.0-279.5] vs. 143.0 [109.5-209.5]; P = 0.010) and a lower rate of hospitalization (39.4% vs. 12.8%; P < 0.001). No differences between the 2 groups were found in the baseline characteristics and biomarkers. Conclusion This study suggests a low utility of BC in previously healthy children with SFS in emergency settings in the post-10/13-valent PCV era

Effect of Sodium Nitroprusside on the Activation of Mouse Osteoblastic Cells

Nitric oxide （NO）, which is produced from arginine by a nitric oxide synthase, is a short-lived free radical that plays crucial role in a variety of tissues. Recently, it has been reported that NO is produced by osteoblast stimulated by lipopolysaccharide and several cytokines. Although NO appears to inhibit osteoclastic differentiation and activation, little is known about its possible role in osteoblastic function. Therefore, the effect of sodium nitroprusside （SNP）, as a donor of nitric oxide, on osteoblastic activation in terms of alkaline phosphatase （ALP） activity and calcified nodule formation in the osteoblastic cells were studied. SNP increased not only the ALP activity but also the calcified nodule formation. ALP activity was enhanced significantly by the addition of SNP （30-300 μM）with 14-31％ magnitude when compared with control. SNP also stimulated calcium phosphate - containing calcified matrix formation. The number of calcified nodules was increased significantly by continuous treatment of cultures with 30 μM SNP for 21 days. In addition to the recently reported inihbition of osteoclast function by NO, these results raise the possibility that NO could promote osteoblastic bone formation by stimulating osteoblastic function and mineralization as well

Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression

The mechanism of inhibitory action of bisphosphonates on bone resorption is not fully elucidated. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-kappaB ligand (RANKL) and the inhibitor, osteoprotegerin (OPG). To investigate in vitro effects of bisphosphonates on mouse osteoblastic cells, we examined the expression levels of RANKL and OPG in the cells treated with alendronate or pamidronate (10(-8)~10(-5) M) alone, or combined with 10 nM of 1,25-(OH)(2)VitD(3) for 24 or 48 h. Various concentrations of alendronate and pamidronate did not change the mRNA expression of RANKL and OPG consistently irrespective of 1,25-(OH)(2)VitD(3) presence. When added into cocultures of mouse osteoblastic cells and bone marrow cells, both alendronate and pamidronate inhibited osteoclast formation and bone resorption but failed to alter the RANKL and OPG mRNA expression. These results indicate that the inhibition of bone resorption by bisphosphonates is not mediated by the regulation of RANKL and OPG expression

Effects of Cell Cycle Inhibitors on Cell Death of Human Cancer Cell Lines

Genotoxic agents are known to cause cell death mostly by apoptosis in susceptible cells and this action plays an important role in tumor regression after chemotherapy. It has been thought that the apoptotic process may be associated with cell cycle. In the present study, the effect of aphidicolin and nocodazole, reversible cell cycle inhibitors, on the apoptosis or cell death induced by actinomycin D was studied in 3 cell lines. HeLa, SiHa and NIH／3T3 cells were treated with actinomycin D（100 nM）alone or in combination with cell cycle inhibitors for 20 hours; or presynchronized cells were treated with actinomycin D for 4 hours and further incubated for 20 hours in fresh medium. The synergistic effect of IBMX（5 or 10 μM）on the apoptosis or cell death induced by actinomycin D was also studied. The results were as follows; Majority of HeLa cells showed apoptotic changes after actinomycin D treatment. Aphidicolin　or nocodazole blocked neither initiation nor progression of actinomycin D-induced apoptosis in HeLa cell. Aphidicolin pretreatment enhanced the actinomycin D-induced apoptosis of HeLa cells and cell death of SiHa cells and nocodazole pretreatment enhanced cell death of SiHa cells. IBMX moderately enhanced actinomycin D-induced cell death of NIH／3T3 and to less extent that of SiHa cells. These results show that neither initiation nor progression of actinomycin D-induced apoptosis or cell death can be blocked by cell cycle arrest, and that cAMP is partly responsible for actinomycin D-induced apoptosis in certain types of cells

Alteration of DNA Synthesis and p53 Level by Modulators of Adenylate Cyclase System and Other Second Messenger Systems in Human Oral Keratinocytes

To study the role of adenylate cyclase system and other mediators of signal transduction pathway in the cell cycle arrest which provides enough time for the cells to repair damaged DNA, we examined the cellular response of primarily cultured human oral keratinocytes (HOKs) to UV irradiation, UV irradiation reduced replicative DNA synthesis of HOKs in a dose-drpendent manner without significant changes in viability. Dibutyryl cAMP(5 uM) and forskotin (5 uM) partly inhibited the reduction in replicative DNA failed to inhibit the reduction in replicative DNA synthesis at 12th hour after irradiation. At 24th hour after irradiation, none of the agents inhibited the suppression of DNA synthesis. The reduction in DNA synthesis was accompanied by marked p53 induction, which was completely blocked by dibutyryl cAMP (5 uM) forskolin (5 uM), IBMX (5 uM) and dibutyryl cGMP (5 uM). Treatment of TPA (5 uM), staurosporin (10 uM) and dexamethasone (1 uM) reduced DNA synthesis in HOKs ana prompted death of the cells. Those drugs, however. did not induce p53 indicating that different mechanism from that for UV irradiation may be involved in the reduction of replicative DNA synthesis. Increased reparative DNA synthesis induced by UV irradiation was suppressed by forskolin. Our results suggest that cAMP may be involved in DNA damage-induced cell cycle arrest and DNA repair, and cAMP and cGMP may be associated with blockade of p53 induction.This work was supported by a grant no.01-94-267 from the Seoul National University Hospital Research Fund

Effects of 5-Lipoxygenase Matabolites on the Osteoclast Fromation

Since 5-iipoxygenase metabolites production is known, to be enhanced in inflammed and malignant tissues which are associated with local bone destruction, ´ it is likely that 5iipoxygenase metabolites play some ro´,es in bone destruction. Therefore .ve performed this study to ´find out the effect of 5lipoxygenase metabolites on the osteoclast formation. Coculture of neonatal mouse calvarial osteoblastic cells and mouse bone ma-row cells was established and cultured for 7 days. After the culture, staining for tartrateresistant ac´ol phosphatase, a marker enzyme of osteoclast, was executed and the number of tartrateresistant acid ´phosphatase positive multinucleated cells was counted. 1,25-dhydroxyvitamn D; and tumor necrosis factor a significantly stimulated the osteoclast formation. When 5lipoxygenase pathway was blocked by caffeic acid, 1,25dihydroxyvitamin D, or tumor ´necrosis factor- a -induced osteoclast formation was significantly inhibited. Furthermore, when both cyclooxygenase and 54poxygenase pathway were blocked by combined treatment of indomethaoin and caffeic acid, 1,25dhydroxyvitamin D, o-tumor necrosis factor a -induced osteoclast formation was almost completely inhibited. Though phospholipase A, or meilitin failed to induce osteoclast formation- sy themselves, they significantly enhanced 1,25dihydroxyvitamin D:induced osteoclast formation. And caffeic acid partly , inh!Lted the osteoclast formation induced by comD´ned t7eatment of phospholipase A, and 125dihydroxyvitarrin D, or meliftur and 1,25dihydroxyvitamin D;. Taken toge her, 5lipoxygenase metabolites seem to have certain role(3) in the osteoc:ast formation induced by 1,25dihydroxyvitamin D; or tumor necrosis factora and in the local bone destruction associated with inflammation by enhancing osteoclast fDrmation