Young-Ae Choi

Secretome analysis of human BMSCs and identification of SMOC1 as an important ECM protein in osteoblast differentiation.

Extracellular matrix proteins have been implicated in the regulation of osteoblast differentiation of bone marrow derived mesenchymal stem cells (BMSCs) through paracrine or autocrine mechanisms. In the current study, we analyzed the secretory protein profiles of BMSCs grown in osteogenic medium (OSM) and identified SPARC-related modular calcium-binding protein 1 (SMOC1), a member of the SPARC family, as a regulator of osteoblast differentiation of BMSCs. BMSCs with high and low osteogenic potential were grouped and stimulated with OSM, after which conditioned medium was collected and analyzed by LC-MS/MS. We identified 410 proteins, 64 of which were selectively secreted by high osteogenic potential BMSCs. Of these 64 secreted proteins, we selected extracellular matrix proteins for validation in BMSCs undergoing osteoblast differentiation and found that SMOC1 is highly expressed and secreted in BMSCs stimulated with OSM. To examine the role of SMOC1 in osteoblast differentiation, we analyzed the effect of SMOC1 knockdown and overexpression using shRNAs and wild-type cDNA, respectively. Knockdown of SMOC1 significantly inhibited mineralization and the expression of osteoblast differentiation markers, while overexpression of SMOC1 substantially increased the expression of osteoblast differentiation-related genes. Thus, validation of secretome profiling data identified SMOC1 as a putative regulator of osteoblast differentiation of BMSCs.

Drug delivery from hydroxyapatite-coated titanium surfaces using biodegradable particle carriers.

The goal of this study was to develop a functional titanium (Ti) implant loaded with bioactive molecules using biodegradable polymeric particles as drug delivery carrier for dental applications. In this study, dexamethasone (DEX)-loaded poly(lactic-co-glycolic acid) (PLGA) particles were electrostatically immobilized on a Ti disc surface coated with hydroxyapatite (HA) nanocrystals using a low temperature high speed collision (LTHSC) method. Resorbable blasting media (RBM) Ti discs (S1), HA-Ti discs (S2), and HA-Ti discs treated with DEX-loaded PLGA particles (S3) were fabricated in this study as sample discs. To facilitate surface immobilization, PLGA particles were coated with polyethyleneimine (PEI) to produce a positive surface charge. This modification of PLGA particle surfaces, allowed DEX-loaded PLGA particles to be immobilized on negatively charged S2 disc surface. It was found that DEX-loaded PLGA particles were well dispersed and immobilized onto the S3 disc surfaces. Release profile studies of DEX from S3 discs in a 4-week immersion study indicated an initial burst release followed by sustained release. In vitro evaluation of bone marrow derived mesenchymal stem cells (BMSCs) cultured for 1 and 2 weeks on S3 discs showed greater BMSC differentiation than on S1 or S2 discs, demonstrating that this innovative delivery platform potently induced BMSC differentiation in vitro, and suggesting that it could be exploited for stem cell therapy purposes or to enhance in vivo osteogenesis. In addition, the results of the present study shows that various bioactive molecules that promote bone regeneration can be efficiently incorporated onto HA-Ti surfaces using biodegradable polymeric particles.

Interleukin-1β stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes

IL-1β is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1β-induced matrix metalloproteinase-2 (MMP- 2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1β promotes the proteolytic process leading to MMP-2 activation. IL-1β-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1β-induced MMP-2 activation. Taken together, these results demonstrated that IL-1β induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.

Methyl-beta-cyclodextrin inhibits cell growth and cell cycle arrest via a prostaglandin E(2) independent pathway.

Methyl-β-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-β-cyclodextrin in Raw264.7 macrophage cells. Methyl-β-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-β-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-β-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-β-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.

Gene expression profiling of oral epithelium during tooth development.

OBJECTIVE Tooth development is regulated by the complex interplay of various regulatory molecules. To identify new regulatory genes released from the dental epithelium, gene expression profiling of dental epithelium was analysed. DESIGN ICR mouse dental epithelia were isolated from the initiation (E10.5) and bell (E16.5) stages, and microarray analysis was performed using Affymetrix GeneChip(®). Microarray data were validated using reverse transcriptase polymerase chain reaction (RT-PCR), and gene ontology and signalling network were analysed. RESULTS Detection signals more than 300 and changes more than two folds were considered as positive signals and were further analysed. Expressions of 193 genes in the E10.5 epithelium and 582 genes in the E16.5 epithelium were significantly increased. Validation of the selected genes using RT-PCR showed a well correlation with microarray data. Subsequent signalling network analysis revealed that at E10.5 and 16.5, nine genes such as histones, signalling molecules and transcription factors were closely related with neighbouring molecules. Moreover, gene ontology analysis showed that seven growth factors/receptors or secreted proteins were highly expressed at E10.5, including the platelet-derived growth factor, C polypeptide (Pdgfc), insulin-like growth factor binding protein 2 (Igfbp2) and Igfbp5. At E16.5, nine growth factors/receptors or secreted proteins, including Igfbp3, Igfbp10/Cyr61 and heparin-binding EGF-like growth factor (Hbegf) were highly expressed. CONCLUSIONS These data suggest that the regulatory genes newly identified in this study may play significant roles in tooth development.

