[Show abstract][Hide abstract] ABSTRACT: Transcription 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.Result: New 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.
Cell communication and signaling : CCS. 05/2014; 12(1):35.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
These data suggest that the regulatory genes newly identified in this study may play significant roles in tooth development.
Archives of oral biology 03/2012; 57(8):1100-7. · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Proteome Research 04/2010; 9(6):2946-56. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
In the present study, for the first time, we detect the secretion of sPLA2 during limb development and suggest that sPLA2 influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA2 promoted cell death by activating MMP-9 (matrix metalloproteinase-9) and increasing type I collagen degradation. The additive chondro-inhibitory actions were induced by co-treatment of mp-BSA (p-aminophenyl-mannopyranoside-BSA), a known ligand of the mannose receptor. Chondro-inhibitory actions by sPLA2 were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA2 (phospholipase A2) receptor and by inhibition of ERK (extracellular-signal-regulated kinase) activity.
Taken together, our results suggest that elevated levels of sPLA2 secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP-9 in a manner typical of receptor-mediated signalling and that these events lead to cell death.
Biology of the Cell 09/2009; 102(2):107-19. · 3.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. We explored the underlying mechanism of BMP-2-induced chondrocyte commitment of C3H10T1/2 cells. Treating cells with exogenous BMP-2 was tied to chondrocyte commitment by inhibiting matrix metalloproteinase-9 activity (MMP-9: 92 kDa type IV collagenase/gelatinase B). Glycogen synthase kinase (GSK)-3beta inhibition by its specific inhibitor blocked BMP-2-induced chondrocyte commitment by stimulating MMP-9 activity. These findings indicate that the downregulation of MMP-9 by BMP-2 is associated with chondrocyte commitment, and that the GSK-3beta signaling pathway is involved in this process.
Cell Biology International 06/2009; 33(8):887-92. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloprotease-2 (MMP-2) has the capacity to degrade cartilage extracellular matrix molecules, the turnover of which is an essential event in chondrogenesis. Here, we investigated the functional role of MMP-2 in chondrogenesis of leg bud mesenchymal cells. Small interference RNA (siRNA)-mediated knockdown of mmp-2 promoted precartilage condensation and chondrogenesis. Treatment with bafilomycin A1, an MMP-2 activator, or GM6001, an MMP inhibitor, at the pre-condensation stage resulted in the inhibition or promotion of chondrogenesis, respectively. By comparison, treatment at the post-condensation stage had little or no effect on chondrogenesis. These results indicate that MMP-2 is involved in the regulation of cell condensation. Inhibition of MMP-2 activity by mmp-2 specific siRNA increased the protein level of fibronectin, and integrins alpha5 and beta1. The interaction between focal adhesion kinase (FAK) and integrin beta1 leading to tyrosine phosphorylation of FAK was also enhanced. Moreover, inactivation of p38MAPK down-regulated the level of MMP-2 mRNA and activity, and increased mesenchymal cell condensation in parallel with enhanced phosphorylation of FAK. Taken together, our data indicate that MMP-2 mediates the inhibitory signals of p38MAPK during mesenchymal cell condensation by functioning as a negative regulator of focal adhesion activity regulated by FAK via interactions with fibronectin through integrin beta1.
[Show abstract][Hide abstract] ABSTRACT: Although transforming growth factors (TGFs) are implicated in the process of endochondral ossification, which is initiated by the differentiation of mesenchymal cells into chondrocytes, it is not clear how TGF-beta 3 regulates the chondrogenic differentiation of limb bud mesenchymal cells. Here, differential display polymerase chain reaction (DD-PCR) screening and RT-PCR analysis revealed that transcripts of A Disintegrin And Metalloprotease 10 (ADAM 10) decreased during the chondro-inhibitory action of TGF-beta 3 on cultured chick leg bud mesenchymal cells. Electroporation of ADAM 10 morpholino antisense oligonucleotides inhibited the ectodomain shedding of delta-1, and cell proliferation and subsequent precartilage condensation, in a manner similar to that caused by TGF-beta3. The suppression of mesenchymal cell proliferation induced by TGF-beta 3 and ADAM 10 morpholino antisense oligonucleotides was reversed by activation of ADAM 10 with phorbol 12-myristate 13-acetate (PMA) or knockdown of Notch-1 with siRNA. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF-beta 3 downregulates ADAM 10 and inhibits cell proliferation and subsequent precartilage condensation by inhibiting the ectodomain shedding of delta-1, and that this results in the activation of Notch signaling.
Molecules and Cells 09/2007; 24(1):139-47. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.
Molecules and Cells 01/2007; 22(3):353-9. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phospholipase A2 proteins are major regulators of the arachidonic acid cascade and are involved in various cellular responses. Previously, we reported that group IB PLA2 proteins stimulate MMP-2 activation and subsequent cell migration. Here, we describe a novel mechanism whereby sPLA2-induced proMMP-2 activation is regulated by intracellular cAMP in HT1080 cells, although sPLA2 itself had no effect on the regulation of cAMP levels. Exogenous dibutyryl cAMP (a cAMP analogue) strongly inhibited proMMP-2 activation, and cAMP elevating agents, namely, cholera toxin (a Gs activator) and forskolin (an adenylyl cyclase activator), abrogated basal and sPLA2-induced proMMP-2 activation. We also found that the down-regulation of TIMP-2 expression and extracellular signal-regulated kinase (ERK)1/2 activation by sPLA2 were blocked by increasing the intracellular cAMP level. Taken together, our data indicate that sPLA2-induced proMMP-2 activation is influenced by intracellular cAMP levels via the modulations of TIMP-2 expression and ERK1/2 activation.
Biochemical and Biophysical Research Communications 03/2006; 340(4):1278-83. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.
Journal of Biological Chemistry 09/2004; 279(35):36579-85. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-1beta 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-1beta-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-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-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-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.
Experimental and Molecular Medicine 07/2004; 36(3):226-32. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methyl-beta-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-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-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-beta-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-beta-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-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.
Experimental and Molecular Medicine 03/2004; 36(1):78-84. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and the production of nitric oxide, prostaglandin E2, which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.
Journal of Biological Chemistry 12/2003; 278(46):45117-27. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.
Journal of Biological Chemistry 09/2003; 278(34):31731-6. · 4.65 Impact Factor