Secretory phospholipase A2 promotes MMP-9-mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis.

Background information. sPLA2 (secretory phospholipase A2) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA2 during chondrogenesis have not been studied.

A genetic variation in microRNA target site of KRT81 gene is associated with survival in early stage non-small cell lung cancer

BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.BACKGROUND MicroRNAs (miRNAs) have a key role in carcinogenesis through negative regulation of their target genes. Therefore, genetic variations in miRNAs or their target sites may affect miRNA-mRNA interactions, thereby result in altered expression of target genes. This study was conducted to investigate the associations between single-nucleotide polymorphisms (SNP) located in the miRNA target sites (poly-miRTSs) and survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS Using public SNP database and miRNA target sites prediction program, 354 poly-miRTSs were selected for genotyping. Among these, 154 SNPs applicable to Sequenoms MassARRAY platform were investigated in 357 patients. A replication study was carried out on an independent patient population (n = 479). Renilla luciferase assay and reverse transcription-polymerase chain reaction were conducted to examine functional relevance of potentially functional poly-miRTSs. RESULTS Of the 154 SNPs analyzed in a discovery set, 14 SNPs were significantly associated with survival outcomes. Among these, KRT81 rs3660G>C was found to be associated with survival outcomes in the validation cohort. In the combined analysis, patients with the rs3660 GC + CC genotype had a significantly better overall survival compared with those with GG genotype [adjusted hazard ratio (aHR) for OS, 0.65; 95% confidence interval (CI) 0.50-0.85; P = 0.001]. An increased expression of the reporter gene for the C allele of rs3660 compared with the G allele was observed by luciferase assay. Consistently, the C allele was associated with higher relative expression level of KRT81 in tumor tissues. CONCLUSION The rs3660G>C affects KRT81 expression and thus influences survival in early-stage NSCLC. The analysis of the rs3660G>C polymorphism may be useful to identify patients at high risk of a poor disease outcome.

Integrin signaling and cell spreading alterations by rottlerin treatment of chick limb bud mesenchymal cells

Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. Prechondrogenic condensation, in which mesenchymal cells aggregate, requires cell migration and proliferation. However, the molecular mechanisms promoting this aggregation remain to be elucidated. Here, we report that rottlerin suppresses migration and cell surface expression of integrin beta1 in chondrogenic progenitors. Perturbation of integrin beta1 function using an anti-integrin beta1 blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by rottlerin treatment. Cell treatment with PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, suppressed cell migration in a wound-healing assay. Cells treated with rottlerin showed decreased phosphorylation of Akt, independent of PKCdelta inhibition. In addition, an Akt inhibitor suppressed the migration of chick limb bud mesenchymal cells. Taken together, our results point to the novel finding that rottlerin may act as a negative regulator for cell migration, an essential step for prechondrogenic condensation, by regulating integrin beta1 signaling at focal adhesion complexes via modulation of Akt activity.

Bobby Sox homology regulates odontoblast differentiation of human dental pulp stem cells/progenitors

BackgroundTranscription factors have been implicated in regulating the differentiation of odontoblasts from dental pulp stem cells/progenitors (DPSCs/progenitors), but their regulatory network is not completely understood.ResultNew transcription factors that control the odontoblast differentiation of human DPSCs/progenitors were analyzed using a microarray. The result revealed bobby sox homolog (BBX) to be expressed most strongly during odontoblast differentiation. Validation using RT-PCR also revealed the strong expression of BBX during the odontoblast differentiation of DPSCs/progenitors. BBX expression was also detected in adult molar odontoblasts and other tissues, including the heart, kidney, testis, and bone marrow. To understand the role of BBX in odontoblast differentiation, BBX variant 1 and 2 cDNA were cloned and overexpressed in DPSCs/progenitors. The results showed that the overexpression of BBX cDNA in DPSCs/progenitors induced substantial mineralization and expression of the odontoblast marker genes, such as ALP, OPN, BSP, DMP1, and DSPP. The knockdown of BBX using shRNA, however, did not affect mineralization, but the expression of ALP and DSPP was decreased substantially. Meanwhile overexpression or knockdown of BBX did not modulate proliferation of DPSCs/progenitors.ConclusionOur results suggest that BBX plays an important role during the odontoblast differentiation of human DPSCs/progenitors.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1–100 μmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.